KR20090056922A - Genetically-modified cell line for producing cloned miniature pigs for xenotransplantation and method for preparing the same - Google Patents
Genetically-modified cell line for producing cloned miniature pigs for xenotransplantation and method for preparing the same Download PDFInfo
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- KR20090056922A KR20090056922A KR1020080120498A KR20080120498A KR20090056922A KR 20090056922 A KR20090056922 A KR 20090056922A KR 1020080120498 A KR1020080120498 A KR 1020080120498A KR 20080120498 A KR20080120498 A KR 20080120498A KR 20090056922 A KR20090056922 A KR 20090056922A
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Abstract
Description
본 출원은 2007년 11월 30일에 출원된, 대한민국 특허출원 제2007-123823호에 대한 우선권의 이익을 향유하며, 상기 출원서 전체는 본 출원에 참조로서 삽입된다.This application enjoys the benefit of priority to Korean Patent Application No. 2007-123823, filed November 30, 2007, the entire application of which is incorporated herein by reference.
본 발명은 이종 장기 이식용 미니 복제돼지 생산을 위한 유전자 조작된 세포주 및 그의 생산 방법에 관한 것이다. The present invention relates to a genetically engineered cell line for producing miniature pigs for xenotransplantation and a method of producing the same.
국립장기이식센터에 따르면 국내에서 한해 2만 여명에 달하는 환자들이 장기이식을 기다리고 있으나, 기증 건수는 수요의 10%에도 못 미치는 것으로 보고 되었다. 또한 미국의 경우 장기를 필요로 하는 대기자는 16분에 한명씩 추가 되고 있으나, 하루에 대기자 중 11명은 수술 받지도 못하고 사망하는 실정이다. 이러한 상황과 더불어 생명과학기술의 발달은 이종 장기이식 기술개발의 배경이 된다.According to the National Organ Transplantation Center, more than 20,000 patients in Korea are waiting for organ transplants, but the number of donations is reported to be less than 10% of the demand. In addition, in the United States, there is an additional waiting time every 16 minutes for the need for organs. However, 11 of the waiting days die without surgery. Together with this situation, the development of life science and technology is the background for the development of xenotransplantation technology.
동물 유전학의 꾸준한 진보는 특정 유전자를 제거 또는 삽입함으로써 각 유전자의 역할 규명과 함께 상업적으로 유용한 형질전환 동물 생산을 가능케 하였다. 형질전환된 동물 제조를 위한 방법으로는 미세주입법(microinjection) 또는 바이러스 감염법(viral infection)등을 이용한 무작위적인 유전자 조작법과 배아줄기세포(embrynic stem cells) 또는 체세포(somatic cells)를 이용하여 특정 유전자를 타겟팅 시킬 수 있는 유전자 적중법이 있다. Steady progress in animal genetics Removal or insertion of specific genes enabled the identification of each gene's role and enabled the production of commercially useful transgenic animals. Methods for preparing transformed animals include random gene manipulation using microinjection or viral infection, and specific genes using embryonic stem cells or somatic cells. There is a gene targeting method that can target.
미세주입법은 외래 DNA를 수정란의 전핵에 삽입하는 고전적인 방법으로 형질전환 동물을 생산하기 위해 널리 이용되어져 왔다(Harbers et al., Nature, 293(5833): 540-2, 1981; Hammer et al., Nature, 315(6021): 680-683, 1985; van Berkel et al., Nat. Biotechnol., 20(5): 484-487, 2002; Damak et al., Biotechnology(NY), 14(2): 185-186, 1996). 그러나 미세주입법으로 외래 DNA가 삽입된 수정란 유래의 형질전환된 산자의 생산 비율은 2 내지 3 % 로 효율이 매우 낮으며(Clark et al., Transgenic Res., 9: 263-275, 2000), 외래 유전자의 삽입 위치의 조절 및 특정 내부 유전자의 제거가 불가능하다. Microinjection has been widely used to produce transgenic animals by the classical method of inserting foreign DNA into the embryonated nucleus (Harbers et al ., Nature , 293 (5833): 540-2, 1981; Hammer et al . , Nature , 315 (6021): 680-683, 1985; van Berkel et al ., Nat. Biotechnol. , 20 (5): 484-487, 2002; Damak et al ., Biotechnology (NY), 14 (2) : 185-186, 1996). However, the production rate of transformed litter derived from fertilized eggs with foreign DNA inserted by microinjection was very low (Clark et al ., Transgenic Res. , 9: 263-275, 2000). It is not possible to control the insertion position of genes and to remove certain internal genes.
바이러스감염법 또한 동물의 유전자 조작을 위해 널리 사용된다(Soriano et al., Genes Dev., 1(4): 366-375, 1987; Hirata et al., Cloning Stem Cells, 6(1): 31-36, 2004). 바이러스 감염법은 삽입하고자 하는 유전자가 바이러스 벡터를 통하여 동물의 유전자로 도입되므로 미세주입법보다 좀더 효율적이나, 여전히 특정 위치로의 외래 유전자의 삽입 및 특정 내부 유전자의 제거가 불가능하다. 또한, 삽입 하고자 하는 유전자의 최대 사이즈는 7 kb로 제한되며, 바이러스에 의해 발현되는 단백질이 문제된다(Wei et al., Annu. Rev. Pharmacol. Toxicol., 37: 119-141, 1997; Yanez et al., Gene Ther., 5(2): 149-159, 1998). Viral infections are also widely used for genetic manipulation of animals (Soriano et al ., Genes Dev. , 1 (4): 366-375, 1987; Hirata et al ., Cloning Stem Cells , 6 (1): 31- 36, 2004). Viral infection is more efficient than microinjection because the gene to be inserted is introduced into the animal's gene through the viral vector, but it is still impossible to insert a foreign gene into a specific position and to remove a specific internal gene. In addition, the maximum size of the gene to be inserted is limited to 7 kb, the protein expressed by the virus is a problem (Wei et al ., Annu. Rev. Pharmacol. Toxicol. , 37: 119-141, 1997; Yanez et. al ., Gene Ther. , 5 (2): 149-159, 1998).
위에서 언급한 문제점들 극복하기 위해서, 특정 유전자를 제거 또는 삽입할 수 있는 유전자 타겟팅 기술이 이용될 수 있다. 유전자 타겟팅 기술은 마우스 배아 줄기세포를 이용한 유전자 기능 연구에서 처음으로 사용되었다. 상동 재조합을 이용하여 특정 유전자가 타겟팅된 마우스 배아 줄기세포를 배반포 단계에 있는 배아에 삽입함으로써 결국 특정 유전자가 조작된 산자 생산이 가능하게 된다. 이러한 마우스 배아줄기세포에 유전자 타겟팅법을 이용하면서, 많은 수의 특정 유전자 타겟팅된 마우스가 생산되었다(Brandon et al., Curr. Biol., 5(6): 625-634, 1995; Capecchi et al., Science, 244(4910): 1288-1292, 1989; Thompson et al., Cell, 56(2): 313-321, 1989; Hamanaka et al., Hum. Mol. Genet., 9(3): 353-361, 2000; Thomas et al., Cell, 51(3): 503-512, 1987; te Riele et al., Proc. Natl. Acad. Sci. USA, 89(11): 5182-5132, 1992; Mansour et al., Nature, 336(6197): 348-352, 1988; Luo et al., Oncogene, 20(3): 320-328, 2001). 유전자 타겟팅법이 가축에 적용될 때, 대량의 치료 단백질을 생산하는 동물생체 반응기(animal bioreactor) 또는 면역거부반응에 관여하는 유전자를 제거 또는 특정 위치에서의 과다발현에 의해 이종간 장기이식에 사용될 수 있는 질병 모델 동물 생산이 가능하며, 이는 산업적으로 큰 경제적인 이익을 가져올 것으로 예상되고 있다. To overcome the problems mentioned above, gene targeting techniques can be used that can remove or insert a particular gene. Gene targeting techniques were used for the first time in the study of gene function using mouse embryonic stem cells. By using homologous recombination, mouse embryonic stem cells targeted to specific genes are inserted into embryos in the blastocyst stage, thereby enabling production of engineered litters of specific genes. Using gene targeting methods on these mouse embryonic stem cells, a large number of specific gene targeted mice have been produced (Brandon et al ., Curr. Biol. , 5 (6): 625-634, 1995; Capecchi et al . , Science , 244 (4910): 1288-1292, 1989; Thompson et al ., Cell , 56 (2): 313-321, 1989; Hamanaka et al ., Hum.Mol.Genet . , 9 (3): 353 -361, 2000; Thomas et al. , Cell , 51 (3): 503-512, 1987; te Riele et al ., Proc. Natl. Acad. Sci. USA , 89 (11): 5182-5132, 1992; Mansour et al ., Nature , 336 (6197): 348-352, 1988; Luo et al ., Oncogene , 20 (3): 320-328, 2001). When genetic targeting is applied to livestock, diseases that can be used for cross-organ transplantation by eliminating genes involved in animal bioreactors or immune rejection reactions that produce large amounts of therapeutic proteins or by overexpression at specific sites Model animal production is possible, which is expected to bring significant economic benefits.
유전자 타겟팅된 동물을 생산하기 위해서, 배아줄기세포의 사용이 필수적인 요소로 여겨져 왔다. 그러나 돼지와 소를 포함한 가축에서 배아줄기세포와 유사한 세포 주들이 보고 되었음에도 불구하고, 현재까지 가축에서 배아 줄기 세포의 사용은 제한된다(Doetschman et al., Dev. Biol., 127(1): 224-227, 1988; Stice et al., Biol. Reprod., 54(1): 100-110, 1996; Sukoyan et al., Mol. Reprod. Dev., 36(2): 148-158, 1993; Iannaccone et al., Dev. Biol., 163(1): 288-292, 1994; Pain et al., Development, 122(8): 2339-2348, 1996; Thomson et al., Proc. Natl. Acad. Sci. USA, 92(17): 7844-7848, 1995; Wheeler et al., Reprod. Fertil. Dev., 6(5): 563-568, 1994). 대신에, 핵 공여 세포로서 일반 체세포가 유전자 타겟팅에 사용될 수 있다는 가능성이 제시되면서, 형질전환된 복제 가축 생산이 가능하게 되었다(Brophy et al., Nat. Biotechnol., 21(2): 157-162, 2003; Cibelli et al., Science, 280(5367): 1256-1258, 1998; Campbell et al., Nature, 380(6569): 64-66, 1996; Akira Onishi et al., Science, 289;1188-1190, 2000 Denning et al., Cloning Stem Cells, 3(4): 221-231, 2001; McCreath et al., Nature, 405(6790): 1066-1069, 2000). In order to produce genetically targeted animals, the use of embryonic stem cells has been considered an essential factor. However, although embryonic stem cell-like cell lines have been reported in livestock, including pigs and cattle, the use of embryonic stem cells in livestock is currently limited (Doetschman et al ., Dev. Biol. , 127 (1): 224) . -227, 1988; Stice et al ., Biol. Reprod. , 54 (1): 100-110, 1996; Sukoyan et al ., Mol. Reprod. Dev. , 36 (2): 148-158, 1993; Iannaccone et al ., Dev. Biol. , 163 (1): 288-292, 1994; Pain et al. , Development , 122 (8): 2339-2348, 1996; Thomson et al ., Proc. Natl. Acad. Sci USA , 92 (17): 7844-7848, 1995; Wheeler et al ., Reprod. Fertil. Dev. , 6 (5): 563-568, 1994). Instead, the possibility of normal somatic cells as nuclear donor cells can be used for gene targeting, thus enabling the production of transformed cloned livestock (Brophy et al ., Nat. Biotechnol ., 21 (2): 157-162 , 2003; Cibelli et al ., Science , 280 (5367): 1256-1258, 1998; Campbell et al ., Nature , 380 (6569): 64-66, 1996; Akira Onishi et al ., Science , 289; 1188 -1190, 2000 Denning et al ., Cloning Stem Cells , 3 (4): 221-231, 2001; McCreath et al ., Nature , 405 (6790): 1066-1069, 2000).
한편, 이종 장기이식의 최적의 제공 원으로는 장기의 크기 및 생리적 특징이 인간과 유사하고 다산성에 의해 장기의 대량 공급이 가능한 미니 돼지가 고려된다. On the other hand, as an optimal source of xenotransplantation, mini pigs, which are similar to humans in organ size and physiological characteristics and which can be supplied in large quantities by fertility, are considered.
돼지 장기의 성공적인 이식은 일련의 면역거부반응(초 급성, 급성 혈관성, 세포 매개성, 및 만성 면역거부반응)의 극복에 의존하다. 이식 후 수분 내에 발생하는 초 급성 면역거부반응은 알파-1,3-갈락토실 항원 결정기 합성에 관여하는 유전자를 제거하고 인체 보체 조절 유전자를 과다발현 시킴으로써 극복될 수 있음이 보고되었다. Successful transplantation of porcine organs relies on overcoming a series of immune rejection reactions (super acute, acute vascular, cell mediated, and chronic immune rejection). It has been reported that hyperacute rejection reactions occurring within minutes after transplantation can be overcome by removing genes involved in the synthesis of alpha-1,3-galactosyl epitopes and overexpressing human complement regulatory genes.
구체적으로, 2002년 영국의 PPL사로부터 세계 최초로 알파-1,3-갈락토실트랜스퍼라아제(alpha-1,3-galactosyltransferase, 이하, 'GT'로 약창힘) 유전자가 이형접합적으로(heterozygous) 제거된 체세포 복제 돼지가 생산되었다 (Yifan Dai et al., Nat. Biotechnology, 20: 251-255, 2002). GT 유전자는 이종간 이식시 급속한 면역거부반응을 일으키는 원인 유전자로서, 이 유전자가 제거될 경우 생체거부반응이 제거된 이종간 장기 이식용 질환모델동물 개발이 가능하다. 2003년에 동 회사에서 GT 유전자가 동형접합적으로(homozygous) 제거된 체세포 복제에 성공함으로써 현 장기부족현상을 해결해 줄 수 있는 장기 이식용 질환모델동물 생산을 위한 획기적인 진보를 가져왔다(Carol J. Phelps, Science, 299: 411-414, 2003). Specifically, in 2002, PPL of the UK, the world's first alpha-1,3-galactosyltransferase (hereinafter, abbreviated as 'GT') gene was heterozygous (heterozygous) Removed somatic cloned pigs were produced (Yifan Dai et al ., Nat. Biotechnology , 20: 251-255, 2002). The GT gene is a gene that causes a rapid immune rejection reaction between xenotransplantation. If this gene is removed, it is possible to develop a disease model animal for xenotransplantation where the biorejection response is removed. In 2003, the company succeeded in replicating somatic cells from which the GT gene was homozygous removed, resulting in breakthroughs in the production of disease model animals for organ transplantation that could address the current shortage of organs (Carol J. Phelps, Science , 299: 411-414, 2003).
2005년 GT 유전자 제거된 복제 돼지 유래의 장기는 원숭이로 이식되었으며, 그 결과 급성 면역 거부반응 없이 이식 후 2-6 개월까지 생존이 지연됨을 보고하였다(Kenji Kuwaki et al., Nature Medicine, 11(1): 29-31, 2005). 그러나 GT 유전자가 제거되었음에도 불구하고 다른 경로를 통하는 이종 항원에 의해 인체 보체 유전자들이 활성화 됨으로써 장기 이식시 심각한 거부반응이 초래될 수 있음이 보고되었다(Tanemura, M. et al., Biochem. Biophys. Res. Commun., 235: 359-364, 1997; Komoda, H. et al., Xenotransplantation, 11: 237-246, 2004). 그러한 장애를 극복하기 위해, GT 유전자 제거와 함께 분해 촉진 인자(decay-accerating factor, 이하, 'DAF'로 약칭함), 막 보조인자 단백질(membrane co-factor protein, 이하, 'MCP'로 약칭함), CD59와 같은 인체 보체 억제 유전자를 과발현하는 복제 돼지를 생산하는 방법이 사용되었다(Yoichi Takahagi, Molecular Reproduction and Development 71: 331-338, 2005 Cozzi, Eb et al., Transplant Proc., 26: 1402-1403, 1994; Fodor, W. L. et al., Proc. Natl. Acad. Sci. USA 91: 11153-11157, 1994; Adams, D. H. et al., Xenotransplantation, 8: 36-40, 2001). In 2005, GT-derived cloned pig-derived organs were transplanted into monkeys, and as a result, survival was delayed until 2-6 months after transplantation without acute immune rejection (Kenji Kuwaki et al ., Nature Medicine , 11 (1). ): 29-31, 2005). However, even though the GT gene has been eliminated, it has been reported that the activation of human complement genes by heterologous antigens via other pathways can lead to severe rejection in organ transplantation (Tanemura, M. et al. , Biochem. Biophys. Res). Commun ., 235: 359-364, 1997; Komoda, H. et al ., Xenotransplantation , 11: 237-246, 2004). In order to overcome such obstacles, along with GT gene removal, decay-accerating factor (abbreviated as 'DAF'), membrane co-factor protein (abbreviated as 'MCP') ), A method of producing cloned pigs that overexpress human complement inhibitory genes such as CD59 was used (Yoichi Takahagi, Molecular Reproduction and Development 71: 331-338, 2005 Cozzi, Eb et al ., Transplant Proc. , 26: 1402). -1403, 1994; Fodor, WL et al ., Proc. Natl. Acad. Sci. USA 91: 11153-11157, 1994; Adams, DH et al ., Xenotransplantation , 8: 36-40, 2001).
한편, 인간을 제외한 대부분의 포유동물에 존재하는 N-글루콜릴뉴라미닌산(N-glycolylneuraminic acid, 이하, 'Neu5Gc'로 약칭함) 항원 결정기 또한 이종장기이식시 면역거부반응을 야기함이 보고되었다(WO20061133356A; Pam Tangvoranuntakul, Proc. Natl. Acad. Soc. USA 100: 12045-12050, 2003; Barbara Bighignoli, BMC genetics, 8: 27, 2007). Neu5Gc는 CMAH에 의해 N-아세틸뉴라미닌산(N-acetylneuraminic acid, 이하, 'Neu5Ac'로 약칭함)로부터 전환된다. Meanwhile, N-glycolylneuraminic acid (hereinafter abbreviated as 'Neu5Gc') antigenic determinant present in most mammals except humans has also been reported to cause immunorejection reactions during xenotransplantation. (WO20061133356A; Pam Tangvoranuntakul, Proc. Natl. Acad. Soc. USA 100: 12045-12050, 2003; Barbara Bighignoli, BMC genetics , 8: 27, 2007). Neu5Gc is converted from N-acetylneuraminic acid (hereinafter abbreviated as 'Neu5Ac') by CMAH.
보체는 생체 내에서 면역작용에 관계하는 단백질들로 구성된 단백질 복합체 (C1-C9)로서 항원-항체 복합체가 형성되면 보체가 세균의 세포막에 결합하여 구멍을 내는 보체고정과 또는 항원-항체 복합체에 보체가 결합되어 식세포작용이 촉진되는 식균작용증진(opsonization)이 있다. 이러한 보체의 활동을 조절할 수 있는 여러가지의 조절 단백질들이 발견되었으며, 보체의 활성화를 막거나 활성화된 보체의 분해를 촉진함으로써 보체의 작용을 조절한다. 숙주의 세포막에 있는 DAF는 C2와 C4b의 결합을 방해할 수 있으며, MCP는 C4b의 분해를 촉진하여 보체가 숙주세포에서 활성화되는 것을 막아줌으로서 보체에 의한 숙주세포 파괴를 방지할 수 있다. 숙주 세포 표면에 있는 CD59는 C7, C8과 C5b6의 결합을 방해하여 막 공격 복합체의 형성을 막을 수 있다. Complement is a protein complex (C1-C9) consisting of proteins involved in immunity in vivo. When an antigen-antibody complex is formed, the complement binds to the bacterial cell membrane and punctures the complement or the antigen-antibody complex. There is opsonization that is combined to promote phagocytosis. A number of regulatory proteins have been found that can modulate the activity of complement, and regulate the actions of complement by preventing the activation of complement or by promoting the degradation of activated complement. DAF in the cell membrane of the host can interfere with the binding of C2 and C4b, MCP promotes the degradation of C4b to prevent the complement from being activated in the host cell to prevent host cell destruction by complement. CD59 on the surface of the host cell can interfere with the binding of C7, C8 and C5b6 to prevent the formation of membrane attack complexes.
상기 보체 작용 조절 유전자 외에도, 인간 CD39 유전자의 과다발현에 의해, 이종장기이식 시 발생하는 혈전증이 억제됨이 보고되어져 있다(US20080003212A, Karren, M. D., The Journal of Clinical Investigation, 113: 1440-1446, 2004). In addition to the complement-regulatory gene, overexpression of human CD39 gene has been reported to inhibit thrombosis caused by xenotransplantation (US20080003212A, Karren, MD, The Journal of Clinical Investigation , 113: 1440-1446, 2004). .
기존의 보고에 의하면, 외래 유전자의 정규장소 외의 발현은 배 발달에 장애를 야기하고 배아 발달 후기와 출생 후 초기에 대부분 발달하는 신경 시스템에 치명적이라는 문제점이 있다(Gao et al., Neurochem. Res., 24(9): 1181-1188, 1999). Existing reports suggest that off-regular expression of foreign genes causes disturbances in embryonic development and is fatal to the nervous system, which develops most during the late embryonic and early postnatal periods (Gao et al ., Neurochem. Res. , 24 (9): 1181-1188, 1999).
이에, 상기 문제점의 극복을 위해, 예의 노력한 결과, 본 발명자들은 알파-1,3-갈락토실 항원 결정기 합성에 관여하는 GT 유전자 또는 Neu5Gc 항원 결정기 합성에 관여하는 CMAH 유전자를 제거함과 동시에 상기 유전자의 좌위에 이종 면역반응 또는 혈전증과 같은 이종장기 이식시 야기되는 부작용을 억제할 수 있는 인체 보체 조절 유전자 또는 혈전억제 유전자를 적중시켜, 상기 유전자를 과발현시킬 수 있는 타겟팅 벡터를 제작하였으며, 이를 이용하여, 상기 GT 유전자 또는 CMAH 유전자가 넉아웃되면서도, 당해 넉아웃된 위치에 삽입된 보체 조절유전자 또는 혈전억제 유전자가 과발현되는 체세포주의 제조에 성공하였다.Therefore, in order to overcome the above problems, the present inventors have made efforts to remove the GT gene involved in the synthesis of alpha-1,3-galactosyl epitopes or the CMAH gene involved in the synthesis of Neu5Gc epitopes. A targeting vector capable of overexpressing the gene was prepared by targeting a human complement regulatory gene or a thrombus suppressor gene capable of suppressing side effects caused by heterologous transplantation such as a heterologous immune response or thrombosis. Even when the GT gene or CMAH gene was knocked out, the somatic cell line was successfully overexpressed with the complement regulatory gene or the thrombus suppressor gene inserted into the knocked out position.
본 발명에 의해 제조된 타켓팅 벡터 및 형질전환 세포주는 면역거부반응에 관여하는 유전자들의 발현을 복합적으로 조절함으로써, 효율적으로 이종장기이식용 복제 돼지생산에 이용될 수 있다.The targeting vector and the transformed cell line prepared by the present invention can be effectively used for the production of cloned pigs for xenotransplantation by efficiently controlling the expression of genes involved in immunorejection.
본 발명의 첫 번째 목적은 이종 항원 결정기 합성 유전자의 넉아웃 및 보체 작용 조절 유전자 또는 항혈전증 유전자의 삽입이 가능한 유전자 타켓팅 벡터를 제공하는 것이다.It is a first object of the present invention to provide a gene targeting vector capable of knocking out a heterologous antigenic determinant synthetic gene and inserting a complement action regulatory gene or an antithrombogenic gene.
본 발명의 두 번째 목적은 상기 유전자 타켓팅 벡터로 형질전환되어, 이종 항원 결정기 합성 유전자가 넉아웃되고, 상기 이종 항원 결정기 합성 유전자가 존재하는 좌위에 상기 보체 작용 조절 유전자 또는 항혈전증 유전자가 삽입되어 과발현되는 것을 특징으로하는 형질전환 체세포주를 제공하는 것이다.A second object of the present invention is transformed with the gene targeting vector, the heterologous antigenic determinant synthetic gene is knocked out, and the complement action regulatory gene or antithrombosis gene is inserted at the locus where the heterologous antigenic determinant synthetic gene is present. It is to provide a transgenic somatic cell line characterized in that.
본 발명의 세 번째 목적은 상기 형질전환 체세포주의 핵 이식을 통해 제조되는 비인간 복제동물을 제공하는 것이다.It is a third object of the present invention to provide a non-human clone produced by nuclear transfer of the transgenic somatic cell line.
본 발며의 네 번째 목적은 상기 비인간 복제동물을 사육한 후, 장기를 적출하는 단계를 포함하는 면역거부반응을 제거한 이식용 이종 장기의 생산방법을 제공하는 것이다.A fourth object of the present invention is to provide a method for producing a heterologous organ for transplantation, wherein the immune rejection reaction including the step of extracting the organ after breeding the non-human clone animal is removed.
용어의 정의Definition of Terms
본 발명에서 용어, "유전자 타겟팅 벡터(gene targeting vector)"는 게놈의 특정 유전자 위치로 목적하는 유전자를 제거 또는 삽입할 수 있는 벡터를 말하며, 상동 재조합(homologous recombination)이 일어나도록 타겟팅하고자 하는 특정 유 전자에 상동 염기 서열을 포함한다. 용어 "벡터"와 "벡터 카세트"는 동일한 의미로 사용되고 있으며, 원형(circular) 또는 선형(linear) 형태일 수 있다.As used herein, the term "gene targeting vector" refers to a vector capable of removing or inserting a gene of interest into a specific gene position of a genome, and the specific gene to be targeted for homologous recombination to occur. The homologous base sequence is included in the former. The terms "vector" and "vector cassette" are used in the same sense and may be of circular or linear form.
본 발명에서 용어 "상동(homologous)"은 제1영역 또는 제2영역과 이에 해당하는 유전자의 핵산 서열과의 동일성 정도를 나타내는 것으로, 적어도 90% 이상 동일하고, 바람직하게는 95% 이상 동일하다. As used herein, the term "homologous" refers to a degree of identity between a first region or a second region and a nucleic acid sequence of a gene corresponding thereto, and is at least 90% or more identical, and preferably 95% or more identical.
본 발명의 용어 "항원 결정기"는 이종 장기의 이식시 수용체의 면역시스템에 의하여 항원으로 인식되는 부위를 의미하는데, 대표적인 것으로는 세포표면 당쇄인 갈락토오스-알파1,3-갈락토오스(galactose-α1,3-galactose, 이하, 'αGal'로 약칭함) 및 N-글리콜릴뉴라민닌산(N-glycolylneuraminic acid, 이하, 'Neu5Gc'라 약칭함)이 있고, "항원 결정기 합성 유전자"는 상기 항원 결정기를 생합성하는 효소를 암호화하는 유전자로서, 상기 효소에는 αGal의 생합성을 담당하는 알파-1,3-갈락토실트랜스퍼라아제(이하, 'GT'로 약칭함)와 Neu5Gc의 생합성을 담당하는 CMP-아세틸뉴라미닌산 히드록실라아제(이하, 'CMAH'로 약칭함)가 대표적이다.The term "antigen determinant" of the present invention refers to a site recognized as an antigen by the immune system of the receptor during transplantation of heterologous organs, representatively galactose-
본 발명에서 용어 "선별마커(selection marker)"란 세포로 유전자 타겟팅 벡터로 형질전환된 세포를 선별하기 위한 것으로, 약물 내성, 영양 요구성, 세포 독성제에 대한 내성 또는 표면 단백질의 발현과 같은 선택가능 표현형을 부여하는 마커들이 사용될 수 있고, 양성 선별마커와 음성 선별마커가 있다. As used herein, the term "selection marker" is for selecting cells transformed with a gene targeting vector into cells, and selecting such drugs as drug resistance, nutritional requirements, resistance to cytotoxic agents or expression of surface proteins. Markers that confer a possible phenotype can be used, with positive and negative screening markers.
용어 "양성 선별 마커"는 선택제(selective agent)가 처리된 환경에서 당해 특정 마커를 발현하는 세포만 생존하도록 하여 양성 선택을 가능하게 하는 마커를 의미하며, "양성 선별 마커 유전자"는 상기 양성 선별 마커를 암호화하는 유전자를 의미하는데, 예를 들어, 네오마이신 포스포트랜스퍼라아제(이하, 'neo'라 약칭함)는 항생제 네오마이신이 첨가된 배지에서 진핵세포가 생존할 수 있도록 하는 항생제 내성을 부여함으로써, 진핵세포에 있어서 안정적 형질감염 세포를 선별하는데 사용된다.The term "positive selection marker" refers to a marker that allows only positive cells to express cells in the environment in which the selective agent is treated to enable positive selection, and "positive selection marker gene" refers to the positive selection marker. Refers to a gene coding for, for example, neomycin phosphotransferase (hereinafter abbreviated as 'neo') endows antibiotic resistance to allow eukaryotic cells to survive in a medium to which the antibiotic neomycin is added. Thus, it is used to select stable transfected cells in eukaryotic cells.
용어 "음성 선별 마커 유전자"는 무작위적 삽입(random insertion)이 일어난 세포를 선별하여 제거하는 음성 선택을 가능하게 하는 마커 유전자로서, 당해 유전자를 발현하는 세포만을 선별적으로 사멸시킴으로써, 무작위적 삽입에 의한 형질도입을 방지하는 역할을 수행한다.The term “negative selection marker gene” is a marker gene that enables negative selection to select and remove cells in which random insertion has occurred, and by selectively killing only cells expressing the gene, Serves to prevent transduction by
용어 "내부 리보좀 개시 부위(internal ribosome entry site, 이하, 'IRES'로 약칭함)"는 진핵생물의 단백질 합성과정에서, 5'-캡 구조 대신 mRNA 중간 부위에서부터 번역이 가능하도록 기능하는 핵산 서열을 의미하며, 이를 이용할 경우, 하나의 mRNA로부터 복수의 다른 기능을 수행하는 단백질을 생산하는 것이 가능하다.The term “internal ribosome entry site (hereinafter abbreviated as 'IRES')” refers to a nucleic acid sequence that functions to allow translation from an intermediate mRNA region instead of the 5'-cap structure in eukaryotic protein synthesis. By means of this, it is possible to produce a protein which performs a plurality of different functions from one mRNA.
용어 "보체 억제 단백질"은 이종이식의 결정적인 장애로 작용하는 보체 활성화 단백질들의 연쇄적인 반응에 의해 활성화된 보체가 숙주세포의 막에 결합하여 급성면역거부반응을 유도하는 것을 억제하는 단백질 의미한다. The term "complement inhibitory protein" refers to a protein that inhibits the complement activated by a chain reaction of complement activating proteins acting as a determinant disorder of xenograft to the host cell's membrane to induce acute immune rejection.
용어 "혈전증 억제 단백질"은 동종타가이식(allograft) 또는 이종이식(xenograft) 후, 수용체의 혈소판이 이식 장기의 내피 또는 내피하 세포와 상호작용을 시작하여, 활성화된 이후, 이식장기의 내피에 혈소판이 응집하여 혈전이 형성됨으로써, 결국 장기 이식을 실패하게 만드는 혈전증을 억제할 수 있는 단백질로 서, 대표적으로 ATP-디포스포하이드롤라아제(ATP-diphosphohydrolase, 이하, 'NTPDase'로 약칭함)가 존재한다.The term "thrombosis inhibiting protein" refers to platelets in the endothelial of the transplanted organ after the platelet of the receptor begins to interact with the endothelial or subendothelial cells of the transplanted organ, after activation, after allograft or xenograft. As a result of this aggregation, a blood clot is formed, and thus, a protein capable of inhibiting thrombosis that causes organ transplantation to fail, typically ATP-diphosphohydrolase (abbreviated as 'NTPDase') is present. do.
용어 "형질전환"은 DNA를 숙주로 도입하여 DNA가 염색체외 인자로서 또는 염색체 통합완성에 의해 복제가능하게 되는 것을 의미한다. 형질전환은 핵산 분자를 유기체, 세포, 조직 또는 기관에 도입하는 어떤 방법도 포함되며, 당 분야에서 공지된 바와 같이 숙주 세포에 따라 적합한 표준 기술을 선택하여 수행할 수 있다. 플라스미드 또는 비플라스미드성 나출(naked DNA)에 의한 진핵세포의 형질전환을 세포의 종양화의 의미로서의 형질전환과 구분하기 위해, '형질감염(transfection)'이라고 부르기도 하는데, 본 문서에서는 동일한 의미로 사용된다.The term "transformation" means the introduction of DNA into a host such that the DNA is replicable as an extrachromosomal factor or by chromosomal integration. Transformation includes any method of introducing a nucleic acid molecule into an organism, cell, tissue, or organ, and can be carried out by selecting appropriate standard techniques according to the host cell as known in the art. In order to distinguish eukaryotic transformation by plasmid or non-plasmid naked DNA from transformation as a means of tumorigenization of cells, it is also referred to as 'transfection'. Used.
발명의 상세한 설명Detailed description of the invention
일 구체예에서, 본 발명은In one embodiment, the present invention
(1) 항원 결정기 합성 유전자에 상동인 2 내지 4 kb 길이의 핵산 서열을 갖는 제1영역; (2) 양성 선별마커 유전자; (3) 내부 리보좀 개시 부위(internal ribosome entry site, 이하, 'IRES'로 약칭함); (4) 보체 억제 단백질 또는 혈전증 억제 단백질을 암호화하는 유전자; 및 (5) 항원 결정기 합성 유전자에 상동인 6 내지 8 kb 길이의 핵산 서열을 갖는 제2영역을 순차적으로 포함하는 세포 내의 이종 항원 결정기 합성 유전자를 제거함과 동시에 보체 억제 단백질 또는 혈전증 억제 단백질을 암호화하는 유전자를 적중시킬 수 있는 유전자 타겟팅 벡터를 제공한다.(1) a first region having a nucleic acid sequence of 2 to 4 kb in length homologous to the antigenic determinant synthetic gene; (2) positive selection marker genes; (3) internal ribosome entry site (abbreviated as 'IRES'); (4) genes encoding complement inhibitory proteins or thrombosis inhibitory proteins; And (5) encoding a complement inhibitory protein or a thrombosis inhibitory protein while simultaneously removing a heterologous antigenic decider synthetic gene in the cell which includes a second region having a nucleic acid sequence of 6 to 8 kb in length homologous to the antigenic determinant synthetic gene. Provide gene targeting vectors capable of targeting genes.
본원발명의 유전자 타겟팅 벡터의 일 구체예에 있어서, 상기 제1영역은 5' 비번역 지역을 포함하는 레프트 암(left arm)에 해당하고, 상기 제2영역은 개방 해독틀(open reading frame, ORF)를 포함하는 엑손 부위를 포함하는 라이트 암(right arm)에 해당한다. 상기 제1영역은 2 내지 4 kb의 크기를 갖는 것이 바람직하고, 더욱 바람직하게는 2.5 내지 3.5 kb의 크기를 갖는다. 상기 제2영역은 5 내지 8 kb 길이의 핵산서열을 갖는 것이 바람직하고, 더욱 바람직하게는 5.5 내지 7.5 kb 길이의 핵산서열을 갖는다. In one embodiment of the gene targeting vector of the present invention, the first region corresponds to a left arm including a 5 'untranslated region, and the second region is an open reading frame (ORF). Corresponds to the right arm including the exon region, including). The first region preferably has a size of 2 to 4 kb, more preferably 2.5 to 3.5 kb. The second region preferably has a nucleic acid sequence of 5 to 8 kb in length, and more preferably has a nucleic acid sequence of 5.5 to 7.5 kb in length.
본 발명의 유전자 타겟팅 벡터의 일 구체예에 있어서, 상기 이종 항원 결정기 합성 유전자는 알파-1,3-갈락토실트랜스퍼라아제(이하, 'GT'로 약칭함)를 암호화하는 유전자 또는 CMP-아세틸뉴라미닌산 히드록실라아제(이하, 'CMAH'로 약칭함)를 암호화하는 유전자인 것이 바람직하나 이에 제한되는 것은 아니다.In one embodiment of the gene targeting vector of the present invention, the heterologous antigenic determinant synthetic gene is a gene encoding alpha-1,3-galactosyltransferase (hereinafter abbreviated as 'GT') or CMP-acetyl It is preferred that the gene encodes neuraminic acid hydroxylase (hereinafter abbreviated as 'CMAH'), but is not limited thereto.
상기 항원 결정기 합성 유전자가 GT인 경우, 상기 제1영역은 바람직하게 GT 유전자의 인트론 2의 일부 또는 전체와 엑손 4를 포함하고, 더욱 바람직하게는 3.1 kb 크기의 서열번호 1의 핵산 서열을 가지며, 상기 제2영역은 바람직하게는 GT 유전자의 인트론 4와 엑손 5 일부 또는 전체를 포함하며, 더욱 바람직하게는 6.9 kb 크기의 서열번호 2의 핵산 서열을 갖는다. 상기 항원 결정기 합성 유전자가 CMAH인 경우에는, 상기 제1영역은 CMAH 유전자의 인트론 3번 일부분과 엑손 4번의 일부 또는 전체를 포함하고, 더욱 바람직하게는 3.5 kb 크기를 가지며, 상기 제2영역은 바람직하게는 CMAH 유전자의 엑손 6번의 일부와 인트론 6번의 일부 또는 전부를 포함하고, 더욱 바람직하게는 6 kb 크기를 갖는다.When the antigenic determinant synthetic gene is GT, the first region preferably comprises part or all of
상기 벡터로 타겟팅되는 GT 유전자는 소, 양, 염소, 돼지, 말, 토끼, 개, 원 숭이 등 포유동물 유래의 GT 유전자인 것이 바람직하고, 돼지 유래의 GT 유전자인 것이 더욱 바람직하며, 미니어쳐 돼지의 GT 유전자인 것이 가장 바람직하나, 이에 제한되는 것은 아니다.The GT gene targeted by the vector is preferably a GT gene derived from a mammal such as a cow, a sheep, a goat, a pig, a horse, a rabbit, a dog, a monkey, more preferably a GT gene derived from a pig, and a miniature pig. The GT gene is most preferably, but is not limited thereto.
아울러, 상기 벡터로 타겟팅되는 CMAH 유전자는 소, 양, 염소, 돼지, 말, 토끼, 개, 원숭이 등 포유동물 유래의 CMAH 유전자인 것이 바람직하고, 돼지 유래의 CMAH 유전자인 것이 더욱 바람직하며, 미니어쳐 돼지의 CMAH 유전자인 것이 가장 바람직하나, 이에 제한되는 것은 아니다. In addition, the CMAH gene targeted by the vector is preferably a CMAH gene derived from a mammal such as cow, sheep, goat, pig, horse, rabbit, dog, monkey, more preferably a CMAH gene derived from pig, and a miniature pig. The CMAH gene is most preferably, but is not limited thereto.
또한, 본 발명의 벡터는 양성 선별마커 유전자를 포함한다. In addition, the vector of the present invention contains a positive selection marker gene.
양성 선별마커 유전자로는 네오마이신 포스포트랜스퍼라아제(neomycin phosphotransferase, Neo), 하이그로마이신 포스포트랜스퍼라아제(hygromycin phosphotransferase, Hyg), 히스티디놀 디하이드로게나제(histidinol dehydrogenase, hisD), 퓨로마이신(puromycin, Puro) 및 구아닌 포스포리보실트랜스퍼라(guanine phosphosribosyltransferase, Gpt) 등이 사용될 수 있으며, 바람직하게는 Neo이지만, 이들로 제한되지 않는다. 상기 양성 선별마커 유전자는 프로모터 트랩 방식에 의해, 즉, 프로모터 없이 내생적 항원 결정기 합성 유전자의 개시 코돈을 이용하여 번역될 수 있도록 넉-인(knock-in)될 수 있고, 아니면, 통상적으로 사용되는 사이토메갈로바이러스(cytomegalovirus, CMV) 프로모터와 같은 항상성 프로모터에 작동가능하도록 연결될 수 있다. Positive selectable marker genes include neomycin phosphotransferase (Neo), hygromycin phosphotransferase (Hyg), histidinol dehydrogenase (hiD), puro Mycin (puromycin, Puro) And guanine phosphosribosyltransferase (Gpt) and the like may be used, preferably Neo, but not limited thereto. The positive selection marker gene can be knock-in so that it can be translated by a promoter trap approach, ie without the promoter, using the initiation codon of the endogenous epitope synthesizing gene, or otherwise It may be operably linked to a homeostatic promoter, such as a cytomegalovirus (CMV) promoter.
또한, 본 발명의 유전자 타겟팅 벡터는 내부 리보좀 개시부위(internal ribosome entry site, IRES)를 포함한다. IRES 요소는 독립적으로 유전자의 5'말 단 메틸구아노시늄 캡(CAP 구조) 업스트림의 해독 개시를 기능적으로 활성화시키고 동물 세포에서는 단일 전사체로부터 2개의 시스트론(개방 판독 프레임)을 해독하는 서열을 포함한다. IRES 요소는 바로 다운스트림에 위치하는 개방 판독 프레임의 해독을 위한 독립적인 리보솜 진입 부위를 제공하는 것으로 알려져 있다. 본 발명의 양성 선별마커인 neo 유전자와 MCS로 삽입되는 인체 보체 억제 유전자 또는 혈전증 억제 유전자는 하나의 mRNA로 발현되지만, IRES로 결합되는 리보좀에 의해 두 개의 독립적인 단백질로 발현될 수 있다. In addition, the gene targeting vector of the present invention includes an internal ribosome entry site (IRES). The IRES element independently functionally activates the translational initiation of the 5'-terminal methylguanosnium cap (CAP structure) upstream of the gene, and in animal cells encodes a sequence that reads two cystrons (open reading frame) from a single transcript. Include. IRES elements are known to provide independent ribosomal entry sites for the reading of open reading frames located immediately downstream. Neo positive gene of the present invention and human complement inhibitory gene or thrombosis inhibitory gene inserted into MCS is expressed as one mRNA, but may be expressed as two independent proteins by ribosomes bound to IRES.
본 발명의 상기 벡터는 넉-인(knock-in)되는 유전자로 보체 억제 단백질 및/또는 혈전증 억제 단백질을 암호화하는 유전자를 포함하는데, 상기 보체 억제 단백질로는 CD59, DAF, MCP 또는 CD46이 사용되는 것이 바람직하고, CD59인 것이 더욱 바람직하나, 이에 제한되는 것은 아니다. 인간 보체 조절 단백질 DAF(decay accelerating factor)는 보체반응경로에서 C2와 C4b의 결합을 방해함으로서 보체의 활성화를 억제하며, MCP(membrane cofactor protein)는 C4b 분해를 촉진하여 보체가 숙주세포에서 활성화되는 것을 억제하며, CD59(homologous restriction factor)는 C7, C8과 C5b6의 결합을 방해함으로서 막 공격복합체(MAC)의 형성을 억제하는 것으로 알려져 있다. 한편, 혈전증 억제 단백질로는 ATP-디포스포하이드롤라아제(ATP-diphosphohydrolase, 이하, 'NTPDase'로 약칭함)가 바람직하고, CD39가 더욱 바람직하나, 이에 제한되는 것은 아니다(혈전증을 억제할 수 있는 유전자로 다른 것이 있으면 알려주시기 바랍니다). 본 발명의 벡터는 보다 효율적인 장기 이식용 동물의 제조를 위해, 보체 억제 단백질과 혈전증 억제 단백질을 암호화하는 유전자가 하나의 벡터를 통해 동시에 넉-인 되도록 제조될 수 있다. 이 경우, 상기 보체 억제 단백질과 혈전증 억제 단백질은 융합단백질의 형태로 제조될 수 있으며, IRES를 이용하여, 하나의 시스트론으로 전사되어 독립적인 두 개의 단백질로 번역될 수도 있다.The vector of the present invention is a knock-in gene, which includes a gene encoding a complement inhibitory protein and / or a thrombosis inhibitory protein, wherein the complement inhibitory protein is CD59, DAF, MCP or CD46. Preferably, it is more preferable that it is CD59, but it is not limited to this. The human complement regulatory protein decay accelerating factor (DAF) inhibits the activation of complement by interfering with the binding of C2 and C4b in the complement response pathway, and the membrane cofactor protein (MCP) promotes C4b degradation to activate complement in host cells. CD59 (homologous restriction factor) is known to inhibit the formation of membrane attack complex (MAC) by inhibiting the binding of C7, C8 and C5b6. On the other hand, as a thrombosis inhibiting protein, ATP-diphosphohydrolase (abbreviated as 'TPDase') is preferable, and CD39 is more preferable, but is not limited thereto (which can suppress thrombosis). Please let me know if there is anything else). The vector of the present invention can be prepared such that the gene encoding the complement inhibitory protein and the thrombosis inhibitory protein is simultaneously knocked-in through one vector, for the production of more efficient organ transplant animals. In this case, the complement inhibitory protein and the thrombosis inhibitory protein may be prepared in the form of a fusion protein, and may be transcribed into one cystron and translated into two independent proteins using IRES.
또한, 본 발명의 벡터는 음성 선별마커를 추가로 포함할 수 있다. 상기 음성 선별마커로는 허피스 심플렉스 바이러스-티미딘 키나아제(Herpes simplex virus-thymidine kinase: HSV-tk), 하이포잔틴 포스포리보실 트랜스퍼라아제(hypoxanthine phosphoribosyl transferase: Hprt), 싸이토신 디아미나아제(cytosine deaminase) 및 디프테리아 톡신(Diphtheria toxin, DT) 등이 있으며 바람직하게는 티미딘 키나제 또는 디프테리아 톡신이지만이지만, 이로 제한되지 않는다. 음성 선별마커는 제1영역의 5' 말단 쪽 또는 제2영역의 3' 말단 쪽에 위치할 수 있다.In addition, the vector of the present invention may further include a negative selection marker. The negative screening markers include Herpes simplex virus-thymidine kinase (HSV-tk), hypoxanthine phosphoribosyl transferase (Hprt), and cytosine deaminase (cytosine). deaminase) and Diphtheria toxin (DT) and the like and are preferably, but are not limited to, thymidine kinase or diphtheria toxin. The negative selection marker may be located at the 5 'end of the first region or at the 3' end of the second region.
본 발명의 유전자 타겟팅 벡터가 숙주 세포 내로 타겟팅되면, 숙주 세포 게놈 상의 내생적(endogeneous) 항원 결정기 합성 유전자와 타겟팅 벡터 사이에 상동 재조합이 일어나면서, 염기 서열이 교체된다. 벡터 상의 양성 선별마커 유전자 및 보체 억제 단백질 또는 혈전증 억제 단백질을 암호화하는 유전자는 내생적 항원 결정기 합성 유전자의 프로모터 또는 양성 선별마커 유전자의 발현을 위해 상기 양성 선별마커 유전자의 5'-말단에 삽입한 프로모터에 의해 발현되는데, 전자의 경우에는 일종의 프로모터 트랩 벡터라 할 수 있다. 세포 내에서 전사되어 기능하는 유전자(발현유전자)를 망라적으로 포착(트랩)하는 방법으로는 프로모터 트랩 이외에 도 인핸서 트랩, 엑손 트랩 등이 있다.When the gene targeting vector of the present invention is targeted into a host cell, homologous recombination occurs between the endogeneous epitope synthesizing gene on the host cell genome and the targeting vector, replacing the base sequence. A positive selection marker gene and a gene encoding a complement inhibitory protein or a thrombosis inhibitory protein on the vector may be a promoter of an endogenous antigenic determinant synthetic gene or a promoter inserted at the 5'-end of the positive selection marker gene for expression of the positive selection marker gene. In the former case, it can be referred to as a promoter trap vector. In addition to a promoter trap, there are an enhancer trap, an exon trap, and the like as a method of capturing and trapping genes (expressed genes) that are transcribed and function in cells.
또 다른 구체예에서, 본 발명은 (1) 항원 결정기 합성 유전자에 상동인 2 내지 4 kb 길이의 핵산 서열을 갖는 제1영역; (2) 보체 억제 단백질 또는 혈전증 억제 단백질을 암호화하는 유전자; (3) 프로모터에 작동가능하게 연결된 양성 선별마커 유전자; 및 (4) 항원 결정기 합성 유전자에 상동인 5 내지 8 kb 길이의 핵산 서열을 갖는 제2영역을 순차적으로 포함하는 세포 내의 이종 항원 결정기 합성 유전자를 제거함과 동시에 보체 억제 단백질 또는 혈전증 억제 단백질을 암호화하는 유전자를 적중시킬 수 있는 유전자 타겟팅 벡터를 제공한다.In another embodiment, the present invention provides a kit comprising: (1) a first region having a nucleic acid sequence of 2 to 4 kb in length homologous to an antigenic determinant synthetic gene; (2) genes encoding complement inhibitory proteins or thrombosis inhibitory proteins; (3) a positive selection marker gene operably linked to a promoter; And (4) encoding a complement inhibitory protein or a thrombosis inhibitory protein while simultaneously removing the heterologous antigenic receptor synthetic gene in the cell which includes a second region having a nucleic acid sequence of 5 to 8 kb in length homologous to the antigenic determinant synthetic gene. Provide gene targeting vectors capable of targeting genes.
상기 유전자 타겟팅 벡터의 일 구체예에 있어서, 상기 제1영역은 5' 비번역 지역을 포함하는 레프트 암(left arm)에 해당하고, 상기 제2영역은 엑손 부위를 포함하는 라이트 암(right arm)에 해당한다. 상기 제1영역은 2 내지 4 kb의 크기를 갖는 것이 바람직하고, 더욱 바람직하게는 2.5 내지 3.5 kb의 크기를 갖는다. 상기 제2영역은 5 내지 8 kb 길이의 핵산서열을 갖는 것이 바람직하고, 더욱 바람직하게는 5.5 내지 7.5 kb 길이의 핵산서열을 갖는다. In one embodiment of the gene targeting vector, the first region corresponds to a left arm including a 5 'untranslated region, and the second region includes a right arm including an exon region. Corresponds to The first region preferably has a size of 2 to 4 kb, more preferably 2.5 to 3.5 kb. The second region preferably has a nucleic acid sequence of 5 to 8 kb in length, and more preferably has a nucleic acid sequence of 5.5 to 7.5 kb in length.
상기 유전자 타겟팅 벡터의 일 구체예에 있어서, 상기 이종 항원 결정기 합성 유전자는 알파-1,3-갈락토실트랜스퍼라아제(이하, 'GT'로 약칭함)를 암호화하는 유전자 또는 CMP-아세틸뉴라미닌산 히드록실라아제(이하, 'CMAH'로 약칭함)를 암호화하는 유전자인 것이 바람직하나 이에 제한되는 것은 아니다. In one embodiment of the gene targeting vector, the heterologous antigenic determinant synthetic gene is a gene encoding alpha-1,3-galactosyltransferase (hereinafter abbreviated as 'GT') or CMP-acetylneuraminin Preferred is a gene encoding acid hydroxylase (hereinafter abbreviated as 'CMAH'), but is not limited thereto.
상기 항원 결정기 합성 유전자가 GT인 경우, 상기 제1영역은 바람직하게 GT 유전자의 인트론 2의 일부 또는 전체와 엑손 4를 포함하고, 더욱 바람직하게는 3.1 kb 크기의 서열번호 1의 핵산 서열을 가지며, 상기 제2영역은 바람직하게는 GT 유전자의 인트론 4와 엑손 5 일부 또는 전체를 포함하며, 더욱 바람직하게는 6.9 kb 크기의 서열번호 2의 핵산 서열을 갖는다. 상기 항원 결정기 합성 유전자가 CMAH인 경우에는, 상기 제1영역은 CMAH 유전자의 인트론 3번 일부분과 엑손 4번의 일부 또는 전체를 포함하고, 더욱 바람직하게는 3.5 kb 크기를 가지며, 상기 제2영역은 바람직하게는 CMAH 유전자의 엑손 6번의 일부와 인트론 6번의 일부 또는 전부를 포함하고, 더욱 바람직하게는 6 kb 크기를 갖는다.When the antigenic determinant synthetic gene is GT, the first region preferably comprises part or all of
상기 벡터로 타겟팅되는 GT 유전자는 소, 양, 염소, 돼지, 말, 토끼, 개, 원숭이 등 포유동물 유래의 GT 유전자인 것이 바람직하고, 돼지 유래의 GT 유전자인 것이 더욱 바람직하며, 미니어쳐 돼지의 GT 유전자인 것이 가장 바람직하나, 이에 제한되는 것은 아니다.The GT gene targeted by the vector is preferably a GT gene derived from a mammal such as a cow, a sheep, a goat, a pig, a horse, a rabbit, a dog, a monkey, more preferably a GT gene derived from a pig, and a GT of miniature pigs. Most preferably, the gene is not limited thereto.
아울러, 상기 벡터로 타겟팅되는 CMAH 유전자는 소, 양, 염소, 돼지, 말, 토끼, 개, 원숭이 등 포유동물 유래의 CMAH 유전자인 것이 바람직하고, 돼지 유래의 CMAH 유전자인 것이 더욱 바람직하며, 미니어쳐 돼지의 CMAH 유전자인 것이 가장 바람직하나, 이에 제한되는 것은 아니다. In addition, the CMAH gene targeted by the vector is preferably a CMAH gene derived from a mammal such as cow, sheep, goat, pig, horse, rabbit, dog, monkey, more preferably a CMAH gene derived from pig, and a miniature pig. The CMAH gene is most preferably, but is not limited thereto.
본 발명의 상기 벡터는 넉-인(knock-in)되는 유전자로 보체 억제 단백질 및/또는 혈전증 억제 단백질을 암호화하는 유전자를 포함하는데, 상기 보체 억제 단백질로는 CD59, DAF, MCP 또는 CD46이 사용되는 것이 바람직하고, CD59인 것이 더욱 바람직하나, 이에 제한되는 것은 아니다. 인간 보체 조절 단백질 DAF(decay accelerating factor)는 보체반응경로에서 C2와 C4b의 결합을 방해함으로서 보체의 활성화를 억제하며, MCP(membrane cofactor protein)는 C4b 분해를 촉진하여 보체가 숙주세포에서 활성화되는 것을 억제하며, CD59(homologous restriction factor)는 C7, C8과 C5b6의 결합을 방해함으로서 막 공격복합체(MAC)의 형성을 억제하는 것으로 알려져 있다. 한편, 혈전증 억제 단백질로는 ATP-디포스포하이드롤라아제(ATP-diphosphohydrolase, 이하, 'NTPDase'로 약칭함)가 바람직하고, CD39가 더욱 바람직하나, 이에 제한되는 것은 아니다(혈전증을 억제할 수 있는 유전자로 다른 것이 있으면 알려주시기 바랍니다). 본 발명의 벡터는 보다 효율적인 장기 이식용 동물의 제조를 위해, 보체 억제 단백질과 혈전증 억제 단백질을 암호화하는 유전자가 하나의 벡터를 통해 동시에 넉-인 되도록 제조될 수 있다. 이 경우, 상기 보체 억제 단백질과 혈전증 억제 단백질은 융합단백질의 형태로 제조될 수 있으며, IRES를 이용하여, 하나의 시스트론으로 전사되어 독립적인 두 개의 단백질로 번역될 수도 있다.The vector of the present invention is a knock-in gene, which includes a gene encoding a complement inhibitory protein and / or a thrombosis inhibitory protein, wherein the complement inhibitory protein is CD59, DAF, MCP or CD46. Preferably, it is more preferable that it is CD59, but it is not limited to this. The human complement regulatory protein decay accelerating factor (DAF) inhibits the activation of complement by interfering with the binding of C2 and C4b in the complement response pathway, and the membrane cofactor protein (MCP) promotes C4b degradation to activate complement in host cells. CD59 (homologous restriction factor) is known to inhibit the formation of membrane attack complex (MAC) by inhibiting the binding of C7, C8 and C5b6. On the other hand, as a thrombosis inhibiting protein, ATP-diphosphohydrolase (abbreviated as 'TPDase') is preferable, and CD39 is more preferable, but is not limited thereto (which can suppress thrombosis). Please let me know if there is anything else). The vector of the present invention can be prepared such that the gene encoding the complement inhibitory protein and the thrombosis inhibitory protein is simultaneously knocked-in through one vector, for the production of more efficient organ transplant animals. In this case, the complement inhibitory protein and the thrombosis inhibitory protein may be prepared in the form of a fusion protein, and may be transcribed into one cystron and translated into two independent proteins using IRES.
이때, 상기 보체 억제 단배질 및/또는 혈전증 억제 단백질을 암호화하는 유전자의 5'-말단에 추가적으로 프로모터가 포함될 수 있는데, 이 때 사용되는 프로모터는 CMV, EF1a, SV40 초기 프로모터 등 통상의 진핵 프로모터가 사용될 수 있으며, 바람직하게는 SV40 초기 프로모터가 사용되며, 프로모터가 추가적으로 포함되지 않을 경우에는 내생적 항원 결정기 합성 유전자의 프로모터를 통해 전사가 이루어질 수 있도록 할 수 있다.At this time, the 5'-terminus of the gene encoding the complement inhibitory protein and / or thrombosis inhibitor protein may be additionally included, the promoter used at this time is a conventional eukaryotic promoter such as CMV, EF1a, SV40 early promoter Preferably, the SV40 initial promoter is used, and if the promoter is not additionally included, transcription may be achieved through a promoter of an endogenous antigenic determinant synthetic gene.
또한, 본 발명의 벡터는 상기에서 기술한 양성 선별마커 유전자를 포함한다. In addition, the vector of the present invention contains the positive selection marker gene described above.
또한, 본 발명의 벡터는 상기에서 기술한 음성 선별마커를 추가로 포함할 수 있다. In addition, the vector of the present invention may further include the negative selection marker described above.
본 발명의 구체적인 실시예에서, 본 발명자는 돼지 알파 1,3-갈락토실트랜스퍼라아제 유전자를 제거함과 동시에 인체 보체 억제 유전자를 적중시킬 수 있는 알파 1,3-갈락토실트랜스퍼라아제 유전자 타겟팅 벡터로서 pGTKOIRESCD59KITK 벡터를 제조하였으며, 상기 벡터의 제조 과정에서 알파 1,3-갈락토실트랜스퍼라아제 유전자 타겟팅이 가능한 pGTKOneoTK 및 pGTKOIRESKITK 벡터를 제조하였다. In a specific embodiment of the present invention, the inventors target an
먼저, 상기 pGTKOIRESCD59KITK 벡터에 포함되는 돼지 알파 1,3-갈락토실트랜스퍼라아제 핵산 서열에 상동인 부분을 얻기 위해, 미니어쳐 돼지의 알파 1,3-갈락토실트랜스퍼라아제 유전자에 적중되어지는 미니어쳐 돼지의 알파 1,3-갈락토실트랜스퍼라아제 유전자 부위의 일부 염기서열을 동정하였다. First, in order to obtain a portion homologous to the
상기 벡터 상의 제1영역 및 제2영역으로 정의되는 미니어쳐 돼지의 알파 1,3-갈락토실트랜스퍼라아제 유전자 부분을 결정하기 위해, 이미 보고된 돼지의 염기서열(Kihiro Koike et al., Transplantation, 70: 1275-1283, 2000)을 바탕으로 프라이머(primers)를 제작하여 교정(proofreading) 기능이 있는 택 폴리머라제(Taq polymerase)로 PCR 반응하였다.In order to determine the
pGTKOneoTK 벡터는 알파 1,3-갈락토실트랜스퍼라아제 유전자의 인트론 2 일부 및 엑손 4 전체 염기서열로 구성되는 크기 3.1kb 정도의 제1영역을 포함한다. 또한, 인트론 4 전체 및 엑손 5 일부 염기서열로 구성되는 크기 6.9kb 정도의 제2 영역을 포함한다. 상기 벡터는 양성 선별마커로서 neo 유전자 영역을 가지며, polyA 유전자 영역 및 음성 선별마커로서 TK 유전자 영역을 가진다. The pGTKOneoTK vector comprises a first region of about 3.1 kb in size consisting of a portion of the
pGTKOIRESKITK 벡터는 상기 pGTKOneoTK 벡터의 구성 외에 IRES 유전자 영역 및 멀티클로닝사이트(MCS)를 포함한다. pGTKOIRESKITK 벡터의 MCS 내로 목적하는 단백질을 코딩하는 유전자를 클로닝시켜, 숙주 세포 내에서 발현되도록 할 수 있다. 본 발명에서는 보체 억제 단백질을 코딩하는 유전자를 삽입시키는 것이 바람직하다.The pGTKOIRESKITK vector contains the IRES gene region and multicloning site (MCS) in addition to the construction of the pGTKOneoTK vector. The gene encoding the protein of interest can be cloned into the MCS of the pGTKOIRESKITK vector for expression in the host cell. In the present invention, it is preferable to insert a gene encoding a complement inhibitory protein.
pGTKOIRESCD59KITK 벡터는 상기 pGTKOIRESKITK 벡터의 MCS 내로 보체 억제 단백질을 코딩하는 유전자의 일종인 CD59 유전자를 삽입시킨 형태이다. pGTKOIRESCD59KITK 벡터는 돼지 알파 1,3-갈락토실트랜스퍼라아제 유전자를 제거함과 동시에 인체 보체 억제 유전자를 적중시킬 수 있는 벡터로서, 본 발명의 목적에 가장 부합하는 벡터라 할 수 있으며, 구체적인 구성을 살펴보면, 5'-말단에서부터 돼지 알파 1,3-갈락토실트랜스퍼라아제 유전자의 인트론 2 일부 및 엑손 4 전체 염기서열로 구성되는 크기 3.1kb 정도의 제1영역, 양성 선별마커로서 SV40 초기 프로모터에 작동가능하도록 연결된 neo 유전자 영역, IRES, Sac I 및 Not I의 제한효소 위치를 가지는 멀티클로닝 사이트, poly A, 인트론 4 전체 및 엑손 5 일부 염기서열로 구성되는 크기 6.9kb 정도의 제2영역을 포함한다. 또한, 상기 제한효소 위치로 보체 억제 단백질을 코딩하는 유전자를 삽입시켰다(도 8).The pGTKOIRESCD59KITK vector is a form in which the CD59 gene, which is a type of gene encoding a complement inhibitory protein, is inserted into the MCS of the pGTKOIRESKITK vector. The pGTKOIRESCD59KITK vector is a vector capable of removing the
상기 벡터 제작은 당해 기술분야에서 잘 알려진 유전자 재조합 기술을 이용하여 제조할 수 있으며, 부위-특이적 DNA 절단 및 연결은 당해 기술 분야에서 일반적으로 알려진 효소 등을 사용할 수 있다. The vector construction can be prepared using genetic recombination techniques well known in the art, site-specific DNA cleavage and ligation can be used enzymes generally known in the art.
본 발명의 또 다른 구체적인 실시예에서, 본 발명자는 돼지 CMAH 유전자를 제거함과 동시에 인체 혈전증 억제 단백질을 암호화하는 유전자를 적중시킬 수 있는 CMAH 유전자 타겟팅 벡터로서 pCMAHKOCD39KIDT 벡터를 제조하였으며, 상기 벡터의 제조 과정에서 CMAH 유전자 타겟팅이 가능한 pCMAHKOneoDT 벡터를 제조하였다. In another specific embodiment of the present invention, the present inventors prepared a pCMAHKOCD39KIDT vector as a CMAH gene targeting vector capable of simultaneously removing a pig CMAH gene and targeting a gene encoding a human thrombosis inhibitory protein. A pCMAHKOneoDT vector capable of CMAH gene targeting was prepared.
먼저, 상기 pCMAHKOCD39KIDT 벡터에 포함되는 돼지 CMAH 유전자 핵산 서열에 상동인 부분을 얻기 위해, 미니어쳐 돼지의 CMAH 유전자에 적중되어지는 미니어쳐 돼지의 CMAH 유전자 부위의 일부 염기서열을 동정하였다. First, in order to obtain a part homologous to the porcine CMAH gene nucleic acid sequence included in the pCMAHKOCD39KIDT vector, a partial base sequence of the CMAH gene region of the miniature pig that was hit by the CMAH gene of the miniature pig was identified.
상기 벡터 상의 제1영역 및 제2영역으로 정의되는 미니어쳐 돼지의 CMAH 유전자 부분을 결정하기 위해, 이미 보고된 돼지의 염기서열(Kihiro Koike et al., Transplantation, 70: 1275-1283, 2000)을 바탕으로 프라이머(primers)를 제작하여 교정(proofreading) 기능이 있는 택 폴리머라제(Taq polymerase)로 PCR 반응하였다.In order to determine the CMAH gene portion of the miniature pig defined as the first region and the second region on the vector, the previously reported pig sequence (Kihiro Koike et al ., Transplantation , 70: 1275-1283, 2000) Primers were prepared and PCR reacted with Taq polymerase with proofreading.
pCMAHKOneoDT 벡터는 CMAH 유전자의 인트론 3번 일부분과 엑손 4번 일부 부분을 포함하는 약 3.5 kb 크기의 제1영역을 포함한다. 또한, 엑손 6번 일부분과 인트론 6번 일부 부분을 포함하는 6.0 kb 크기의 제2영역을 포함한다. 상기 벡터는 양성 선별마커로서 neo 유전자 영역을 가지며, polyA 유전자 영역 및 음성 선별마커로서 TK 유전자 영역을 가진다. The pCMAHKOneoDT vector comprises a first region of about 3.5 kb size comprising a portion of
그 다음으로 상기 pCMAHKOneoDT 벡터에 혈전증 억제 단백질인 CD39를 암호화하는 유전자를 neo 유전자 앞부분에 삽입하여 pCMAHKOCD39KIDT 벡터를 제조하였다. 이때, 상기 CD39를 암호화하는 유전자는 CMAH 유전자와 개시코돈을 중첩하여, CMAH 유전자의 프로모터를 통해 CD39 유전자가 발현되도록 고안하였다. Next, a pCMAHKOCD39KIDT vector was prepared by inserting a gene encoding CD39, a thrombosis inhibitory protein, in the neo gene at the front of the pCMAHKOneoDT vector. In this case, the gene encoding the CD39 overlaps the CMAH gene and the start codon, and is designed to express the CD39 gene through a promoter of the CMAH gene.
pCMAHKOCD39KIDT 벡터는 돼지 CMAH 유전자를 제거함과 동시에 인체 CD39 유전자를 적중시킬 수 있는 벡터로서, 본 발명의 목적에 부합하는 벡터라 할 수 있으며, 구체적인 구성을 살펴보면, 인트론 3번 일부분과 엑손 4번 일부 부분을 포함하는 약 3.5 kb 크기의 제1영역, CMAH 유전자의 개시코돈에 프레임이 맞게 연결된 CD39 단백질을 암호화하는 유전자, SV40 초기 프로모터에 작동가능하도록 연결된 neo 유전자 영역, poly A, 엑손 6번 일부분과 인트론 6번 일부 부분을 포함하는 6.0 kb 크기의 제2영역 및 음성 선별마커로서 DT 유전자를 포함한다. The pCMAHKOCD39KIDT vector is a vector capable of hitting the human CD39 gene while removing the porcine CMAH gene. The pCMAHKOCD39KIDT vector can be referred to as a vector meeting the object of the present invention. A first region of about 3.5 kb in size, a gene encoding the CD39 protein framed to the start codon of the CMAH gene, a neo gene region operably linked to the SV40 early promoter, poly A, a portion of
상기 벡터 제작은 당해 기술분야에서 잘 알려진 유전자 재조합 기술을 이용하여 제조할 수 있으며, 부위-특이적 DNA 절단 및 연결은 당해 기술 분야에서 일반적으로 알려진 효소 등을 사용할 수 있다. The vector construction can be prepared using genetic recombination techniques well known in the art, site-specific DNA cleavage and ligation can be used enzymes generally known in the art.
또 다른 양태로서, 본 발명은 상기 유전자 타겟팅 벡터가 도입된 형질전환체에 관한 것이다. In another aspect, the present invention relates to a transformant into which the gene targeting vector is introduced.
상기 형질전환은 핵산 분자를 유기체, 세포, 조직 또는 기관에 도입하는 어떤 방법도 포함되며, 당 분야에서 공지된 바와 같이 숙주 세포에 따라 적합한 표준 기술을 선택하여 수행할 수 있다. 이런 방법에는 전기천공법(electroporation), 인산칼슘(CaPO4) 침전, 염화칼슘(CaCl2) 침전, 미세주입법(microinjection), 폴리에틸렌글리콜(PEG)법, DEAE-덱스트란법, 양이온성 리포좀법, 및 초산 리튬-DMSO법 등이 포함되나, 이들로 제한되지 않는다. The transformation may include any method of introducing a nucleic acid molecule into an organism, cell, tissue or organ, and may be carried out by selecting a suitable standard technique according to the host cell as known in the art. These methods include electroporation, calcium phosphate (CaPO 4 ) precipitation, calcium chloride (CaCl 2 ) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and Lithium acetate-DMSO method and the like, but are not limited thereto.
본 발명의 구체적인 실시예에서 제작된 타겟팅 벡터인 pCMAHKOCD39KIDT를 미니어쳐 돼지 귀조직 유래 섬유아세포(pESF)로 도입하여 적중된 2개의 세포주 (CMAHKI#1, CMAHKI#2)를 제조하였으며, 그 중 CMAHKI#1 세포주를 2008년 11월 24일에 KCTC(Korean Collection for Type Cultures, 한국 대전광역시 유성구 과학로 111번지 한국생명공학연구원)에 기탁번호 KCTC 11433BP로 기탁하였다. Two targeting cell lines (
상기 수탁기관은 미생물 기탁에 관한 부다페스트 조약에 의해 지정된 수탁기관으로서, 본 발명자들은 상기 기탁이 부타페스트 조약에서 정한 조건을 충족함을 보증한다.The trustee is a trustee designated by the Budapest Treaty on the Deposition of Microorganisms, and the inventors ensure that the deposit meets the conditions laid down in the Butapest Treaty.
또한, 본 발명은 상기 형질전환 체세포주의 핵 이식을 통해 제조되는 비인간 복제동물을 제공한다.The present invention also provides a non-human clone produced by nuclear transfer of the transgenic somatic cell line.
상기 비인간 복제동물은 양, 산양, 돼지, 개 등 인간과 체구가 유사한 포유동물이라도 가능하나, 돼지인 것이 더욱 바람직하며, 그 중 미니어처 돼지인 것이 가장 바람직하다. The non-human clone may be a mammal similar in size to a human such as sheep, goats, pigs, and dogs, but more preferably pigs, and most preferably, miniature pigs.
상기 복제동물의 제조에 이용되는 핵이식 방법은 당업계에 잘 알려진 방법을 사용하는 것이 가능하나, 보다 바람직하게는 US6,781,030B, US6,603,059B, US6,235,969B, US7,355,094B, US7,071,372B, KR862298B, KR500412B, KR807644B, JP4153878B, US6,700,037B, US7,291,764B, US6,258,998B, US6,548,741B, WO03/089632A, US7,371,922B 등이 사용될 수 있고, 특히 돼지의 경우에는 KR500412B, KR807644, JP4153878B, US6,700,037B, US7,291,764B, US6,258,998B, US6,548,741B, WO03/089632A 또는 US7,371,922B에 기재된 방법을 사용하는 것이 더욱 바람직하다. 상기 특허문헌들은 모두 본 문서에 참조로 삽입된다.The nuclear transfer method used for the production of the cloned animal can be used in the art well known in the art, more preferably US6,781,030B, US6,603,059B, US6,235,969B, US7,355,094B, US7 , 071,372B, KR862298B, KR500412B, KR807644B, JP4153878B, US6,700,037B, US7,291,764B, US6,258,998B, US6,548,741B, WO03 / 089632A, US7,371,922B and the like can be used. More preferred is the use of the methods described in KR500412B, KR807644, JP4153878B, US6,700,037B, US7,291,764B, US6,258,998B, US6,548,741B, WO03 / 089632A or US7,371,922B. All of these patent documents are incorporated herein by reference.
아울러, 본 발명은 상기 비인간 복제동물을 사육한 후, 이식에 필요한 장기를 적출하는 단계를 포함하는 이식용 이종 장기의 생산방법을 제공한다. 상기 장기는 수용 대상의 성별, 나이, 체중, 신장 등을 고려하여, 사육시기를 조절하여 공여체 복제동물을 사육한 다음, 통상의 외과적 수술을 통해 적출할 수 있으며, 적출 후 수용 대상에게 바록 이식되거나, 신속하게 냉장보관될 수 있다. In addition, the present invention provides a method for producing a heterologous organ for transplantation, comprising the step of extracting the organ for transplantation after breeding the non-human clone animal. The organs may be harvested by adjusting the breeding time in consideration of the sex, age, weight, height of the recipient, breeding donor clone animals, and then performing a conventional surgical operation. Or can be refrigerated quickly.
본 발명의 유전자 타겟팅 벡터는 장기 공여 동물의 내생적 항원 결정기 합성 유전자를 적중함과 동시에, 적중되는 내생적 항원 결정기 합성 유전자의 좌위에 발현하고자 하는 보체 억제 단백질 및/또는 혈전증 억제 단백질을 암호화하는 유전자를 넉-인시킴으로써, 도입 대상 유전자의 무작위적 삽입에 따른 유전자 침묵, 발아유전자의 활성화로 인한 암유발과 같은 문제점을 해소함으로써, 보다 안전한 이종간 장기 이식을 위한 공여 동물을 제조하는데 매우 효과적으로 이용될 수 있다.The gene targeting vector of the present invention targets an endogenous antigenic determinant synthetic gene of an organ donor animal and simultaneously encodes a complement inhibitory protein and / or a thrombosis inhibitory protein to be expressed at the locus of the endogenous antigenic determinant synthetic gene. By knock-in can solve the problems such as gene silencing following the random insertion of the gene to be introduced, cancer-causing caused by the activation of the germination gene, it can be very effectively used to prepare donor animals for safer cross-organ transplantation have.
이하 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하기 위한 것으로, 본 발명의 내용이 이에 한정되는 것은 아니다. Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are provided to illustrate the present invention, but the content of the present invention is not limited thereto.
실시예 1 : Example 1: GTGT 유전자의 Gene 타겟팅Targeting 벡터 제조 Vector manufacturing
1-1: 미니어쳐 돼지의 알파 1,3-갈락토실트랜스퍼라아제 유전자 엑손 4번 전체와 인트론 2번 일부분의 염기서열 동정1-1: Identification of the base sequence of the
돼지의 알파 1,3-갈락토실트랜스퍼라아제 유전자는 9개의 엑손으로 이루어져 있으며, 엑손의 전체 염기서열과 인트론의 염기서열 일부분이 보고되어져 있다 (Kihiro Koike et al., Transplantation, 70: 1275-1283, 2000). 본 발명에서는 미니어쳐 돼지의 알파 1,3-갈락토실트랜스퍼라아제 유전자를 적중시키기 위해서 적중되는 미니어쳐 돼지의 알파 1,3-갈락토실트랜스퍼라아제 유전자 부위의 일부 염기서열을 동정하였다. 미니어쳐 돼지의 인트론 2번 일부분과 엑손 4번 전체의 염기서열을 결정하기 위해서 이미 보고된 돼지의 염기서열(Kihiro Koike et al., Transplantation, 70: 1275-1283, 2000)을 바탕으로 서열번호 3(5'-TCCATGAACAACTTCGATTGCATG-3')의 포워드 프라이머 및 서열번호 4(5'-TGGCTGATAACTAGGAGATTAGAGGAG-3')의 리버스 프라이머를 제작하여 교정(proofreading) 기능이 있는 Taq DNA 폴리머라제(Taq polymerase, Intron Biotechnology, Korea)로 94℃에서 2분간 예열, 94℃ 20초, 60℃ 45초 및 68℃ 4분 35회 및 68℃ 10분의 조건으로 PCR 반응을 수행하였다. The
합성된 PCR 산물은 T-easy vector(Promega, USA)로 클로닝 되었으며, 총 4개의 클론에 대해 시퀀싱한 결과 공통적인 염기 서열을 동정할 수 있었다(서열번호 5 및 도 1). 미니어쳐 돼지의 알파 1,3-갈락토실트랜스퍼라아제 유전자의 인트론 2 영역을 서열번호 6에 나타냈으며, 인트론 4 영역을 서열번호 7에 나타냈다.The synthesized PCR product was cloned into a T-easy vector (Promega, USA) and sequenced for a total of four clones to identify common nucleotide sequences (SEQ ID NO: 5 and Figure 1). The
타겟팅 벡터에 사용되어질 레프트 암(left arm)부분은 EcoR I제한효소 위치가 연결된 서열번호 8의 포워드 프라이머(5'-GAATTCATGATTATTATCCTCCCAAGC-3') 및 서열번호 9(5'-CATGAATTCCATTATTTTCTCCTGGG-3')의 리버스 프라이머를 제작하여 교정 기능이 있는 Taq DNA 폴리머라제(Intron Biotechnology, Korea)로 94℃에서 2분간 예열, 94℃ 20초, 60℃ 45초 및 68℃ 3분 30초 35회 및 68℃ 10분의 조건으로 PCR 반응을 수행하였다. The left arm portion to be used in the targeting vector is a reverse primer of SEQ ID NO: 8 (5'-GAATTCATGATTATTATCCTCCCAAGC-3 ') and SEQ ID NO: 9 (5'-CATGAATTCCATTATTTTCTCCTGGG-3') linked to the Eco RI restriction enzyme position. A primer was prepared to pre-correct Taq DNA polymerase (Intron Biotechnology, Korea) for 2 minutes at 94 ° C, 94 °
합성된 PCR 산물은 T-easy vector(Promega, USA)로 클로닝 되었으며, 획득된 클론은 제한효소 EcoR I으로 자른 후, 타겟팅 벡터의 제1영역(서열번호 1)을 구성하는 레프트 암(left arm)으로 사용되었다(도 5 및 6). The synthesized PCR product was cloned into a T-easy vector (Promega, USA), and the obtained clone was cut with the restriction enzyme Eco RI, and then left arm that constitutes the first region (SEQ ID NO: 1) of the targeting vector. Was used (FIGS. 5 and 6).
1-2: 1-2:
미니어쳐
미니어쳐 돼지 알파 1,3-갈락토실트랜스퍼라아제 유전자의 인트론 4번 전체와 엑손 5번 일부분의 염기서열을 결정하기 위해서 이미 보고된 돼지의 염기서열 (Kihiro Koike et al., Transplantation, 70: 1275-1283, 2000)을 바탕으로 Sal I과 Xho I 제한효소위치가 각각 연결된 서열번호 10(5'-GTCGACCTGTCAATGCTGCTTG-3')의 포워드 프라이머 및 서열번호 11(5'-CTCGAGCCAGAACAAAGAACCTTC-3')의 리버스 프라이머를 제작하여 교정 기능이 있는 Taq DNA 폴리머라제(Intron Biotechnology, Korea)로 94℃에서 2분간 예열, 94℃ 20초, 60℃ 45초 및 68℃ 7분 35회 및 68℃ 10분의 조건으로 PCR 반응하였다.
합성된 PCR 산물은 T-easy vector(Promega, USA)로 클로닝 되었으며, 총 4개의 클론에 대해 시퀀싱한 결과 공통적인 염기 서열을 동정할 수 있었다(서열번호 12 및 도 2). 획득된 클론은 제한효소 Sal I, Xho I으로 자른 후, 타겟팅 벡터의 제2영역(서열번호 2)을 구성하는 라이트 암(right arm)으로 사용되었다(도 5 및 6). The synthesized PCR product was cloned into a T-easy vector (Promega, USA), and the sequencing of a total of four clones was able to identify a common base sequence (SEQ ID NO: 12 and Figure 2). The obtained clone was cut with restriction enzymes Sal I, Xho I and used as a right arm constituting the second region of the targeting vector (SEQ ID NO: 2) (FIGS. 5 and 6).
1-3: pGTKOneoTK 벡터의 제작 1-3: Construction of pGTKOneoTK Vector
실시예 1-1 및 1-2에서 각각 제작된 EcoR I제한효소 위치를 가지고 있는 레프트 암과 Sal I, Xho I제한효소 위치를 가지고 있는 라이트 암은 음성 선별 마커인 TK(thymidine kinase) 유전자를 가지고 있는 pBst-TK 벡터의 각각 EcoR I과 Sal I 위치로 삽입되었다(도 5). TK 유전자는 배지에 간시클로버(gancyclovir)라는 물질이 존재하면, 이를 분해하여 세포에 독성이 있는 효소 산물을 만들어 낸다. 상동 재조합이 일어나게 되어 타겟팅 벡터의 TK 유전자가 탈락된(도 3, 4 및 8) 세포주는 간시클로버(gancyclovir)가 첨가된 배지에서 자랄 수 있으나, 무작위적인 삽입에 의해 타겟팅 벡터가 염색체의 임의의 위치에 끼워들어가면 TK 유전자가 함께 들어가 발현되기 때문에 간시클로버(gancyclovir)가 함유된 배지에서 성장하지 못하고 죽게 된다. 양성 선별마커인 neo 유전자는 프로모터와 poly A 부분과 함께 pcDNA3.1 벡터(Invitrogen, USA)에서 EcoR I, EcoR V 제한효소 위치가 연결된 서열번호 13(5'-CTAGGAATTCCTTCGCGATGTACGGGCC-3')의 포워드 프라이머 및 서열번호 14(5'-CAGTGATATCCTTATCGCTATCGATTCACAC-3')의 리버스 프라이머를 제작하여 교정 기능이 있는 Taq DNA 폴리머라제(Intron Biotechnology, Korea)로 94℃에서 2분간 예열, 94℃ 20초, 60℃ 45초 및 68℃ 1분 30초 35회 및 68℃ 10분의 조건으로 PCR 반응하였다. The left arm having the Eco R I restriction enzyme positions prepared in Examples 1-1 and 1-2 and the light arm having the Sal I and Xho I restriction enzyme positions had the thymidine kinase (TK) gene, which is a negative screen And inserted into the Eco R I and Sal I positions, respectively, of the pBst-TK vector (FIG. 5). When the TK gene is present in the medium called gancyclovir, it breaks down to produce enzyme products that are toxic to cells. Homologous recombination occurs so that the cell line from which the TK gene of the targeting vector has been eliminated (FIGS. 3, 4 and 8) can be grown in a medium to which gancyclovir is added, but the randomization of the targeting vector causes the targeting vector to be located at an arbitrary position of the chromosome. When inserted into the TK gene is expressed together to die in the growth medium containing gancyclovir (gancyclovir). The neo gene, a positive selection marker, has a forward primer of SEQ ID NO: 13 (5'-CTAGGAATTCCTTCGCGATGTACGGGCC-3 ') linked with the Eco R I, Eco RV restriction enzyme position in the pcDNA3.1 vector (Invitrogen, USA) with a promoter and poly A moiety. Reverse primer of SEQ ID NO: 14 (5'-CAGTGATATCCTTATCGCTATCGATTCACAC-3 ') was prepared and pre-warmed with Taq DNA polymerase (Intron Biotechnology, Korea) for 2 minutes at 94 ° C for 20 minutes, 60 ° C for 45 seconds, and PCR reaction was performed at 68 ° C for 1
합성된 PCR 산물은 pBst-TK 벡터의 각각 EcoR I과 EcoR V위치로 삽입되었다(도 5). 그 결과, pGTKOneoTK 벡터가 완성되었으며, 제한효소 Not I또는 Xho I에 의해 선형화된 후, 체세포 내로 도입될 수 있다. 상동 재조합이 일어나게 되면 TK 유전자는 탈락하게 된다. 타겟팅 여부는 서열번호 15(5'-GGTCTGCCTACATCTCTCTGATGAAC-3')의 포워드 프라이머 및 서열번호 16(5'-GGCATCAGAGCAGCCGATTG-3')의 리버스 프라이머로 구성된 프라이머쌍 및 서열번호 17(5'-CCTTCTATCGCCTTCTTGACGAG-3')의 포워드 프라이머 및 서열번호 18(5'-CTAGAGATATTTGGAATTCAAAGCACTTAC-3')의 리버스 프라이머로 구성된 프라이머쌍에 의해 Taq DNA 폴리머라제(Takara, Japan)로 확인하게 된다. 서열번호 15 및 16로 기재되는 프라이머쌍을 이용한 PCR 반응에는 94℃에서 2분간 예열, 94℃ 20초, 60℃ 45초 및 68 ℃ 3분 35회 및 68℃ 10분의 조건이 사용되었고, 서열번호 17 및 18로 기재되는 프라이머쌍을 이용한 PCR 반응에는 94℃에서 2분간 예열, 94℃ 20초, 60℃ 45초 및 68℃ 8분 35회 및 68℃ 10분의 조건이 사용되었다(도 3). The synthesized PCR product was inserted into the Eco R I and Eco R V positions of the pBst-TK vector, respectively (FIG. 5). As a result, the pGTKOneoTK vector is complete and can be linearized by restriction enzyme Not I or Xho I and then introduced into somatic cells. When homologous recombination occurs, the TK gene is eliminated. Targeting was carried out with a primer pair consisting of a forward primer of SEQ ID NO: 15 (5'-GGTCTGCCTACATCTCTCTGATGAAC-3 ') and a reverse primer of SEQ ID NO: 16 (5'-GGCATCAGAGCAGCCGATTG-3') and SEQ ID NO: 17 (5'-CCTTCTATCGCCTTCTTGACGAG-3 '). ) Was identified as Taq DNA polymerase (Takara, Japan) by a pair of primers consisting of a forward primer of s) and a reverse primer of SEQ ID NO: 18 (5'-CTAGAGATATTTGGAATTCAAAGCACTTAC-3 '). PCR reactions using primer pairs as set forth in SEQ ID NOs: 15 and 16 used conditions of preheating at 94 ° C. for 2 minutes, 94 ° C. 20 seconds, 60 ° C. 45 seconds, and 68 ° C. 3 minutes 35 times and 68 ° C. 10 minutes. PCR reactions using primer pairs described in Nos. 17 and 18 were preheated at 94 ° C. for 2 minutes, 94 ° C. 20 seconds, 60 ° C. 45 seconds, and 68 ° C. 8 minutes 35 times and 68 ° C. 10 minutes conditions (FIG. 3). ).
내부 리보좀 개시부위(Internal ribosome entry site, IRES), 멀티 클로닝 사이트(MCS, Sal I, Not I), Poly A(pA) 부분은 pIRES(Clontech, USA) 벡터에서 EcoR V, Xho I 제한효소 위치가 연결된 서열번호 19(5'-GATATCCGCGTCGAGCATGC-3') 의 포워드 프라이머 및 서열번호 20(5'-CTCGAGTCGCCATTCAGGC-3')의 리버스 프라이머를 제작하여 교정 기능이 있는 Taq DNA 폴리머라제(Intron Biotechnology, Korea)로 94℃에서 2분간 예열, 94℃ 20초, 60℃ 45초 및 68 ℃ 1분 30초 35회 및 68℃ 10분의 조건으로 PCR 반응하였다. IRES 염기서열에 의해 양성 선별 마커인 neo 유전자와 MCS로 삽입되는 인체 보체 억제 유전자는 하나의 mRNA로 발현되나 IRES로 결합되는 리보좀(ribosome)에 의해 두 개의 독립적인 단백질로 발현될 수 있다(Louis Marie Houdebine et al., Transgenic research, 8: 157-177, 1999). Internal ribosome entry site (IRS), multicloning site (MCS, Sal I, Not I), and Poly A (pA) moiety are located in Eco R V and Xho I restriction enzymes in pIRES (Clontech, USA) vector. A forward primer of linked SEQ ID NO: 19 (5'-GATATCCGCGTCGAGCATGC-3 ') and a reverse primer of SEQ ID NO: 20 (5'-CTCGAGTCGCCATTCAGGC-3') were prepared to provide a calibration of Ta q DNA polymerase (Intron Biotechnology, Korea) PCR reaction was carried out under the conditions of preheating at 94 ° C. for 2 minutes, 94 ° C. 20 seconds, 60 ° C. 45 seconds, and 68 ° C. 1
합성된 PCR 산물은 pBst-TK 벡터를 Hind III로 자른 후 Klenow 효소 처리하고 다시 Sal I으로 자른 위치로 삽입되었다(도 6). 그 결과, 인체 보체 억제 유전자가 삽입될 수 있는 MCS(Sal I, Not I)를 가지는 pGTKOIRESKITK 벡터 카세트가 완성되었으며, 제한효소 Xho I에 의해 선형화 된 후, 체세포 내로 도입될 수 있다. 상동 재조합이 일어나게 되면 TK 유전자는 탈락하게 된다(도 4). The synthesized PCR product was inserted into a position where the pBst-TK vector was cut with Hin d III, treated with Klenow enzyme, and cut with Sal I again (FIG. 6). As a result, a pGTKOIRESKITK vector cassette having a MCS ( Sal I, Not I) into which a human complement inhibitory gene can be inserted is completed, linearized by restriction enzyme Xho I, and then introduced into somatic cells. When homologous recombination occurs, the TK gene is eliminated (FIG. 4).
1-4: pGTKOIRESCD59KITK 벡터 카세트의 제작1-4: Construction of pGTKOIRESCD59KITK Vector Cassette
pGTKOIRESKITK 벡터 카세트의 MCS(Sal I, Not I)위치로 인체 보체 억제 유전자 CD59가 삽입되었다(도 7). CD59 유전자는 Sal I, Not I 제한효소 위치가 연결된 서열번호 21(5'-CTGAGTCGACATGGGAATCCAAGGAGGGTCTG-3')의 포워드 프라이머 및 서열번호 22(5'-CTGAGCGGCCGCTTAGGGATGAAGGCTCCAGG-3')의 리버스 프라이머를 제작하여 교정 기능이 있는 택 폴리머라제(Intron Biotechnology, Korea)로 인체 cDNA 라이브러리에서 94℃에서 2분간 예열, 94℃ 20초, 60℃ 45초 및 68℃ 40초 35회 및 68℃ 10분의 조건으로 PCR 반응하였다. The human complement inhibitory gene CD59 was inserted into the MCS ( Sal I, Not I) position of the pGTKOIRESKITK vector cassette (FIG. 7). The CD59 gene produced a forward primer of SEQ ID NO: 21 (5'-CTGAGTCGACATGGGAATCCAAGGAGGGTCTG-3 ') and a reverse primer of SEQ ID NO: 22 (5'-CTGAGCGGCCGCTTAGGGATGAAGGCTCCAGG-3') with Sal I and Not I restriction enzymes linked thereto. PCR was carried out in a human cDNA library with a tack polymerase (Intron Biotechnology, Korea) at 94 ° C for 2 minutes, 94 °
합성된 PCR 산물은 pGTKOIRESKITK 벡터 카세트의 MCS를 Sal I, Not I으로 자른 위치로 삽입되었다(도 7). 그 결과, 알파 1,3-갈락토실트랜스퍼라아제 유전자를 제거함과 동시에 CD59 유전자를 적중시킬 수 있는 pGTKOIRESCD59KITK 벡터가 완성되었다. pGTKOIRESCD59KITK 벡터는 제한효소 Xho I에 의해 선형화 된 후, 체세포내로 도입될 수 있다. 상동 재조합이 일어나게 되면 TK 유전자는 탈락하게 된다(도 8). The synthesized PCR product was inserted into the position where the MCS of the pGTKOIRESKITK vector cassette was cut into Sal I, Not I (FIG. 7). As a result, a pGTKOIRESCD59KITK vector was completed that could remove the
타겟팅 여부는 상기 실시예 1-3에 기재된 서열번호 17 및 18로 구성되는 프라이머쌍 및 서열번호 19 및 20으로 구성된 프라이머쌍에 의해 Taq DNA 폴리머라아제(Takara, Japan)로 확인하게 된다(도 8). PCR 반응의 조건 역시 실시예 1-3에 기재된 것과 동일하다.Targeting is confirmed by Taq DNA polymerase (Takara, Japan) by the primer pair consisting of SEQ ID NOs: 17 and 18 and the primer pair consisting of SEQ ID NOs: 19 and 20 described in Example 1-3 (FIG. 8). ). The conditions of the PCR reaction were also the same as those described in Examples 1-3.
실시예Example 2: 2: CMAHCMAH 유전자 gene 타겟팅Targeting 벡터의 제조 Manufacture of vector
2-1: pCMAHKOneoDT 벡터의 제작 2-1: Construction of pCMAHKOneoDT Vector
미니어쳐 돼지의 CMAH 유전자 염기서열(www.ncbi.nlm.nih.gov, NW_001886419.1) 중에서 인트론 3번 일부분과 엑손 4번 일부 부분을 타겟팅 벡터의 레프트 암(left arm)으로 사용하고자 Sac I과 Not I 제한효소위치가 연결된 서열번호 23(5'-GAGCTCCATGACAGGAACTCCTGAGATGAATATC-3')의 포워드 프라이머 및 서열번호 24(5'-GCGGCCGCTCGTTGCCTCTCTCCAGGTATTAAG-3')의 리버스 프라이머를 제작하여 교정(proofreading) 기능이 있는 Taq DNA 폴리머라제(ExTaq polymerase, Takara, Japan)로 94℃에서 2분간 예열, 94℃ 20초, 60℃ 45초 및 72℃ 4분 35회 및 72℃ 10분의 조건으로 PCR 반응하였다. Sac I and Not to use the
합성된 PCR 산물은 제한효소 Sac I과 Not I 으로 자른 후, pBCKIDT(AU 2005256120 B2) 벡터의 Sac I과 Not I 위치로 삽입되었다(도 10). The synthesized PCR product was cut into restriction enzymes Sac I and Not I, and then inserted into the Sac I and Not I positions of the pBCKIDT (AU 2005256120 B2) vector (FIG. 10).
구체적으로, 타겟팅 벡터의 라이트 암(Right arm)으로 사용하기 위해, CMAH 유전자 염기서열 중에서 엑손 6번 일부분과 인트론 6번 일부 부분을 EcoR V와 Sal I 제한효소위치가 연결된 서열번호 25(5'-GATATCACCATCAATACTGATCAATGTTTTCTG-3')의 포워드 프라이머 및 서열번호 26(5'-GTCGACAGCAGGAAAAACTGAGTCTGCAGTTC-3')의 리버스 프라이머를 제작하여 교정(proofreading) 기능이 있는 Taq DNA 폴리머라제(ExTaq polymerase, Takara, Japan)로 94℃에서 2분간 예열, 94℃ 20초, 60℃ 45초 및 72℃ 6분 35회 및 72℃ 10분의 조건으로 PCR 반응하였다. 합성된 PCR 산물은 EcoR V와 Sal I 제한효소으로 자른 후, pBCKIDT 벡터의 EcoR V와 Sal I 위치로 연결함으로써, pCMAHneoDT 벡터를 제작하였다(도 10). Specifically, in order to use as a right arm of the targeting vector, a part of
2-2: pCMAHKOCD39KIDT 벡터의 제작 2-2: Construction of pCMAHKOCD39KIDT Vector
pCMAHKOneoDT벡터 Xho I과 Pac I위치로 인간 CD39 유전자를 연결하였다(도 11). 인간 cDNA 라이브러리(Openbiosystems, USA)에 있는 CD39 유전자를 Sal I, Kpn I 제한효소로 자르고, 동시에 Kpn I, BamH I 위치가 연결된 서열번호 27(5'A-TGCAATTTCGCCTCTTGGC-3')의 포워드 프라이머 및 서열번호 28(5'-GAGGATCCCTATACCATATCTTTCCAGAAATATGAAGG-3')의 리버스 프라이머를 제작하여 교정 기능이 있는 Taq DNA 폴리머라제(Genotech, Korea)로 cDNA 라이브러리에서 94℃에서 4분간 예열, 94℃ 20초, 57℃ 45초 및 72℃ 1분 35회 및 72℃ 10분의 조건으로 PCR 반응하여, 제한효소 Kpn I, BamH I로 자른 후, 두 개의 DNA 조각을 pDsRed2-C1 벡터(Clontech, USA) 에 연결하였다. The human CD39 gene was linked to the pCMAHKOneoDT vectors Xho I and Pac I (FIG. 11). Human cDNA libraries cutting the CD39 gene in the (Openbiosystems, USA) with Sal I, Kpn I restriction enzymes, and at the same time forward primer sequence of the Kpn I, Bam HI position is SEQ ID NO: 27 (5'A-TGCAATTTCGCCTCTTGGC-3 ' ) connected Reverse primer of No. 28 (5'-GAGGATCCCTATACCATATCTTTCCAGAAATATGAAGG-3 ') was prepared and pre-warmed in a cDNA library at 94 ° C for 4 minutes in 94 ° C, 94 ° C, 20 seconds, 57 ° C and 45 seconds with Taq DNA polymerase (Genotech, Korea). And PCR reactions at 72 ° C. for 1 minute 35 times and 72 ° C. for 10 minutes to cut the restriction enzymes Kpn I, Bam H I and connect two DNA fragments to the pDsRed2-C1 vector (Clontech, USA).
pDsRed2-C1 벡터에 연결되어져 있는 CD39 유전자는 polyA 염기서열과 함께 Xho I, Pac I 위치가 연결된 서열번호 29(5'-GACTCGAGCATGGAAGATACAAAGGAGTCTAACG-3')의 포워드 프라이머 및 서열번호 30(5'-GCATTAATTAAGATTTAACAAAAATTTAACGCG-3')의 리버스 프라이머를 제작하여 교정 기능이 있는 Taq DNA 폴리머라제(Genotech, Korea)로 94℃에서 2분간 예열, 94℃ 20초, 60℃ 45초 및 72 ℃ 2분 35회 및 72℃ 10분의 조건으로 PCR 반응하여, 제한효소 Kpn I, BamH I로 자른 후, pCMAHKOneoDT벡터 Xho I과 Pac I 위치로 연결하였다. The CD39 gene linked to the pDsRed2-C1 vector is a forward primer of SEQ ID NO: 29 (5'-GACTCGAGCATGGAAGATACAAAGGAGTCTAACG-3 ') linked to the Xho I, Pac I position with a polyA sequence, and SEQ ID NO: 30 (5'-GCATTAATTAAGATTTAACAAAAATTTAACGCC-3). Taq DNA polymerase (Genotech, Korea) with a reverse primer of ') was prepared and preheated at 94 ° C for 2 minutes, 94 ° C for 20 seconds, 60 ° C for 45 seconds, and 72 ° C for 2 minutes 35 and 72 ° C for 10 minutes. PCR reaction under the condition of, and cut with restriction enzymes Kpn I, Bam H I, and then connected to the pCMAHKOneoDT vectors Xho I and Pac I position.
연결된 CD39유전자는 CMAH 유전자의 엑손 4번에 있는 ATG 뒤쪽에 위치함으로써, CMAH 유전자의 프로모터에 의해 발현하게 된다. 그 결과, CMAH 유전자를 제거함과 동시에 CD39 유전자를 적중에 의해 과발현시킬 수 있는 pCMAHKOCD39KIDT 벡터가 완성되었다. pCMAHKOCD39KIDT 벡터는 제한효소 Pml I에 의해 선형화 된 후, 체세포내로 도입될 수 있다. 상동 재조합이 일어나게 되면 DT 유전자는 탈락하게 된다(도 11). 타겟팅 여부는 서열번호 31(5'-ACCCCAGCTCACAATGAGCAACACCAGAT-3')의 포워드 프라이머 및 서열번호 32(5'-AGAGGGACCCCAATGACAGTAACAGCAGA-3')의 리버스 프라이머를 이용하여 LA Taq polymerase(Takara, Japan)로 94℃에서 2분간 예열, 94℃ 20초, 60℃ 45초 및 72℃ 2분 35회 및 72℃ 10분의 조건으로 PCR 반응하여 확 인하였다(도 12). The linked CD39 gene is located behind the ATG at
실시예Example 3: 유전자 3: gene 타겟팅Targeting 세포주 제조 Cell line manufacture
3-1: 3-1: CMAHCMAH 유전자 적중 세포주 제조 Gene targeting cell line production
본 발명자들은 실시예 2에서 제조한 pCMAHKOCD39KIDT 플라즈미드를 플라스미드 분리 키트(QIAfilter Plasmid Midi kits, Qiagen, USA)를 이용하여 정제한 후, 제한효소 Pml I로 선형화 한 후, 미니어쳐 돼지 유래의 귀조직 섬유아세포(pESF)에 도입하였다. pESF 세포는 DMEM(Gibco, Invitrogen corporation, USA), 10% FBS (Hyclone, USA), 0.001% 젠타마이신(Gibco, Invitrogen corporation, USA), 1% MEM 비필수 아미노산(Gibco, Invitrogen corporation, USA) 조성의 배양액으로 5% CO2, 37℃ 조건에서 배양하고 배양액은 2-3일 마다 교환하였다. 계대 2 또는 3인 2ⅹ106 pESF 세포에 Nucleofector(Amaxa Biosystems, USA) 방법으로 5 ㎍의 선형화된 DNA를 도입한 후, G418 300 ㎍/ml(Gibco, USA)를 포함한 배양액에서 배양하였다. 배양 12-14일이 지나면 직경 6-10mm 가량 크기의 세포 주를 관찰할 수 있었으며(도 13), 이들은 96 웰 배양 용기로 계대 배양하였다. 배양 용기에서 85-95% 정도로 자란 세포 주의 1/2은 다시 96well 로 계대배양 되었으며, 1/2은 타겟팅 여부 확인을 위해 사용되었다. 타겟팅 확인을 위한 세포들은 1M KCl, 1M Tris pH8.3, 1M MgCl2, 0.45% NP40, 0.45% Tween 20, 10 ㎍/ml Proteinase K로 구성된 용해 완충액(lysis buffer)에서 55℃, 1시간 동안 용해시킨 후, 100에서 5분간 처리한 후, 서열번호 31의 포워드 프라이머 및 서열번호 32의 리버스 프라이머를 이용하여 상기 실시에 2-2에 기재된 조건으로 PCR 반응을 수행하였다(도 14). 총 132개의 세포주에 대하여 상동재조합을 시도하였으며, 그 결과 2개의 세포주가 적중되어져 있음을 확인하였다. 이렇게 확립된 CMAH 유전자 적중 세포주를 CMAHKI#1과 CMAHKI#2라고 명명하고, 이중 CMAHKI#1을 한국세포주은행에 2008년 11월 24일자로 수탁번호 KCTC 11433BP로 기탁하였다.The present inventors purified the pCMAHKOCD39KIDT plasmid prepared in Example 2 using a plasmid separation kit (QIAfilter Plasmid Midi kits, Qiagen, USA), linearized with restriction enzyme Pml I, and then obtained ear tissue fibroblasts derived from miniature pigs ( pESF). pESF cells consisted of DMEM (Gibco, Invitrogen corporation, USA), 10% FBS (Hyclone, USA), 0.001% gentamycin (Gibco, Invitrogen corporation, USA), 1% MEM non-essential amino acids (Gibco, Invitrogen corporation, USA) The culture solution was cultured at 5% CO 2 , 37 ° C. conditions and the culture solution was changed every 2-3 days. 5 μg of linearized DNA was introduced into 2 × 10 6 pESF cells of
본 발명의 유전자 타겟팅 벡터는 장기 공여 동물의 내생적 항원 결정기 합성 유전자를 적중함과 동시에, 적중되는 내생적 항원 결정기 합성 유전자의 좌위에 발현하고자 하는 보체 억제 단백질 및/또는 혈전증 억제 단백질을 암호화하는 유전자를 넉-인시킴으로써, 도입 대상 유전자의 무작위적 삽입에 따른 유전자 침묵, 발아유전자의 활성화로 인한 암유발과 같은 문제점을 해소함으로써, 보다 안전한 이종간 장기 이식을 위한 공여 동물을 제조하는데 매우 효과적으로 이용될 수 있다.The gene targeting vector of the present invention targets an endogenous antigenic determinant synthetic gene of an organ donor animal and simultaneously encodes a complement inhibitory protein and / or a thrombosis inhibitory protein to be expressed at the locus of the endogenous antigenic determinant synthetic gene. By knock-in can solve the problems such as gene silencing following the random insertion of the gene to be introduced, cancer-causing caused by the activation of the germination gene, it can be very effectively used to prepare donor animals for safer cross-organ transplantation have.
도 1은 미니어쳐 돼지의 GT 유전자의 엑손 4번 전체(밑줄) 및 인트론 2번의 일부분을 포함하는 폴리뉴클레오티드의 핵산서열(서열번호 5)을 나타낸다.Figure 1 shows the nucleic acid sequence (SEQ ID NO: 5) of the polynucleotide containing the entire exon 4 (underscore) and part of the
도 2는 미니어쳐 돼지의 GT 유전자의 인트론 4번 전체와 엑손 5번의 일부분 (밑줄)을 포함하는 폴리뉴클레오티드의 핵산서열(서열번호 12)을 나타낸다.Figure 2 shows the nucleic acid sequence (SEQ ID NO: 12) of the polynucleotide containing the
도 3은 pGTKOneoTK 벡터를 이용한 GT 유전자의 적중을 도식화한 개요도(diagram)이다.Fig. 3 is a diagram illustrating the targeting of the GT gene using the pGTKOneoTK vector.
도 4는 pGTKOIRESKITK 벡터를 이용한 GT 유전자의 적중을 도식화한 개요도이다.4 is a schematic diagram illustrating the GT gene hit using the pGTKOIRESKITK vector.
도 5는 pGTKOneoTK 벡터의 제작과정을 나타내는 개요도이다.Figure 5 is a schematic diagram showing the manufacturing process of the pGTKOneoTK vector.
도 6은 pGTKOIRESKITK 벡터의 제작과정을 나타내는 개요도이다.6 is a schematic diagram illustrating a manufacturing process of a pGTKOIRESKITK vector.
도 7은 본 발명의 pGTKOIRESCD59KITK 벡터의 제작과정을 나타내는 개요도이다.7 is a schematic diagram showing the manufacturing process of the pGTKOIRESCD59KITK vector of the present invention.
도 8은 pGTKOIRESCD59KITK 벡터를 이용한 GT 유전자의 적중을 도식화한 개요도이다.Fig. 8 is a schematic diagram illustrating the GT gene hit using the pGTKOIRESCD59KITK vector.
도 9는 pCMAHKODT 벡터를 이용한 CMAH 유전자의 적중을 도식화한 개요도이다.9 is a schematic diagram illustrating the targeting of the CMAH gene using the pCMAHKODT vector.
도 10은 pCMAHKODT 벡터의 제작과정을 나타내는 개요도이다.10 is a schematic diagram showing a manufacturing process of a pCMAHKODT vector.
도 11은 pCMAHKOCD39KIDT 벡터의 제작과정을 나타내는 개요도이다.11 is a schematic diagram showing a manufacturing process of a pCMAHKOCD39KIDT vector.
도 12는 본 발명의 pCMAHKOCD39KIDT 벡터를 이용한 CMAH 유전자의 적중을 도 식화한 개요도이다.12 is a schematic diagram illustrating the targeting of the CMAH gene using the pCMAHKOCD39KIDT vector of the present invention.
도 13은 pCMAHKOCD39KIDT의 도입되기 전의 pESF 세포 상태와 CMAH 유전자 도입 후, 형성된 세포주의 사진이다.Fig. 13 is a photograph of pESF cell state before the introduction of pCMAHKOCD39KIDT and the cell line formed after the CMAH gene introduction.
도 14는 상기 세포주에서 실제 CMAH 유전자의 적중이 일어났는지 PCR 반응을 통해 확인한 실험결과를 나타내는 사진이다: Figure 14 is a photograph showing the results of experiments confirmed by PCR reaction whether the actual CMAH gene hit in the cell line:
V, 타겟팅 벡터; V, targeting vector;
WG, 유전자 도입하지 않은 세포주; WG, non-transgenic cell line;
#1 및 #2, 적중된 세포주; # 1 and # 2, targeted cell lines;
#3-#7, 유전자가 무작위적으로 삽입된 세포주; 및# 3- # 7, cell line with randomly inserted gene; And
M, 마커 DNA. M, marker DNA.
<110> Korea Research Institute of Bioscience and Biotechnology <120> Genetically-modified cell line for producing cloned miniature pigs for xenotransplantation and method for preparing the same <130> 8P-11-47 <150> KR2007-123823 <151> 2007-11-30 <160> 32 <170> KopatentIn 1.71 <210> 1 <211> 2972 <212> DNA <213> Sus scrofa <400> 1 gaattcatga ttattatcct ccaagcctgt tcctcctcca gcccatctga gaaaatacta 60 caacccccct gcttaagcag aaatcttggg tcttccttgt ctcatctctg ataacaaaat 120 taccaaccac gtcctatcaa ttctctctcc aaagtatata tatatatatt tttttttaat 180 tttttcccgc tgtacagcat ggggatcaag ttattcttac atgtatattt tccccccacc 240 ctttgttccg ttgcaatatg agtatctaga catagttctc aatgctactc agcaggatct 300 ccttgtaaat ctaagttgta tctgataacc ccaagctccc gatccctccc actccctccc 360 tctcctgtcg ggcagccaca agtctattct ccaagtccat gattttcttt tctgtggaga 420 tggtcatttg tgctggatat tagattccag ttataagtga tatcatatgg tatttgtcaa 480 agtatatatt ttatttttct ttgtcttttt gtcttttgtc tttttttttt ttgttgttgt 540 tgttgttgtt gttgttgttg ctattacttg ggccgctccc gcggcatatg gaggttccca 600 ggctaggagt tgaatcggag ctgtagccac cggcctacgc cagagccaca gcaacgcggg 660 atccgagccg cgtctgcaac ctacaccaca gctcacggca acgctggatc cttaacccac 720 tgagcaaggg cagggaccga acccgcaacc tcatggttcc tagtcggatt cgttaaccac 780 tgcgccacga cgggaactcc caaagtatat tttgaatcaa gccacccttt gagccaggcc 840 acctcctctt tatggtcatg agaacggtct gcccttgtcc ttttctccat tctccacact 900 cagcacccag atgggtctct ctaggtgaag ttggatcagg ggattctcca gctttagatg 960 ctttttggga ttccccaccc tactttccat acctttccag gttctgactg cctctgcccc 1020 ccttctgact gcctagcacc agccactcaa gggggacagt gtcagtcact atttttttct 1080 tgtccaggtt ttttgctttt gtttttttca aacacgagca gctctttctc ttgtctgcct 1140 ggtatagatg ctgtttccaa aatattctca tcccttctca cggcccttgt catcctttcc 1200 catcctatct tcatcccttg ggaagctcta aagtcatctc cccaaattga agggtgacta 1260 aagagtttcc cagaaggaaa aactgagttt ccaactacta cactgacttg caagaaatgt 1320 ttgtgtcttc attaaatgaa aaagaaaaaa ctgtaacaag atatgagaaa atacagaaag 1380 gaaataataa gactagaaaa gtcaaatata tagtgaaggt gttgcatcaa acacttaaat 1440 aaactagtac agatgttaaa agactaaatt atatagttga aggatagctg tgaagatgta 1500 aactatgaca tctaaaacac aaaatgttgg cgttcccgtc acggcacagt ggaaacgaat 1560 ccgactagga accatgaggt tgcaggttca attcctgccc ttgctcagtg ggttaaggat 1620 ccggtgttgc cgtgagctgt ggtgtaggta gccaatgagg cttggatccc gcgttgctgt 1680 ggctctggtg taggccggtg gctacagctc cgattcgacc cctagcctgg gaacctccat 1740 atgccgcggg agcggcccta aaaagacaaa agaccaaaaa aaaaaaaaaa caaaaaaccc 1800 acaaaatgtt gggaatcagt cctctactag tattatgtta ttgtcaagtt ttccttttat 1860 gtctgttaat atttgcgttc tagatgtagg tgctctgata tcgtgtgcat atatgttaac 1920 caatgttatg tcttcctctg gtattgatcc ctttgttatt atgtaatgcc ctactttatc 1980 ttttgttaca ttctttgttt atgagtattg ctgatatgtg gctagctgcc acacttttct 2040 tgtcctttcc atttacaata aatatctttc tatctccacc caaattaaag tactccgcaa 2100 cctgttattc cacccagcat cccttccctc ttcaactaca atttcatgca gcgatcaaga 2160 aatacgaatg taccgactgt ttgccacttg tgtgggtgca ttggggaaaa gctgggtggg 2220 aagtggcaga gcctagatta taaaggacca gggtgagagt tcccattgtg gctcagctga 2280 aatgaatctg actagcatcc atgaggacga aggtttgatc cctggcctca atcagtgggt 2340 taaggatctg gcgttgctgt ccgtgagttg tggtgtagtt cgcagacaag gcgtggactt 2400 agtgtggctg tggctgtggc ataggctagt ggctacagct ctgattcgac ccctagcctg 2460 ggaatctcta tatgctgtga gtgtggccct aaaatttaaa tgaaattaaa taaaggacca 2520 gggtatattt ttctttgagg ataaggtaca tagtcagtat atcagggaca gtagacctag 2580 gaaacggatg cttcctctag tctgtgatgc gaggtggggc atctgagttg ggggcggctg 2640 gagcccttag ggaccattaa ctaaacccgt cactctccca catctcggtg gaccttggga 2700 tcagtcagga tgcttcccct ttgagcctca aaatggcctt agtatccttc ccaacccaga 2760 cggccctgtc agttcattga cttggctaat ttgccagtgt aggcctatgc aaattaaggt 2820 agaacgcact ccttagcgct cgttgactat tcatcaactt ttccttttag aaaagatatt 2880 ggtataagca cttcttaaaa aaccatattc cactctgggt gtatttaatc taattttccc 2940 ttctcctttt cttttcccag gagaaaataa tg 2972 <210> 2 <211> 6846 <212> DNA <213> Sus scrofa <400> 2 ctgtcaatgc tgcttgtctc aactgtaatg gttgtgtttt gggaatacat caacaggtag 60 ttatgaaaca tgatgaaatg atgttgatga aagtctcctc taatctccta gttatcagcc 120 aagtcaccag cttgcattaa aagtaggatt cactgacacc gtaaagaaag cattccagag 180 agttgccgtt gtggctcagg ggcagcaaac ccaattagga tccaagagga ggtgggtttg 240 atccctggcc ttgctctttg gcttaaggat ccggcattgc cgtgacctgt ggtgtaggtt 300 gcagatgcag ctcggatctg gcattgctgt ggctgtggcg taggctggtg gcttcagctc 360 cagtttgacc cctagcctgg gaacttccat atcccacact tgcggcccta aaaagcaaag 420 aaagaaagaa aatattctac ccttcctgta tccctgagcc cttaaatacc gtctttaaag 480 tcattagatc ttcaagtacc ttccagctaa ttaattatct tccttcctgc catgttgcca 540 ttgtcctgat ttttatacct ctgcagttct gggtagacta gagccagaaa taataaggtc 600 atgttaagac caagatataa tattaaatta tttatatgac cagatatgga agttaccttg 660 agaactttca gacaggaatt ccatgagaaa tacaccctga tttttgcaat cctaaaatat 720 ttgcagagtt taaaggaaca actcaagttg ttgacttttg ctgcaaaaca cactgagtcg 780 ctggtgattc atttgtgcct ggctaaactt ttgggtgttt tgtctctttt tttttaactc 840 tggaaagcaa aatgaattaa acatttctga gttttcaaat tcatcagtgg attcacccca 900 aatatttgag ctgcttcttt gcttttggaa actacgatgc cttggagatt ccagctggag 960 acgcttctga cagaaagaaa tgtctgcaag cagctacaaa aatgcatgat ggctttgact 1020 taagaggcat tgataccgct tggcctttct ttcaaaaagg ccaccttaca acttggcctg 1080 aaggcattcc cgtggtggtg cagcggaaaa tgaatctgac taggaacccc gaggttgtgg 1140 gttcaatccc tggccttgct cagtggctta aggatcgggt gttgaagtaa gctgtggtgt 1200 agattgcaga cgcagcttgg atctggtgtt gctgtggctt tggtgtaggc cggcagctac 1260 agctccactt ggacccctag tctgggaacc tccatatgcc acaggtgtgg ccctaaaagg 1320 aaaaaagaca acaaacaaac aaaaaaccaa aaaacaactt ggcctggaga gctatgtcat 1380 caccattgat attttgatgg gtagtgtttt agtagcccct caagttcagg atgatggcct 1440 ggattaacgt tagaatgtct cttaaattct aagacttgat gagccagcag gaccattttg 1500 gccacttaga aaggaactgc atcttcaggt ccatcagtag aaggaggatt ctctagggag 1560 ttctctctta gctcagcggg ttcaagaatt cagtcttgtc cctacagcag ctcaggtgac 1620 tgctatggct tggctttgat ccctggccca ggaatttctg catgctgcag gtgcagccaa 1680 aaaaaaaaaa aaaaaggagg aggtggattc cctagaataa gaagctgtca ttcctttgga 1740 tgcttcatag atctaaccac ttctggaaca gttattccct ctcattctga agaactcatt 1800 ttaagaaaaa caagacgagc tagagagtga acaaatgtct acaaaccaac cttttcgaat 1860 tgaggaaact gtggtacttc ctctgaagaa aagatgacag cgttggatgc agagaccctg 1920 gggctccctt aggtacttga ggactgagga gatattctca gtggaggctg gagctaggct 1980 gcctggggtt ggtcctgtgc caccacttcc ctcctctgtg actttgggca agtttcccta 2040 tctttaaaaa tggggatgat agtagtacct gcttcatagg gttgttggat aaaataagtt 2100 gtgaataaag cactaagggc aacgtactta gtaagcgctg gctgccatca ccaccaccac 2160 tatcaccatc tgtccggagg gcagcatagg acaggagatt ttggcaaata gaaggaagag 2220 ttctaggagt tcccgttgtg gtgcagggga aatgaatcca actaggaact aggaggtttc 2280 gggttcaatc ccgcgcctcg ctcagtgggt taaggatcca gtgttgccat gagctgtggt 2340 gtagattgca gacatggcta ggatctggag ttgctatggc tgtggtgtaa gctggcagct 2400 gtagctcgga ttctacccct agcctgggaa tttccgtatg ccacaggttt ggccctacaa 2460 agaaaaaaga aaaagaaaaa gaaaaaattc taggggctga aagaatctaa cagaagagca 2520 agttccccat ggggttcctg acctgagttg agatgcttgt gtaggcaacc ttcaagctct 2580 gaactcttga ttgttttgaa ttgcagccag agttatactt ccatattttg ggtacttcac 2640 aaaattaaaa cacagaagcc aaaggcccag aagtgcatat tggtgctggc ctcccataaa 2700 gagggttgtt ttgcagtgct gggcacactc tctcttcaca gtaactggag cagattctgg 2760 ctgctcttca gggccgtagt ctggcaccca gactgcagcc acatcattct tcaatgtgag 2820 gaatctattt gaacatctgc aaggggttta aaaggcagga gattctttgc caccttgtga 2880 attggtctga ggtgagctga gggcactaac cttagacagg tgggtagcac tgtagctaaa 2940 gaggattaca ggagttcctg ttgtggctta gtggtaacaa atccaactag tatccatgag 3000 gattcaggtt cgatccctgg cctcgctcag tgggtcaggt atccggtgtt gctgtggctg 3060 tggtgtaggc tggcagcttc agctctgatt tgacccctag cctgggaact tccatgtgct 3120 gtaggtaagg cccttgaaaa aaaaaaaaaa gagatttaca aaataactcc atcaaacaca 3180 tacagctgtt taagaatgtc atccaggaca gcatttggtt aaaggctaga tgaaaaaaaa 3240 aaaaaaaatc ttagaatttt atttatttat tttttctttt tagggccaga cctgtggcct 3300 atggaaatgc ctgggctagg ggtggaatca gagctgccta caccacagcc atagccacgc 3360 cagatccaag ccccgtctgt gacctacacc acagctcatg gcaaacactg gatccttaat 3420 ccactgagtg aggccaggaa ttgaacccac attctcatgg atgctagttg ggttcttaag 3480 ccactgagcc acaagcttag aattttagag gtggaagaaa ctttaagagc tataataaag 3540 taatgatggt gatggtgatt ttgatgttag cggctactag ttattgagtg tttgcttgtg 3600 ccaggaactc cactgttcat tccctcctgt ttttaaaaca gccctggaag gtcagtgtta 3660 gtccacattt ctagatgagg aatactgagt ttccacaata ttaaatgtga acgttcaagg 3720 tcacattttt aggaagattt aggtccaggg ctgtctgact tgggtaacct gggtaaccct 3780 tcctttagtc aaggtttcca ttgttcaggc gatggacaag taggtgaaat gccttaacag 3840 tgaacttatg tctaacttct aattagaact cagatcttct gattcatcat ctggggctcc 3900 ttctggagct ggttgttcat gccaaatgct gcgaggggta cagtgtgcgc caaggagaat 3960 ccctaccctc aaggggttat gctgtagatg gagcaggcag aggtacccat gaaagcccaa 4020 caacacaggc tagaaggagg atgtcagaga gagagagcaa aggaacgtga gagttcaggg 4080 agggcaagat tatgtttggc ttggagatgg atctatgttt tgcatttatt tttttggggg 4140 gggggtcttt ttgctacttc ttgggctgct cccgaggcat atggaggttc ccaggctagg 4200 ggtctaattg gagccgcagc caccagccta tgccagagcc acagcaacgc aggatctgag 4260 ccgcgtctgc aaccttcacc acagctcacg gcaacgccag atcgttaacc cactgagcaa 4320 gggcagggac cgaacctgca acctcatggt tcctagtcag attcgttaag cactgcgcca 4380 cgacgggaac tccctcattt agaaatattt attgagcacc tactgtatgc caggcattgt 4440 gctaggttca taccaaagaa ggctcagaag agatggcatc cgagctgtgc cttgaaggat 4500 gaatatgtgt taaatgccgt acacttcagg gtggttgttg ctgtgacctg aggtgttgaa 4560 ggcttctggg aaaggagggt gagatgagga agagggaggg gttactaaaa agatgggacg 4620 aggtggcaaa tccaaatcta taaattgatg ccctgagtgc ctcggcagga gggtggggct 4680 cctgagtgct gggtggcacg ggccctcccc ctcctcttgc ccctttccct tccccctctt 4740 gtaggatctg aagtcagatt ccccaggttc aaatactgtt tcttccctta gcagtatgac 4800 cttgggcaaa ataatttatt gcctctgtcc ctctgaggag gaatagaacc tccttcattg 4860 actgttatta gaatttaatg agctaataca tgtcagttgc ttagaaaggt ccccagccaa 4920 ctattagcta ttatgaatat tatcagatca atagacagat ttagaaacaa gggactttag 4980 agctgggtcc atgggtactg agcttagagg ggaaaccata ggtggtagga aggcatgtat 5040 ttcattccta ccaggagatg tggactccca gctggggcag aaggcagagg gaggagatcg 5100 gggctttggc agaatctcaa acaaatatta gtggttagtg gttttttgtt tctgttttaa 5160 gagatgaggg caggcgtttc cgatgtggcg cagtggaaac gaatctgact agtatccatg 5220 aggatgcagg ttccatccct ggactcactc agtgagttaa ggatccggca ttgccgtgag 5280 ttgtggtgta ggtcacagac acagctcaga tctggcattg ctgtggctgt ggtgtaggct 5340 agcagctgta gctccaattc aacccctagc ctgggaactt ccacatgccg caggtgcaac 5400 cccaaaagat aaatgaataa ataaataaat atgcgacctt cctttcttgg ggcctttgca 5460 tgtttttctc tctgttaggc acactcttgc taatccctct tcactgggcc tcctatgtat 5520 ccttcagaac tcagctaaaa catcatcccc tcccctgggg agccttcgag gtcttcctgt 5580 taagtgctcc tatgctttct tggagttttg aagtcctata atgatgtgtt tatcaaaata 5640 gggtccaccc tccctgccag cttctctaca ccacagacac atggtgtctg tttcagtcaa 5700 cactgtatgt ctggcacttg acatgtaacg catgctcagc aggtatttgt tgaatgaatg 5760 gaggcggtct gctagagtcg tcatatattt actgatcccg tcttgtagga tggtctcact 5820 gcttttgtta gcttaagaag tacctttttt tttttttttt aatggccaca cccatggcat 5880 atagaaattc cacgaaggaa ggaagaaaga aagaaagaaa gaaggaaatt cctgggtcag 5940 ggattgaatc caagccacag gtgcaacctg agctgcagtt gcggcaacac cacatctttt 6000 aacccactgt gctgggccag ggatcatacc tgtgcatcta cagcgaccca agccacggca 6060 gtcagattct ttttctgcct ttctttcttt cttttctttt tttttttttt ttttttttgt 6120 ctttttgcct tttctaggtg cggcatatgg aggttcccag gctaggtgtc gaatcagagc 6180 tgtagacgcc ggcctaaacc acagccacag caacacagga tccaagcctt gtctgtgacc 6240 tacaccacag ctcacggcaa cgctggatcc ttaacccgct gagcgaggcc agggattgaa 6300 cccgcaacct catggttctt agttggattc gttaaccact gagccatgat gggaactcct 6360 gcagtcagat tcttaaccca ctatgccaca gcaggaactc ctagaagtgc cctttgaggc 6420 tactctgtag acagctttga gccagcgagg caagacctgt ttttctggag gaagataaat 6480 cctgggtgag ggatgggtgg gctgtggtct tcctgggacc catctctgga gcctctctcc 6540 ctcagcaaag ccaccttgga caataagagc tgccatctat tttttttttc tttaaactaa 6600 gatttgatat tttccagaga cctccctccc accgtttgat ctgagtaatt ctgaaatgac 6660 gagagtcccg tgatatcatt ttttcgatct cgaaggtgga aacctgggag tagccacaac 6720 ccaggctctc agctcagcct agggtttcaa tgataatgat tgcaaaatag cttttctctg 6780 cattccaagt aacatgatat gtttttattt ccatttgctt ttagcccaga aggttctttg 6840 ttctgg 6846 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward for GT partial intron 2 and exon 4 <400> 3 tccatgaaca acttcgattg catg 24 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Reverse for GT partial intron 2 and exon 4 <400> 4 tggctgataa ctaggagatt agaggag 27 <210> 5 <211> 3242 <212> DNA <213> Sus scrofa <400> 5 tccatgaaca acttcgattg catggtctgc ctacatctct ctgatgaact ttagacttga 60 atgtccactt gtctccctgt ccccttttag gtattcgcac actccccgac attcacacgt 120 ccaaaaggga attcatgatt attatcctcc aagcctgttc ctcctccagc ccatctgaga 180 aaatactaca acccccctgc ttaagcagaa atcttgggtc ttccttgtct catctctgat 240 aacaaaatta ccaaccacgt cctatcaatt ctctctccaa agtatatata tatatatttt 300 tttttaattt tttcccgctg tacagcatgg ggatcaagtt attcttacat gtatattttc 360 cccccaccct ttgttccgtt gcaatatgag tatctagaca tagttctcaa tgctactcag 420 caggatctcc ttgtaaatct aagttgtatc tgataacccc aagctcccga tccctcccac 480 tccctccctc tcctgtcggg cagccacaag tctattctcc aagtccatga ttttcttttc 540 tgtggagatg gtcatttgtg ctggatatta gattccagtt ataagtgata tcatatggta 600 tttgtcaaag tatatatttt atttttcttt gtctttttgt cttttgtctt tttttttttt 660 gttgttgttg ttgttgttgt tgttgttgct attacttggg ccgctcccgc ggcatatgga 720 ggttcccagg ctaggagttg aatcggagct gtagccaccg gcctacgcca gagccacagc 780 aacgcgggat ccgagccgcg tctgcaacct acaccacagc tcacggcaac gctggatcct 840 taacccactg agcaagggca gggaccgaac ccgcaacctc atggttccta gtcggattcg 900 ttaaccactg cgccacgacg ggaactccca aagtatattt tgaatcaagc caccctttga 960 gccaggccac ctcctcttta tggtcatgag aacggtctgc ccttgtcctt ttctccattc 1020 tccacactca gcacccagat gggtctctct aggtgaagtt ggatcagggg attctccagc 1080 tttagatgct ttttgggatt ccccacccta ctttccatac ctttccaggt tctgactgcc 1140 tctgcccccc ttctgactgc ctagcaccag ccactcaagg gggacagtgt cagtcactat 1200 ttttttcttg tccaggtttt ttgcttttgt ttttttcaaa cacgagcagc tctttctctt 1260 gtctgcctgg tatagatgct gtttccaaaa tattctcatc ccttctcacg gcccttgtca 1320 tcctttccca tcctatcttc atcccttggg aagctctaaa gtcatctccc caaattgaag 1380 ggtgactaaa gagtttccca gaaggaaaaa ctgagtttcc aactactaca ctgacttgca 1440 agaaatgttt gtgtcttcat taaatgaaaa agaaaaaact gtaacaagat atgagaaaat 1500 acagaaagga aataataaga ctagaaaagt caaatatata gtgaaggtgt tgcatcaaac 1560 acttaaataa actagtacag atgttaaaag actaaattat atagttgaag gatagctgtg 1620 aagatgtaaa ctatgacatc taaaacacaa aatgttggcg ttcccgtcac ggcacagtgg 1680 aaacgaatcc gactaggaac catgaggttg caggttcaat tcctgccctt gctcagtggg 1740 ttaaggatcc ggtgttgccg tgagctgtgg tgtaggtagc caatgaggct tggatcccgc 1800 gttgctgtgg ctctggtgta ggccggtggc tacagctccg attcgacccc tagcctggga 1860 acctccatat gccgcgggag cggccctaaa aagacaaaag accaaaaaaa aaaaaaaaca 1920 aaaaacccac aaaatgttgg gaatcagtcc tctactagta ttatgttatt gtcaagtttt 1980 ccttttatgt ctgttaatat ttgcgttcta gatgtaggtg ctctgatatc gtgtgcatat 2040 atgttaacca atgttatgtc ttcctctggt attgatccct ttgttattat gtaatgccct 2100 actttatctt ttgttacatt ctttgtttat gagtattgct gatatgtggc tagctgccac 2160 acttttcttg tcctttccat ttacaataaa tatctttcta tctccaccca aattaaagta 2220 ctccgcaacc tgttattcca cccagcatcc cttccctctt caactacaat ttcatgcagc 2280 gatcaagaaa tacgaatgta ccgactgttt gccacttgtg tgggtgcatt ggggaaaagc 2340 tgggtgggaa gtggcagagc ctagattata aaggaccagg gtgagagttc ccattgtggc 2400 tcagctgaaa tgaatctgac tagcatccat gaggacgaag gtttgatccc tggcctcaat 2460 cagtgggtta aggatctggc gttgctgtcc gtgagttgtg gtgtagttcg cagacaaggc 2520 gtggacttag tgtggctgtg gctgtggcat aggctagtgg ctacagctct gattcgaccc 2580 ctagcctggg aatctctata tgctgtgagt gtggccctaa aatttaaatg aaattaaata 2640 aaggaccagg gtatattttt ctttgaggat aaggtacata gtcagtatat cagggacagt 2700 agacctagga aacggatgct tcctctagtc tgtgatgcga ggtggggcat ctgagttggg 2760 ggcggctgga gcccttaggg accattaact aaacccgtca ctctcccaca tctcggtgga 2820 ccttgggatc agtcaggatg cttccccttt gagcctcaaa atggccttag tatccttccc 2880 aacccagacg gccctgtcag ttcattgact tggctaattt gccagtgtag gcctatgcaa 2940 attaaggtag aacgcactcc ttagcgctcg ttgactattc atcaactttt ccttttagaa 3000 aagatattgg tataagcact tcttaaaaaa ccatattcca ctctgggtgt atttaatcta 3060 attttccctt ctccttttct tttcccagga gaaaataatg aatgtcaaag gaagagtggt 3120 tctgtcaatg ctgcttgtct caactgtaat ggttgtgttt tgggaataca tcaacaggta 3180 attatgaaac atgatgaaat gatgttgatg aaagtctcct ctaatctcct agttatcagc 3240 ca 3242 <210> 6 <211> 3088 <212> DNA <213> Sus scrofa <400> 6 tccatgaaca acttcgattg catggtctgc ctacatctct ctgatgaact ttagacttga 60 atgtccactt gtctccctgt ccccttttag gtattcgcac actccccgac attcacacgt 120 ccaaaaggga attcatgatt attatcctcc aagcctgttc ctcctccagc ccatctgaga 180 aaatactaca acccccctgc ttaagcagaa atcttgggtc ttccttgtct catctctgat 240 aacaaaatta ccaaccacgt cctatcaatt ctctctccaa agtatatata tatatatttt 300 tttttaattt tttcccgctg tacagcatgg ggatcaagtt attcttacat gtatattttc 360 cccccaccct ttgttccgtt gcaatatgag tatctagaca tagttctcaa tgctactcag 420 caggatctcc ttgtaaatct aagttgtatc tgataacccc aagctcccga tccctcccac 480 tccctccctc tcctgtcggg cagccacaag tctattctcc aagtccatga ttttcttttc 540 tgtggagatg gtcatttgtg ctggatatta gattccagtt ataagtgata tcatatggta 600 tttgtcaaag tatatatttt atttttcttt gtctttttgt cttttgtctt tttttttttt 660 gttgttgttg ttgttgttgt tgttgttgct attacttggg ccgctcccgc ggcatatgga 720 ggttcccagg ctaggagttg aatcggagct gtagccaccg gcctacgcca gagccacagc 780 aacgcgggat ccgagccgcg tctgcaacct acaccacagc tcacggcaac gctggatcct 840 taacccactg agcaagggca gggaccgaac ccgcaacctc atggttccta gtcggattcg 900 ttaaccactg cgccacgacg ggaactccca aagtatattt tgaatcaagc caccctttga 960 gccaggccac ctcctcttta tggtcatgag aacggtctgc ccttgtcctt ttctccattc 1020 tccacactca gcacccagat gggtctctct aggtgaagtt ggatcagggg attctccagc 1080 tttagatgct ttttgggatt ccccacccta ctttccatac ctttccaggt tctgactgcc 1140 tctgcccccc ttctgactgc ctagcaccag ccactcaagg gggacagtgt cagtcactat 1200 ttttttcttg tccaggtttt ttgcttttgt ttttttcaaa cacgagcagc tctttctctt 1260 gtctgcctgg tatagatgct gtttccaaaa tattctcatc ccttctcacg gcccttgtca 1320 tcctttccca tcctatcttc atcccttggg aagctctaaa gtcatctccc caaattgaag 1380 ggtgactaaa gagtttccca gaaggaaaaa ctgagtttcc aactactaca ctgacttgca 1440 agaaatgttt gtgtcttcat taaatgaaaa agaaaaaact gtaacaagat atgagaaaat 1500 acagaaagga aataataaga ctagaaaagt caaatatata gtgaaggtgt tgcatcaaac 1560 acttaaataa actagtacag atgttaaaag actaaattat atagttgaag gatagctgtg 1620 aagatgtaaa ctatgacatc taaaacacaa aatgttggcg ttcccgtcac ggcacagtgg 1680 aaacgaatcc gactaggaac catgaggttg caggttcaat tcctgccctt gctcagtggg 1740 ttaaggatcc ggtgttgccg tgagctgtgg tgtaggtagc caatgaggct tggatcccgc 1800 gttgctgtgg ctctggtgta ggccggtggc tacagctccg attcgacccc tagcctggga 1860 acctccatat gccgcgggag cggccctaaa aagacaaaag accaaaaaaa aaaaaaaaca 1920 aaaaacccac aaaatgttgg gaatcagtcc tctactagta ttatgttatt gtcaagtttt 1980 ccttttatgt ctgttaatat ttgcgttcta gatgtaggtg ctctgatatc gtgtgcatat 2040 atgttaacca atgttatgtc ttcctctggt attgatccct ttgttattat gtaatgccct 2100 actttatctt ttgttacatt ctttgtttat gagtattgct gatatgtggc tagctgccac 2160 acttttcttg tcctttccat ttacaataaa tatctttcta tctccaccca aattaaagta 2220 ctccgcaacc tgttattcca cccagcatcc cttccctctt caactacaat ttcatgcagc 2280 gatcaagaaa tacgaatgta ccgactgttt gccacttgtg tgggtgcatt ggggaaaagc 2340 tgggtgggaa gtggcagagc ctagattata aaggaccagg gtgagagttc ccattgtggc 2400 tcagctgaaa tgaatctgac tagcatccat gaggacgaag gtttgatccc tggcctcaat 2460 cagtgggtta aggatctggc gttgctgtcc gtgagttgtg gtgtagttcg cagacaaggc 2520 gtggacttag tgtggctgtg gctgtggcat aggctagtgg ctacagctct gattcgaccc 2580 ctagcctggg aatctctata tgctgtgagt gtggccctaa aatttaaatg aaattaaata 2640 aaggaccagg gtatattttt ctttgaggat aaggtacata gtcagtatat cagggacagt 2700 agacctagga aacggatgct tcctctagtc tgtgatgcga ggtggggcat ctgagttggg 2760 ggcggctgga gcccttaggg accattaact aaacccgtca ctctcccaca tctcggtgga 2820 ccttgggatc agtcaggatg cttccccttt gagcctcaaa atggccttag tatccttccc 2880 aacccagacg gccctgtcag ttcattgact tggctaattt gccagtgtag gcctatgcaa 2940 attaaggtag aacgcactcc ttagcgctcg ttgactattc atcaactttt ccttttagaa 3000 aagatattgg tataagcact tcttaaaaaa ccatattcca ctctgggtgt atttaatcta 3060 attttccctt ctccttttct tttcccag 3088 <210> 7 <211> 89 <212> DNA <213> Sus scrofa <400> 7 gagaaaataa tgaatgtcaa aggaagagtg gttctgtcaa tgctgcttgt ctcaactgta 60 atggttgtgt tttgggaata catcaacag 89 <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Forward for left arm <400> 8 gaattcatga ttattatcct cccaagc 27 <210> 9 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Reverse for left arm <400> 9 catgaattcc attattttct cctggg 26 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward for GT intron 4 and partial exon 5 <400> 10 gtcgacctgt caatgctgct tg 22 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Reverse for GT intron 4 and partial exon 5 <400> 11 ctcgagccag aacaaagaac cttc 24 <210> 12 <211> 6858 <212> DNA <213> Sus scrofa <400> 12 gtcgacctgt caatgctgct tgtctcaact gtaatggttg tgttttggga atacatcaac 60 aggtagttat gaaacatgat gaaatgatgt tgatgaaagt ctcctctaat ctcctagtta 120 tcagccaagt caccagcttg cattaaaagt aggattcact gacaccgtaa agaaagcatt 180 ccagagagtt gccgttgtgg ctcaggggca gcaaacccaa ttaggatcca agaggaggtg 240 ggtttgatcc ctggccttgc tctttggctt aaggatccgg cattgccgtg acctgtggtg 300 taggttgcag atgcagctcg gatctggcat tgctgtggct gtggcgtagg ctggtggctt 360 cagctccagt ttgaccccta gcctgggaac ttccatatcc cacacttgcg gccctaaaaa 420 gcaaagaaag aaagaaaata ttctaccctt cctgtatccc tgagccctta aataccgtct 480 ttaaagtcat tagatcttca agtaccttcc agctaattaa ttatcttcct tcctgccatg 540 ttgccattgt cctgattttt atacctctgc agttctgggt agactagagc cagaaataat 600 aaggtcatgt taagaccaag atataatatt aaattattta tatgaccaga tatggaagtt 660 accttgagaa ctttcagaca ggaattccat gagaaataca ccctgatttt tgcaatccta 720 aaatatttgc agagtttaaa ggaacaactc aagttgttga cttttgctgc aaaacacact 780 gagtcgctgg tgattcattt gtgcctggct aaacttttgg gtgttttgtc tctttttttt 840 taactctgga aagcaaaatg aattaaacat ttctgagttt tcaaattcat cagtggattc 900 accccaaata tttgagctgc ttctttgctt ttggaaacta cgatgccttg gagattccag 960 ctggagacgc ttctgacaga aagaaatgtc tgcaagcagc tacaaaaatg catgatggct 1020 ttgacttaag aggcattgat accgcttggc ctttctttca aaaaggccac cttacaactt 1080 ggcctgaagg cattcccgtg gtggtgcagc ggaaaatgaa tctgactagg aaccccgagg 1140 ttgtgggttc aatccctggc cttgctcagt ggcttaagga tcgggtgttg aagtaagctg 1200 tggtgtagat tgcagacgca gcttggatct ggtgttgctg tggctttggt gtaggccggc 1260 agctacagct ccacttggac ccctagtctg ggaacctcca tatgccacag gtgtggccct 1320 aaaaggaaaa aagacaacaa acaaacaaaa aaccaaaaaa caacttggcc tggagagcta 1380 tgtcatcacc attgatattt tgatgggtag tgttttagta gcccctcaag ttcaggatga 1440 tggcctggat taacgttaga atgtctctta aattctaaga cttgatgagc cagcaggacc 1500 attttggcca cttagaaagg aactgcatct tcaggtccat cagtagaagg aggattctct 1560 agggagttct ctcttagctc agcgggttca agaattcagt cttgtcccta cagcagctca 1620 ggtgactgct atggcttggc tttgatccct ggcccaggaa tttctgcatg ctgcaggtgc 1680 agccaaaaaa aaaaaaaaaa aggaggaggt ggattcccta gaataagaag ctgtcattcc 1740 tttggatgct tcatagatct aaccacttct ggaacagtta ttccctctca ttctgaagaa 1800 ctcattttaa gaaaaacaag acgagctaga gagtgaacaa atgtctacaa accaaccttt 1860 tcgaattgag gaaactgtgg tacttcctct gaagaaaaga tgacagcgtt ggatgcagag 1920 accctggggc tcccttaggt acttgaggac tgaggagata ttctcagtgg aggctggagc 1980 taggctgcct ggggttggtc ctgtgccacc acttccctcc tctgtgactt tgggcaagtt 2040 tccctatctt taaaaatggg gatgatagta gtacctgctt catagggttg ttggataaaa 2100 taagttgtga ataaagcact aagggcaacg tacttagtaa gcgctggctg ccatcaccac 2160 caccactatc accatctgtc cggagggcag cataggacag gagattttgg caaatagaag 2220 gaagagttct aggagttccc gttgtggtgc aggggaaatg aatccaacta ggaactagga 2280 ggtttcgggt tcaatcccgc gcctcgctca gtgggttaag gatccagtgt tgccatgagc 2340 tgtggtgtag attgcagaca tggctaggat ctggagttgc tatggctgtg gtgtaagctg 2400 gcagctgtag ctcggattct acccctagcc tgggaatttc cgtatgccac aggtttggcc 2460 ctacaaagaa aaaagaaaaa gaaaaagaaa aaattctagg ggctgaaaga atctaacaga 2520 agagcaagtt ccccatgggg ttcctgacct gagttgagat gcttgtgtag gcaaccttca 2580 agctctgaac tcttgattgt tttgaattgc agccagagtt atacttccat attttgggta 2640 cttcacaaaa ttaaaacaca gaagccaaag gcccagaagt gcatattggt gctggcctcc 2700 cataaagagg gttgttttgc agtgctgggc acactctctc ttcacagtaa ctggagcaga 2760 ttctggctgc tcttcagggc cgtagtctgg cacccagact gcagccacat cattcttcaa 2820 tgtgaggaat ctatttgaac atctgcaagg ggtttaaaag gcaggagatt ctttgccacc 2880 ttgtgaattg gtctgaggtg agctgagggc actaacctta gacaggtggg tagcactgta 2940 gctaaagagg attacaggag ttcctgttgt ggcttagtgg taacaaatcc aactagtatc 3000 catgaggatt caggttcgat ccctggcctc gctcagtggg tcaggtatcc ggtgttgctg 3060 tggctgtggt gtaggctggc agcttcagct ctgatttgac ccctagcctg ggaacttcca 3120 tgtgctgtag gtaaggccct tgaaaaaaaa aaaaaagaga tttacaaaat aactccatca 3180 aacacataca gctgtttaag aatgtcatcc aggacagcat ttggttaaag gctagatgaa 3240 aaaaaaaaaa aaaatcttag aattttattt atttattttt tctttttagg gccagacctg 3300 tggcctatgg aaatgcctgg gctaggggtg gaatcagagc tgcctacacc acagccatag 3360 ccacgccaga tccaagcccc gtctgtgacc tacaccacag ctcatggcaa acactggatc 3420 cttaatccac tgagtgaggc caggaattga acccacattc tcatggatgc tagttgggtt 3480 cttaagccac tgagccacaa gcttagaatt ttagaggtgg aagaaacttt aagagctata 3540 ataaagtaat gatggtgatg gtgattttga tgttagcggc tactagttat tgagtgtttg 3600 cttgtgccag gaactccact gttcattccc tcctgttttt aaaacagccc tggaaggtca 3660 gtgttagtcc acatttctag atgaggaata ctgagtttcc acaatattaa atgtgaacgt 3720 tcaaggtcac atttttagga agatttaggt ccagggctgt ctgacttggg taacctgggt 3780 aacccttcct ttagtcaagg tttccattgt tcaggcgatg gacaagtagg tgaaatgcct 3840 taacagtgaa cttatgtcta acttctaatt agaactcaga tcttctgatt catcatctgg 3900 ggctccttct ggagctggtt gttcatgcca aatgctgcga ggggtacagt gtgcgccaag 3960 gagaatccct accctcaagg ggttatgctg tagatggagc aggcagaggt acccatgaaa 4020 gcccaacaac acaggctaga aggaggatgt cagagagaga gagcaaagga acgtgagagt 4080 tcagggaggg caagattatg tttggcttgg agatggatct atgttttgca tttatttttt 4140 tggggggggg gtctttttgc tacttcttgg gctgctcccg aggcatatgg aggttcccag 4200 gctaggggtc taattggagc cgcagccacc agcctatgcc agagccacag caacgcagga 4260 tctgagccgc gtctgcaacc ttcaccacag ctcacggcaa cgccagatcg ttaacccact 4320 gagcaagggc agggaccgaa cctgcaacct catggttcct agtcagattc gttaagcact 4380 gcgccacgac gggaactccc tcatttagaa atatttattg agcacctact gtatgccagg 4440 cattgtgcta ggttcatacc aaagaaggct cagaagagat ggcatccgag ctgtgccttg 4500 aaggatgaat atgtgttaaa tgccgtacac ttcagggtgg ttgttgctgt gacctgaggt 4560 gttgaaggct tctgggaaag gagggtgaga tgaggaagag ggaggggtta ctaaaaagat 4620 gggacgaggt ggcaaatcca aatctataaa ttgatgccct gagtgcctcg gcaggagggt 4680 ggggctcctg agtgctgggt ggcacgggcc ctccccctcc tcttgcccct ttcccttccc 4740 cctcttgtag gatctgaagt cagattcccc aggttcaaat actgtttctt cccttagcag 4800 tatgaccttg ggcaaaataa tttattgcct ctgtccctct gaggaggaat agaacctcct 4860 tcattgactg ttattagaat ttaatgagct aatacatgtc agttgcttag aaaggtcccc 4920 agccaactat tagctattat gaatattatc agatcaatag acagatttag aaacaaggga 4980 ctttagagct gggtccatgg gtactgagct tagaggggaa accataggtg gtaggaaggc 5040 atgtatttca ttcctaccag gagatgtgga ctcccagctg gggcagaagg cagagggagg 5100 agatcggggc tttggcagaa tctcaaacaa atattagtgg ttagtggttt tttgtttctg 5160 ttttaagaga tgagggcagg cgtttccgat gtggcgcagt ggaaacgaat ctgactagta 5220 tccatgagga tgcaggttcc atccctggac tcactcagtg agttaaggat ccggcattgc 5280 cgtgagttgt ggtgtaggtc acagacacag ctcagatctg gcattgctgt ggctgtggtg 5340 taggctagca gctgtagctc caattcaacc cctagcctgg gaacttccac atgccgcagg 5400 tgcaacccca aaagataaat gaataaataa ataaatatgc gaccttcctt tcttggggcc 5460 tttgcatgtt tttctctctg ttaggcacac tcttgctaat ccctcttcac tgggcctcct 5520 atgtatcctt cagaactcag ctaaaacatc atcccctccc ctggggagcc ttcgaggtct 5580 tcctgttaag tgctcctatg ctttcttgga gttttgaagt cctataatga tgtgtttatc 5640 aaaatagggt ccaccctccc tgccagcttc tctacaccac agacacatgg tgtctgtttc 5700 agtcaacact gtatgtctgg cacttgacat gtaacgcatg ctcagcaggt atttgttgaa 5760 tgaatggagg cggtctgcta gagtcgtcat atatttactg atcccgtctt gtaggatggt 5820 ctcactgctt ttgttagctt aagaagtacc tttttttttt ttttttaatg gccacaccca 5880 tggcatatag aaattccacg aaggaaggaa gaaagaaaga aagaaagaag gaaattcctg 5940 ggtcagggat tgaatccaag ccacaggtgc aacctgagct gcagttgcgg caacaccaca 6000 tcttttaacc cactgtgctg ggccagggat catacctgtg catctacagc gacccaagcc 6060 acggcagtca gattcttttt ctgcctttct ttctttcttt tctttttttt tttttttttt 6120 ttttgtcttt ttgccttttc taggtgcggc atatggaggt tcccaggcta ggtgtcgaat 6180 cagagctgta gacgccggcc taaaccacag ccacagcaac acaggatcca agccttgtct 6240 gtgacctaca ccacagctca cggcaacgct ggatccttaa cccgctgagc gaggccaggg 6300 attgaacccg caacctcatg gttcttagtt ggattcgtta accactgagc catgatggga 6360 actcctgcag tcagattctt aacccactat gccacagcag gaactcctag aagtgccctt 6420 tgaggctact ctgtagacag ctttgagcca gcgaggcaag acctgttttt ctggaggaag 6480 ataaatcctg ggtgagggat gggtgggctg tggtcttcct gggacccatc tctggagcct 6540 ctctccctca gcaaagccac cttggacaat aagagctgcc atctattttt tttttcttta 6600 aactaagatt tgatattttc cagagacctc cctcccaccg tttgatctga gtaattctga 6660 aatgacgaga gtcccgtgat atcatttttt cgatctcgaa ggtggaaacc tgggagtagc 6720 cacaacccag gctctcagct cagcctaggg tttcaatgat aatgattgca aaatagcttt 6780 tctctgcatt ccaagtaaca tgatatgttt ttatttccat ttgcttttag cccagaaggt 6840 tctttgttct ggctcgag 6858 <210> 13 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Forward for cloning neo <400> 13 ctaggaattc cttcgcgatg tacgggcc 28 <210> 14 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Reverse for cloning neo <400> 14 cagtgatatc cttatcgcta tcgattcaca c 31 <210> 15 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Forward for validation of homologous recombination of GT left arm <400> 15 ggtctgccta catctctctg atgaac 26 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse for validation of homologous recombination of GT left arm <400> 16 ggcatcagag cagccgattg 20 <210> 17 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Forward for validation of homologous recombination of GT right arm <400> 17 ccttctatcg ccttcttgac gag 23 <210> 18 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Reverse for validation of homologous recombination of GT right arm <400> 18 ctagagatat ttggaattca aagcacttac 30 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward for cloning IRES <400> 19 gatatccgcg tcgagcatgc 20 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Reverse for cloning IRES <400> 20 ctcgagtcgc cattcaggc 19 <210> 21 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Forward for cloning CD59 <400> 21 ctgagtcgac atgggaatcc aaggagggtc tg 32 <210> 22 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Reverse for cloning CD59 <400> 22 ctgagcggcc gcttagggat gaaggctcca gg 32 <210> 23 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Forward for CMAH partial intron 3 and partial exon 4 <400> 23 gagctccatg acaggaactc ctgagatgaa tatc 34 <210> 24 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Reverse for CMAH partial intron 3 and partial exon 4 <400> 24 gcggccgctc gttgcctctc tccaggtatt aag 33 <210> 25 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Forward for CMAH partial exon 6 and partial intron 6 <400> 25 gatatcacca tcaatactga tcaatgtttt ctg 33 <210> 26 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Reverse for CMAH partial exon 6 and partial intron 6 <400> 26 gtcgacagca ggaaaaactg agtctgcagt tc 32 <210> 27 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Forward for cloning CD39 <400> 27 tgcaatttcg cctcttggc 19 <210> 28 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Reverse for cloning CD39 <400> 28 gaggatccct ataccatatc tttccagaaa tatgaagg 38 <210> 29 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Forward for cloning CD39 + polyA <400> 29 gactcgagca tggaagatac aaaggagtct aacg 34 <210> 30 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Reverse for cloning CD39 + polyA <400> 30 gcattaatta agatttaaca aaaatttaac gcg 33 <210> 31 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Forward for validation of homologous recombination of CMAH <400> 31 accccagctc acaatgagca acaccagat 29 <210> 32 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Reverse for validation of homologous recombination of CMAH <400> 32 agagggaccc caatgacagt aacagcaga 29 <110> Korea Research Institute of Bioscience and Biotechnology <120> Genetically-modified cell line for producing cloned miniature pigs for xenotransplantation and method for preparing the same <130> 8P-11-47 <150> KR2007-123823 <151> 2007-11-30 <160> 32 <170> KopatentIn 1.71 <210> 1 <211> 2972 <212> DNA <213> Sus scrofa <400> 1 gaattcatga ttattatcct ccaagcctgt tcctcctcca gcccatctga gaaaatacta 60 caacccccct gcttaagcag aaatcttggg tcttccttgt ctcatctctg ataacaaaat 120 taccaaccac gtcctatcaa ttctctctcc aaagtatata tatatatatt tttttttaat 180 tttttcccgc tgtacagcat ggggatcaag ttattcttac atgtatattt tccccccacc 240 ctttgttccg ttgcaatatg agtatctaga catagttctc aatgctactc agcaggatct 300 ccttgtaaat ctaagttgta tctgataacc ccaagctccc gatccctccc actccctccc 360 tctcctgtcg ggcagccaca agtctattct ccaagtccat gattttcttt tctgtggaga 420 tggtcatttg tgctggatat tagattccag ttataagtga tatcatatgg tatttgtcaa 480 agtatatatt ttatttttct ttgtcttttt gtcttttgtc tttttttttt ttgttgttgt 540 tgttgttgtt gttgttgttg ctattacttg ggccgctccc gcggcatatg gaggttccca 600 ggctaggagt tgaatcggag ctgtagccac cggcctacgc cagagccaca gcaacgcggg 660 atccgagccg cgtctgcaac ctacaccaca gctcacggca acgctggatc cttaacccac 720 tgagcaaggg cagggaccga acccgcaacc tcatggttcc tagtcggatt cgttaaccac 780 tgcgccacga cgggaactcc caaagtatat tttgaatcaa gccacccttt gagccaggcc 840 acctcctctt tatggtcatg agaacggtct gcccttgtcc ttttctccat tctccacact 900 cagcacccag atgggtctct ctaggtgaag ttggatcagg ggattctcca gctttagatg 960 ctttttggga ttccccaccc tactttccat acctttccag gttctgactg cctctgcccc 1020 ccttctgact gcctagcacc agccactcaa gggggacagt gtcagtcact atttttttct 1080 tgtccaggtt ttttgctttt gtttttttca aacacgagca gctctttctc ttgtctgcct 1140 ggtatagatg ctgtttccaa aatattctca tcccttctca cggcccttgt catcctttcc 1200 catcctatct tcatcccttg ggaagctcta aagtcatctc cccaaattga agggtgacta 1260 aagagtttcc cagaaggaaa aactgagttt ccaactacta cactgacttg caagaaatgt 1320 ttgtgtcttc attaaatgaa aaagaaaaaa ctgtaacaag atatgagaaa atacagaaag 1380 gaaataataa gactagaaaa gtcaaatata tagtgaaggt gttgcatcaa acacttaaat 1440 aaactagtac agatgttaaa agactaaatt atatagttga aggatagctg tgaagatgta 1500 aactatgaca tctaaaacac aaaatgttgg cgttcccgtc acggcacagt ggaaacgaat 1560 ccgactagga accatgaggt tgcaggttca attcctgccc ttgctcagtg ggttaaggat 1620 ccggtgttgc cgtgagctgt ggtgtaggta gccaatgagg cttggatccc gcgttgctgt 1680 ggctctggtg taggccggtg gctacagctc cgattcgacc cctagcctgg gaacctccat 1740 atgccgcggg agcggcccta aaaagacaaa agaccaaaaa aaaaaaaaaa caaaaaaccc 1800 acaaaatgtt gggaatcagt cctctactag tattatgtta ttgtcaagtt ttccttttat 1860 gtctgttaat atttgcgttc tagatgtagg tgctctgata tcgtgtgcat atatgttaac 1920 caatgttatg tcttcctctg gtattgatcc ctttgttatt atgtaatgcc ctactttatc 1980 ttttgttaca ttctttgttt atgagtattg ctgatatgtg gctagctgcc acacttttct 2040 tgtcctttcc atttacaata aatatctttc tatctccacc caaattaaag tactccgcaa 2100 cctgttattc cacccagcat cccttccctc ttcaactaca atttcatgca gcgatcaaga 2160 aatacgaatg taccgactgt ttgccacttg tgtgggtgca ttggggaaaa gctgggtggg 2220 aagtggcaga gcctagatta taaaggacca gggtgagagt tcccattgtg gctcagctga 2280 aatgaatctg actagcatcc atgaggacga aggtttgatc cctggcctca atcagtgggt 2340 taaggatctg gcgttgctgt ccgtgagttg tggtgtagtt cgcagacaag gcgtggactt 2400 agtgtggctg tggctgtggc ataggctagt ggctacagct ctgattcgac ccctagcctg 2460 ggaatctcta tatgctgtga gtgtggccct aaaatttaaa tgaaattaaa taaaggacca 2520 gggtatattt ttctttgagg ataaggtaca tagtcagtat atcagggaca gtagacctag 2580 gaaacggatg cttcctctag tctgtgatgc gaggtggggc atctgagttg ggggcggctg 2640 gagcccttag ggaccattaa ctaaacccgt cactctccca catctcggtg gaccttggga 2700 tcagtcagga tgcttcccct ttgagcctca aaatggcctt agtatccttc ccaacccaga 2760 cggccctgtc agttcattga cttggctaat ttgccagtgt aggcctatgc aaattaaggt 2820 agaacgcact ccttagcgct cgttgactat tcatcaactt ttccttttag aaaagatatt 2880 ggtataagca cttcttaaaa aaccatattc cactctgggt gtatttaatc taattttccc 2940 ttctcctttt cttttcccag gagaaaataa tg 2972 <210> 2 <211> 6846 <212> DNA <213> Sus scrofa <400> 2 ctgtcaatgc tgcttgtctc aactgtaatg gttgtgtttt gggaatacat caacaggtag 60 ttatgaaaca tgatgaaatg atgttgatga aagtctcctc taatctccta gttatcagcc 120 aagtcaccag cttgcattaa aagtaggatt cactgacacc gtaaagaaag cattccagag 180 agttgccgtt gtggctcagg ggcagcaaac ccaattagga tccaagagga ggtgggtttg 240 atccctggcc ttgctctttg gcttaaggat ccggcattgc cgtgacctgt ggtgtaggtt 300 gcagatgcag ctcggatctg gcattgctgt ggctgtggcg taggctggtg gcttcagctc 360 cagtttgacc cctagcctgg gaacttccat atcccacact tgcggcccta aaaagcaaag 420 aaagaaagaa aatattctac ccttcctgta tccctgagcc cttaaatacc gtctttaaag 480 tcattagatc ttcaagtacc ttccagctaa ttaattatct tccttcctgc catgttgcca 540 ttgtcctgat ttttatacct ctgcagttct gggtagacta gagccagaaa taataaggtc 600 atgttaagac caagatataa tattaaatta tttatatgac cagatatgga agttaccttg 660 agaactttca gacaggaatt ccatgagaaa tacaccctga tttttgcaat cctaaaatat 720 ttgcagagtt taaaggaaca actcaagttg ttgacttttg ctgcaaaaca cactgagtcg 780 ctggtgattc atttgtgcct ggctaaactt ttgggtgttt tgtctctttt tttttaactc 840 tggaaagcaa aatgaattaa acatttctga gttttcaaat tcatcagtgg attcacccca 900 aatatttgag ctgcttcttt gcttttggaa actacgatgc cttggagatt ccagctggag 960 acgcttctga cagaaagaaa tgtctgcaag cagctacaaa aatgcatgat ggctttgact 1020 taagaggcat tgataccgct tggcctttct ttcaaaaagg ccaccttaca acttggcctg 1080 aaggcattcc cgtggtggtg cagcggaaaa tgaatctgac taggaacccc gaggttgtgg 1140 gttcaatccc tggccttgct cagtggctta aggatcgggt gttgaagtaa gctgtggtgt 1200 agattgcaga cgcagcttgg atctggtgtt gctgtggctt tggtgtaggc cggcagctac 1260 agctccactt ggacccctag tctgggaacc tccatatgcc acaggtgtgg ccctaaaagg 1320 aaaaaagaca acaaacaaac aaaaaaccaa aaaacaactt ggcctggaga gctatgtcat 1380 caccattgat attttgatgg gtagtgtttt agtagcccct caagttcagg atgatggcct 1440 ggattaacgt tagaatgtct cttaaattct aagacttgat gagccagcag gaccattttg 1500 gccacttaga aaggaactgc atcttcaggt ccatcagtag aaggaggatt ctctagggag 1560 ttctctctta gctcagcggg ttcaagaatt cagtcttgtc cctacagcag ctcaggtgac 1620 tgctatggct tggctttgat ccctggccca ggaatttctg catgctgcag gtgcagccaa 1680 aaaaaaaaaa aaaaaggagg aggtggattc cctagaataa gaagctgtca ttcctttgga 1740 tgcttcatag atctaaccac ttctggaaca gttattccct ctcattctga agaactcatt 1800 ttaagaaaaa caagacgagc tagagagtga acaaatgtct acaaaccaac cttttcgaat 1860 tgaggaaact gtggtacttc ctctgaagaa aagatgacag cgttggatgc agagaccctg 1920 gggctccctt aggtacttga ggactgagga gatattctca gtggaggctg gagctaggct 1980 gcctggggtt ggtcctgtgc caccacttcc ctcctctgtg actttgggca agtttcccta 2040 tctttaaaaa tggggatgat agtagtacct gcttcatagg gttgttggat aaaataagtt 2100 gtgaataaag cactaagggc aacgtactta gtaagcgctg gctgccatca ccaccaccac 2160 tatcaccatc tgtccggagg gcagcatagg acaggagatt ttggcaaata gaaggaagag 2220 ttctaggagt tcccgttgtg gtgcagggga aatgaatcca actaggaact aggaggtttc 2280 gggttcaatc ccgcgcctcg ctcagtgggt taaggatcca gtgttgccat gagctgtggt 2340 gtagattgca gacatggcta ggatctggag ttgctatggc tgtggtgtaa gctggcagct 2400 gtagctcgga ttctacccct agcctgggaa tttccgtatg ccacaggttt ggccctacaa 2460 agaaaaaaga aaaagaaaaa gaaaaaattc taggggctga aagaatctaa cagaagagca 2520 agttccccat ggggttcctg acctgagttg agatgcttgt gtaggcaacc ttcaagctct 2580 gaactcttga ttgttttgaa ttgcagccag agttatactt ccatattttg ggtacttcac 2640 aaaattaaaa cacagaagcc aaaggcccag aagtgcatat tggtgctggc ctcccataaa 2700 gagggttgtt ttgcagtgct gggcacactc tctcttcaca gtaactggag cagattctgg 2760 ctgctcttca gggccgtagt ctggcaccca gactgcagcc acatcattct tcaatgtgag 2820 gaatctattt gaacatctgc aaggggttta aaaggcagga gattctttgc caccttgtga 2880 attggtctga ggtgagctga gggcactaac cttagacagg tgggtagcac tgtagctaaa 2940 gaggattaca ggagttcctg ttgtggctta gtggtaacaa atccaactag tatccatgag 3000 gattcaggtt cgatccctgg cctcgctcag tgggtcaggt atccggtgtt gctgtggctg 3060 tggtgtaggc tggcagcttc agctctgatt tgacccctag cctgggaact tccatgtgct 3120 gtaggtaagg cccttgaaaa aaaaaaaaaa gagatttaca aaataactcc atcaaacaca 3180 tacagctgtt taagaatgtc atccaggaca gcatttggtt aaaggctaga tgaaaaaaaa 3240 aaaaaaaatc ttagaatttt atttatttat tttttctttt tagggccaga cctgtggcct 3300 atggaaatgc ctgggctagg ggtggaatca gagctgccta caccacagcc atagccacgc 3360 cagatccaag ccccgtctgt gacctacacc acagctcatg gcaaacactg gatccttaat 3420 ccactgagtg aggccaggaa ttgaacccac attctcatgg atgctagttg ggttcttaag 3480 ccactgagcc acaagcttag aattttagag gtggaagaaa ctttaagagc tataataaag 3540 taatgatggt gatggtgatt ttgatgttag cggctactag ttattgagtg tttgcttgtg 3600 ccaggaactc cactgttcat tccctcctgt ttttaaaaca gccctggaag gtcagtgtta 3660 gtccacattt ctagatgagg aatactgagt ttccacaata ttaaatgtga acgttcaagg 3720 tcacattttt aggaagattt aggtccaggg ctgtctgact tgggtaacct gggtaaccct 3780 tcctttagtc aaggtttcca ttgttcaggc gatggacaag taggtgaaat gccttaacag 3840 tgaacttatg tctaacttct aattagaact cagatcttct gattcatcat ctggggctcc 3900 ttctggagct ggttgttcat gccaaatgct gcgaggggta cagtgtgcgc caaggagaat 3960 ccctaccctc aaggggttat gctgtagatg gagcaggcag aggtacccat gaaagcccaa 4020 caacacaggc tagaaggagg atgtcagaga gagagagcaa aggaacgtga gagttcaggg 4080 agggcaagat tatgtttggc ttggagatgg atctatgttt tgcatttatt tttttggggg 4140 gggggtcttt ttgctacttc ttgggctgct cccgaggcat atggaggttc ccaggctagg 4200 ggtctaattg gagccgcagc caccagccta tgccagagcc acagcaacgc aggatctgag 4260 ccgcgtctgc aaccttcacc acagctcacg gcaacgccag atcgttaacc cactgagcaa 4320 gggcagggac cgaacctgca acctcatggt tcctagtcag attcgttaag cactgcgcca 4380 cgacgggaac tccctcattt agaaatattt attgagcacc tactgtatgc caggcattgt 4440 gctaggttca taccaaagaa ggctcagaag agatggcatc cgagctgtgc cttgaaggat 4500 gaatatgtgt taaatgccgt acacttcagg gtggttgttg ctgtgacctg aggtgttgaa 4560 ggcttctggg aaaggagggt gagatgagga agagggaggg gttactaaaa agatgggacg 4620 aggtggcaaa tccaaatcta taaattgatg ccctgagtgc ctcggcagga gggtggggct 4680 cctgagtgct gggtggcacg ggccctcccc ctcctcttgc ccctttccct tccccctctt 4740 gtaggatctg aagtcagatt ccccaggttc aaatactgtt tcttccctta gcagtatgac 4800 cttgggcaaa ataatttatt gcctctgtcc ctctgaggag gaatagaacc tccttcattg 4860 actgttatta gaatttaatg agctaataca tgtcagttgc ttagaaaggt ccccagccaa 4920 ctattagcta ttatgaatat tatcagatca atagacagat ttagaaacaa gggactttag 4980 agctgggtcc atgggtactg agcttagagg ggaaaccata ggtggtagga aggcatgtat 5040 ttcattccta ccaggagatg tggactccca gctggggcag aaggcagagg gaggagatcg 5100 gggctttggc agaatctcaa acaaatatta gtggttagtg gttttttgtt tctgttttaa 5160 gagatgaggg caggcgtttc cgatgtggcg cagtggaaac gaatctgact agtatccatg 5220 aggatgcagg ttccatccct ggactcactc agtgagttaa ggatccggca ttgccgtgag 5280 ttgtggtgta ggtcacagac acagctcaga tctggcattg ctgtggctgt ggtgtaggct 5340 agcagctgta gctccaattc aacccctagc ctgggaactt ccacatgccg caggtgcaac 5400 cccaaaagat aaatgaataa ataaataaat atgcgacctt cctttcttgg ggcctttgca 5460 tgtttttctc tctgttaggc acactcttgc taatccctct tcactgggcc tcctatgtat 5520 ccttcagaac tcagctaaaa catcatcccc tcccctgggg agccttcgag gtcttcctgt 5580 taagtgctcc tatgctttct tggagttttg aagtcctata atgatgtgtt tatcaaaata 5640 gggtccaccc tccctgccag cttctctaca ccacagacac atggtgtctg tttcagtcaa 5700 cactgtatgt ctggcacttg acatgtaacg catgctcagc aggtatttgt tgaatgaatg 5760 gaggcggtct gctagagtcg tcatatattt actgatcccg tcttgtagga tggtctcact 5820 gcttttgtta gcttaagaag tacctttttt tttttttttt aatggccaca cccatggcat 5880 atagaaattc cacgaaggaa ggaagaaaga aagaaagaaa gaaggaaatt cctgggtcag 5940 ggattgaatc caagccacag gtgcaacctg agctgcagtt gcggcaacac cacatctttt 6000 aacccactgt gctgggccag ggatcatacc tgtgcatcta cagcgaccca agccacggca 6060 gtcagattct ttttctgcct ttctttcttt cttttctttt tttttttttt ttttttttgt 6120 ctttttgcct tttctaggtg cggcatatgg aggttcccag gctaggtgtc gaatcagagc 6180 tgtagacgcc ggcctaaacc acagccacag caacacagga tccaagcctt gtctgtgacc 6240 tacaccacag ctcacggcaa cgctggatcc ttaacccgct gagcgaggcc agggattgaa 6300 cccgcaacct catggttctt agttggattc gttaaccact gagccatgat gggaactcct 6360 gcagtcagat tcttaaccca ctatgccaca gcaggaactc ctagaagtgc cctttgaggc 6420 tactctgtag acagctttga gccagcgagg caagacctgt ttttctggag gaagataaat 6480 cctgggtgag ggatgggtgg gctgtggtct tcctgggacc catctctgga gcctctctcc 6540 ctcagcaaag ccaccttgga caataagagc tgccatctat tttttttttc tttaaactaa 6600 gatttgatat tttccagaga cctccctccc accgtttgat ctgagtaatt ctgaaatgac 6660 gagagtcccg tgatatcatt ttttcgatct cgaaggtgga aacctgggag tagccacaac 6720 ccaggctctc agctcagcct agggtttcaa tgataatgat tgcaaaatag cttttctctg 6780 cattccaagt aacatgatat gtttttattt ccatttgctt ttagcccaga aggttctttg 6840 ttctgg 6846 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward for GT partial intron 2 and exon 4 <400> 3 tccatgaaca acttcgattg catg 24 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> Reverse for GT partial intron 2 and exon 4 <400> 4 tggctgataa ctaggagatt agaggag 27 <210> 5 <211> 3242 <212> DNA <213> Sus scrofa <400> 5 tccatgaaca acttcgattg catggtctgc ctacatctct ctgatgaact ttagacttga 60 atgtccactt gtctccctgt ccccttttag gtattcgcac actccccgac attcacacgt 120 ccaaaaggga attcatgatt attatcctcc aagcctgttc ctcctccagc ccatctgaga 180 aaatactaca acccccctgc ttaagcagaa atcttgggtc ttccttgtct catctctgat 240 aacaaaatta ccaaccacgt cctatcaatt ctctctccaa agtatatata tatatatttt 300 tttttaattt tttcccgctg tacagcatgg ggatcaagtt attcttacat gtatattttc 360 cccccaccct ttgttccgtt gcaatatgag tatctagaca tagttctcaa tgctactcag 420 caggatctcc ttgtaaatct aagttgtatc tgataacccc aagctcccga tccctcccac 480 tccctccctc tcctgtcggg cagccacaag tctattctcc aagtccatga ttttcttttc 540 tgtggagatg gtcatttgtg ctggatatta gattccagtt ataagtgata tcatatggta 600 tttgtcaaag tatatatttt atttttcttt gtctttttgt cttttgtctt tttttttttt 660 gttgttgttg ttgttgttgt tgttgttgct attacttggg ccgctcccgc ggcatatgga 720 ggttcccagg ctaggagttg aatcggagct gtagccaccg gcctacgcca gagccacagc 780 aacgcgggat ccgagccgcg tctgcaacct acaccacagc tcacggcaac gctggatcct 840 taacccactg agcaagggca gggaccgaac ccgcaacctc atggttccta gtcggattcg 900 ttaaccactg cgccacgacg ggaactccca aagtatattt tgaatcaagc caccctttga 960 gccaggccac ctcctcttta tggtcatgag aacggtctgc ccttgtcctt ttctccattc 1020 tccacactca gcacccagat gggtctctct aggtgaagtt ggatcagggg attctccagc 1080 tttagatgct ttttgggatt ccccacccta ctttccatac ctttccaggt tctgactgcc 1140 tctgcccccc ttctgactgc ctagcaccag ccactcaagg gggacagtgt cagtcactat 1200 ttttttcttg tccaggtttt ttgcttttgt ttttttcaaa cacgagcagc tctttctctt 1260 gtctgcctgg tatagatgct gtttccaaaa tattctcatc ccttctcacg gcccttgtca 1320 tcctttccca tcctatcttc atcccttggg aagctctaaa gtcatctccc caaattgaag 1380 ggtgactaaa gagtttccca gaaggaaaaa ctgagtttcc aactactaca ctgacttgca 1440 agaaatgttt gtgtcttcat taaatgaaaa agaaaaaact gtaacaagat atgagaaaat 1500 acagaaagga aataataaga ctagaaaagt caaatatata gtgaaggtgt tgcatcaaac 1560 acttaaataa actagtacag atgttaaaag actaaattat atagttgaag gatagctgtg 1620 aagatgtaaa ctatgacatc taaaacacaa aatgttggcg ttcccgtcac ggcacagtgg 1680 aaacgaatcc gactaggaac catgaggttg caggttcaat tcctgccctt gctcagtggg 1740 ttaaggatcc ggtgttgccg tgagctgtgg tgtaggtagc caatgaggct tggatcccgc 1800 gttgctgtgg ctctggtgta ggccggtggc tacagctccg attcgacccc tagcctggga 1860 acctccatat gccgcgggag cggccctaaa aagacaaaag accaaaaaaa aaaaaaaaca 1920 aaaaacccac aaaatgttgg gaatcagtcc tctactagta ttatgttatt gtcaagtttt 1980 ccttttatgt ctgttaatat ttgcgttcta gatgtaggtg ctctgatatc gtgtgcatat 2040 atgttaacca atgttatgtc ttcctctggt attgatccct ttgttattat gtaatgccct 2100 actttatctt ttgttacatt ctttgtttat gagtattgct gatatgtggc tagctgccac 2160 acttttcttg tcctttccat ttacaataaa tatctttcta tctccaccca aattaaagta 2220 ctccgcaacc tgttattcca cccagcatcc cttccctctt caactacaat ttcatgcagc 2280 gatcaagaaa tacgaatgta ccgactgttt gccacttgtg tgggtgcatt ggggaaaagc 2340 tgggtgggaa gtggcagagc ctagattata aaggaccagg gtgagagttc ccattgtggc 2400 tcagctgaaa tgaatctgac tagcatccat gaggacgaag gtttgatccc tggcctcaat 2460 cagtgggtta aggatctggc gttgctgtcc gtgagttgtg gtgtagttcg cagacaaggc 2520 gtggacttag tgtggctgtg gctgtggcat aggctagtgg ctacagctct gattcgaccc 2580 ctagcctggg aatctctata tgctgtgagt gtggccctaa aatttaaatg aaattaaata 2640 aaggaccagg gtatattttt ctttgaggat aaggtacata gtcagtatat cagggacagt 2700 agacctagga aacggatgct tcctctagtc tgtgatgcga ggtggggcat ctgagttggg 2760 ggcggctgga gcccttaggg accattaact aaacccgtca ctctcccaca tctcggtgga 2820 ccttgggatc agtcaggatg cttccccttt gagcctcaaa atggccttag tatccttccc 2880 aacccagacg gccctgtcag ttcattgact tggctaattt gccagtgtag gcctatgcaa 2940 attaaggtag aacgcactcc ttagcgctcg ttgactattc atcaactttt ccttttagaa 3000 aagatattgg tataagcact tcttaaaaaa ccatattcca ctctgggtgt atttaatcta 3060 attttccctt ctccttttct tttcccagga gaaaataatg aatgtcaaag gaagagtggt 3120 tctgtcaatg ctgcttgtct caactgtaat ggttgtgttt tgggaataca tcaacaggta 3180 attatgaaac atgatgaaat gatgttgatg aaagtctcct ctaatctcct agttatcagc 3240 ca 3242 <210> 6 <211> 3088 <212> DNA <213> Sus scrofa <400> 6 tccatgaaca acttcgattg catggtctgc ctacatctct ctgatgaact ttagacttga 60 atgtccactt gtctccctgt ccccttttag gtattcgcac actccccgac attcacacgt 120 ccaaaaggga attcatgatt attatcctcc aagcctgttc ctcctccagc ccatctgaga 180 aaatactaca acccccctgc ttaagcagaa atcttgggtc ttccttgtct catctctgat 240 aacaaaatta ccaaccacgt cctatcaatt ctctctccaa agtatatata tatatatttt 300 tttttaattt tttcccgctg tacagcatgg ggatcaagtt attcttacat gtatattttc 360 cccccaccct ttgttccgtt gcaatatgag tatctagaca tagttctcaa tgctactcag 420 caggatctcc ttgtaaatct aagttgtatc tgataacccc aagctcccga tccctcccac 480 tccctccctc tcctgtcggg cagccacaag tctattctcc aagtccatga ttttcttttc 540 tgtggagatg gtcatttgtg ctggatatta gattccagtt ataagtgata tcatatggta 600 tttgtcaaag tatatatttt atttttcttt gtctttttgt cttttgtctt tttttttttt 660 gttgttgttg ttgttgttgt tgttgttgct attacttggg ccgctcccgc ggcatatgga 720 ggttcccagg ctaggagttg aatcggagct gtagccaccg gcctacgcca gagccacagc 780 aacgcgggat ccgagccgcg tctgcaacct acaccacagc tcacggcaac gctggatcct 840 taacccactg agcaagggca gggaccgaac ccgcaacctc atggttccta gtcggattcg 900 ttaaccactg cgccacgacg ggaactccca aagtatattt tgaatcaagc caccctttga 960 gccaggccac ctcctcttta tggtcatgag aacggtctgc ccttgtcctt ttctccattc 1020 tccacactca gcacccagat gggtctctct aggtgaagtt ggatcagggg attctccagc 1080 tttagatgct ttttgggatt ccccacccta ctttccatac ctttccaggt tctgactgcc 1140 tctgcccccc ttctgactgc ctagcaccag ccactcaagg gggacagtgt cagtcactat 1200 ttttttcttg tccaggtttt ttgcttttgt ttttttcaaa cacgagcagc tctttctctt 1260 gtctgcctgg tatagatgct gtttccaaaa tattctcatc ccttctcacg gcccttgtca 1320 tcctttccca tcctatcttc atcccttggg aagctctaaa gtcatctccc caaattgaag 1380 ggtgactaaa gagtttccca gaaggaaaaa ctgagtttcc aactactaca ctgacttgca 1440 agaaatgttt gtgtcttcat taaatgaaaa agaaaaaact gtaacaagat atgagaaaat 1500 acagaaagga aataataaga ctagaaaagt caaatatata gtgaaggtgt tgcatcaaac 1560 acttaaataa actagtacag atgttaaaag actaaattat atagttgaag gatagctgtg 1620 aagatgtaaa ctatgacatc taaaacacaa aatgttggcg ttcccgtcac ggcacagtgg 1680 aaacgaatcc gactaggaac catgaggttg caggttcaat tcctgccctt gctcagtggg 1740 ttaaggatcc ggtgttgccg tgagctgtgg tgtaggtagc caatgaggct tggatcccgc 1800 gttgctgtgg ctctggtgta ggccggtggc tacagctccg attcgacccc tagcctggga 1860 acctccatat gccgcgggag cggccctaaa aagacaaaag accaaaaaaa aaaaaaaaca 1920 aaaaacccac aaaatgttgg gaatcagtcc tctactagta ttatgttatt gtcaagtttt 1980 ccttttatgt ctgttaatat ttgcgttcta gatgtaggtg ctctgatatc gtgtgcatat 2040 atgttaacca atgttatgtc ttcctctggt attgatccct ttgttattat gtaatgccct 2100 actttatctt ttgttacatt ctttgtttat gagtattgct gatatgtggc tagctgccac 2160 acttttcttg tcctttccat ttacaataaa tatctttcta tctccaccca aattaaagta 2220 ctccgcaacc tgttattcca cccagcatcc cttccctctt caactacaat ttcatgcagc 2280 gatcaagaaa tacgaatgta ccgactgttt gccacttgtg tgggtgcatt ggggaaaagc 2340 tgggtgggaa gtggcagagc ctagattata aaggaccagg gtgagagttc ccattgtggc 2400 tcagctgaaa tgaatctgac tagcatccat gaggacgaag gtttgatccc tggcctcaat 2460 cagtgggtta aggatctggc gttgctgtcc gtgagttgtg gtgtagttcg cagacaaggc 2520 gtggacttag tgtggctgtg gctgtggcat aggctagtgg ctacagctct gattcgaccc 2580 ctagcctggg aatctctata tgctgtgagt gtggccctaa aatttaaatg aaattaaata 2640 aaggaccagg gtatattttt ctttgaggat aaggtacata gtcagtatat cagggacagt 2700 agacctagga aacggatgct tcctctagtc tgtgatgcga ggtggggcat ctgagttggg 2760 ggcggctgga gcccttaggg accattaact aaacccgtca ctctcccaca tctcggtgga 2820 ccttgggatc agtcaggatg cttccccttt gagcctcaaa atggccttag tatccttccc 2880 aacccagacg gccctgtcag ttcattgact tggctaattt gccagtgtag gcctatgcaa 2940 attaaggtag aacgcactcc ttagcgctcg ttgactattc atcaactttt ccttttagaa 3000 aagatattgg tataagcact tcttaaaaaa ccatattcca ctctgggtgt atttaatcta 3060 attttccctt ctccttttct tttcccag 3088 <210> 7 <211> 89 <212> DNA <213> Sus scrofa <400> 7 gagaaaataa tgaatgtcaa aggaagagtg gttctgtcaa tgctgcttgt ctcaactgta 60 atggttgtgt tttgggaata catcaacag 89 <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Forward for left arm <400> 8 gaattcatga ttattatcct cccaagc 27 <210> 9 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Reverse for left arm <400> 9 catgaattcc attattttct cctggg 26 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward for GT intron 4 and partial exon 5 <400> 10 gtcgacctgt caatgctgct tg 22 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> Reverse for GT intron 4 and partial exon 5 <400> 11 ctcgagccag aacaaagaac cttc 24 <210> 12 <211> 6858 <212> DNA <213> Sus scrofa <400> 12 gtcgacctgt caatgctgct tgtctcaact gtaatggttg tgttttggga atacatcaac 60 aggtagttat gaaacatgat gaaatgatgt tgatgaaagt ctcctctaat ctcctagtta 120 tcagccaagt caccagcttg cattaaaagt aggattcact gacaccgtaa agaaagcatt 180 ccagagagtt gccgttgtgg ctcaggggca gcaaacccaa ttaggatcca agaggaggtg 240 ggtttgatcc ctggccttgc tctttggctt aaggatccgg cattgccgtg acctgtggtg 300 taggttgcag atgcagctcg gatctggcat tgctgtggct gtggcgtagg ctggtggctt 360 cagctccagt ttgaccccta gcctgggaac ttccatatcc cacacttgcg gccctaaaaa 420 gcaaagaaag aaagaaaata ttctaccctt cctgtatccc tgagccctta aataccgtct 480 ttaaagtcat tagatcttca agtaccttcc agctaattaa ttatcttcct tcctgccatg 540 ttgccattgt cctgattttt atacctctgc agttctgggt agactagagc cagaaataat 600 aaggtcatgt taagaccaag atataatatt aaattattta tatgaccaga tatggaagtt 660 accttgagaa ctttcagaca ggaattccat gagaaataca ccctgatttt tgcaatccta 720 aaatatttgc agagtttaaa ggaacaactc aagttgttga cttttgctgc aaaacacact 780 gagtcgctgg tgattcattt gtgcctggct aaacttttgg gtgttttgtc tctttttttt 840 taactctgga aagcaaaatg aattaaacat ttctgagttt tcaaattcat cagtggattc 900 accccaaata tttgagctgc ttctttgctt ttggaaacta cgatgccttg gagattccag 960 ctggagacgc ttctgacaga aagaaatgtc tgcaagcagc tacaaaaatg catgatggct 1020 ttgacttaag aggcattgat accgcttggc ctttctttca aaaaggccac cttacaactt 1080 ggcctgaagg cattcccgtg gtggtgcagc ggaaaatgaa tctgactagg aaccccgagg 1140 ttgtgggttc aatccctggc cttgctcagt ggcttaagga tcgggtgttg aagtaagctg 1200 tggtgtagat tgcagacgca gcttggatct ggtgttgctg tggctttggt gtaggccggc 1260 agctacagct ccacttggac ccctagtctg ggaacctcca tatgccacag gtgtggccct 1320 aaaaggaaaa aagacaacaa acaaacaaaa aaccaaaaaa caacttggcc tggagagcta 1380 tgtcatcacc attgatattt tgatgggtag tgttttagta gcccctcaag ttcaggatga 1440 tggcctggat taacgttaga atgtctctta aattctaaga cttgatgagc cagcaggacc 1500 attttggcca cttagaaagg aactgcatct tcaggtccat cagtagaagg aggattctct 1560 agggagttct ctcttagctc agcgggttca agaattcagt cttgtcccta cagcagctca 1620 ggtgactgct atggcttggc tttgatccct ggcccaggaa tttctgcatg ctgcaggtgc 1680 agccaaaaaa aaaaaaaaaa aggaggaggt ggattcccta gaataagaag ctgtcattcc 1740 tttggatgct tcatagatct aaccacttct ggaacagtta ttccctctca ttctgaagaa 1800 ctcattttaa gaaaaacaag acgagctaga gagtgaacaa atgtctacaa accaaccttt 1860 tcgaattgag gaaactgtgg tacttcctct gaagaaaaga tgacagcgtt ggatgcagag 1920 accctggggc tcccttaggt acttgaggac tgaggagata ttctcagtgg aggctggagc 1980 taggctgcct ggggttggtc ctgtgccacc acttccctcc tctgtgactt tgggcaagtt 2040 tccctatctt taaaaatggg gatgatagta gtacctgctt catagggttg ttggataaaa 2100 taagttgtga ataaagcact aagggcaacg tacttagtaa gcgctggctg ccatcaccac 2160 caccactatc accatctgtc cggagggcag cataggacag gagattttgg caaatagaag 2220 gaagagttct aggagttccc gttgtggtgc aggggaaatg aatccaacta ggaactagga 2280 ggtttcgggt tcaatcccgc gcctcgctca gtgggttaag gatccagtgt tgccatgagc 2340 tgtggtgtag attgcagaca tggctaggat ctggagttgc tatggctgtg gtgtaagctg 2400 gcagctgtag ctcggattct acccctagcc tgggaatttc cgtatgccac aggtttggcc 2460 ctacaaagaa aaaagaaaaa gaaaaagaaa aaattctagg ggctgaaaga atctaacaga 2520 agagcaagtt ccccatgggg ttcctgacct gagttgagat gcttgtgtag gcaaccttca 2580 agctctgaac tcttgattgt tttgaattgc agccagagtt atacttccat attttgggta 2640 cttcacaaaa ttaaaacaca gaagccaaag gcccagaagt gcatattggt gctggcctcc 2700 cataaagagg gttgttttgc agtgctgggc acactctctc ttcacagtaa ctggagcaga 2760 ttctggctgc tcttcagggc cgtagtctgg cacccagact gcagccacat cattcttcaa 2820 tgtgaggaat ctatttgaac atctgcaagg ggtttaaaag gcaggagatt ctttgccacc 2880 ttgtgaattg gtctgaggtg agctgagggc actaacctta gacaggtggg tagcactgta 2940 gctaaagagg attacaggag ttcctgttgt ggcttagtgg taacaaatcc aactagtatc 3000 catgaggatt caggttcgat ccctggcctc gctcagtggg tcaggtatcc ggtgttgctg 3060 tggctgtggt gtaggctggc agcttcagct ctgatttgac ccctagcctg ggaacttcca 3120 tgtgctgtag gtaaggccct tgaaaaaaaa aaaaaagaga tttacaaaat aactccatca 3180 aacacataca gctgtttaag aatgtcatcc aggacagcat ttggttaaag gctagatgaa 3240 aaaaaaaaaa aaaatcttag aattttattt atttattttt tctttttagg gccagacctg 3300 tggcctatgg aaatgcctgg gctaggggtg gaatcagagc tgcctacacc acagccatag 3360 ccacgccaga tccaagcccc gtctgtgacc tacaccacag ctcatggcaa acactggatc 3420 cttaatccac tgagtgaggc caggaattga acccacattc tcatggatgc tagttgggtt 3480 cttaagccac tgagccacaa gcttagaatt ttagaggtgg aagaaacttt aagagctata 3540 ataaagtaat gatggtgatg gtgattttga tgttagcggc tactagttat tgagtgtttg 3600 cttgtgccag gaactccact gttcattccc tcctgttttt aaaacagccc tggaaggtca 3660 gtgttagtcc acatttctag atgaggaata ctgagtttcc acaatattaa atgtgaacgt 3720 tcaaggtcac atttttagga agatttaggt ccagggctgt ctgacttggg taacctgggt 3780 aacccttcct ttagtcaagg tttccattgt tcaggcgatg gacaagtagg tgaaatgcct 3840 taacagtgaa cttatgtcta acttctaatt agaactcaga tcttctgatt catcatctgg 3900 ggctccttct ggagctggtt gttcatgcca aatgctgcga ggggtacagt gtgcgccaag 3960 gagaatccct accctcaagg ggttatgctg tagatggagc aggcagaggt acccatgaaa 4020 gcccaacaac acaggctaga aggaggatgt cagagagaga gagcaaagga acgtgagagt 4080 tcagggaggg caagattatg tttggcttgg agatggatct atgttttgca tttatttttt 4140 tggggggggg gtctttttgc tacttcttgg gctgctcccg aggcatatgg aggttcccag 4200 gctaggggtc taattggagc cgcagccacc agcctatgcc agagccacag caacgcagga 4260 tctgagccgc gtctgcaacc ttcaccacag ctcacggcaa cgccagatcg ttaacccact 4320 gagcaagggc agggaccgaa cctgcaacct catggttcct agtcagattc gttaagcact 4380 gcgccacgac gggaactccc tcatttagaa atatttattg agcacctact gtatgccagg 4440 cattgtgcta ggttcatacc aaagaaggct cagaagagat ggcatccgag ctgtgccttg 4500 aaggatgaat atgtgttaaa tgccgtacac ttcagggtgg ttgttgctgt gacctgaggt 4560 gttgaaggct tctgggaaag gagggtgaga tgaggaagag ggaggggtta ctaaaaagat 4620 gggacgaggt ggcaaatcca aatctataaa ttgatgccct gagtgcctcg gcaggagggt 4680 ggggctcctg agtgctgggt ggcacgggcc ctccccctcc tcttgcccct ttcccttccc 4740 cctcttgtag gatctgaagt cagattcccc aggttcaaat actgtttctt cccttagcag 4800 tatgaccttg ggcaaaataa tttattgcct ctgtccctct gaggaggaat agaacctcct 4860 tcattgactg ttattagaat ttaatgagct aatacatgtc agttgcttag aaaggtcccc 4920 agccaactat tagctattat gaatattatc agatcaatag acagatttag aaacaaggga 4980 ctttagagct gggtccatgg gtactgagct tagaggggaa accataggtg gtaggaaggc 5040 atgtatttca ttcctaccag gagatgtgga ctcccagctg gggcagaagg cagagggagg 5100 agatcggggc tttggcagaa tctcaaacaa atattagtgg ttagtggttt tttgtttctg 5160 ttttaagaga tgagggcagg cgtttccgat gtggcgcagt ggaaacgaat ctgactagta 5220 tccatgagga tgcaggttcc atccctggac tcactcagtg agttaaggat ccggcattgc 5280 cgtgagttgt ggtgtaggtc acagacacag ctcagatctg gcattgctgt ggctgtggtg 5340 taggctagca gctgtagctc caattcaacc cctagcctgg gaacttccac atgccgcagg 5400 tgcaacccca aaagataaat gaataaataa ataaatatgc gaccttcctt tcttggggcc 5460 tttgcatgtt tttctctctg ttaggcacac tcttgctaat ccctcttcac tgggcctcct 5520 atgtatcctt cagaactcag ctaaaacatc atcccctccc ctggggagcc ttcgaggtct 5580 tcctgttaag tgctcctatg ctttcttgga gttttgaagt cctataatga tgtgtttatc 5640 aaaatagggt ccaccctccc tgccagcttc tctacaccac agacacatgg tgtctgtttc 5700 agtcaacact gtatgtctgg cacttgacat gtaacgcatg ctcagcaggt atttgttgaa 5760 tgaatggagg cggtctgcta gagtcgtcat atatttactg atcccgtctt gtaggatggt 5820 ctcactgctt ttgttagctt aagaagtacc tttttttttt ttttttaatg gccacaccca 5880 tggcatatag aaattccacg aaggaaggaa gaaagaaaga aagaaagaag gaaattcctg 5940 ggtcagggat tgaatccaag ccacaggtgc aacctgagct gcagttgcgg caacaccaca 6000 tcttttaacc cactgtgctg ggccagggat catacctgtg catctacagc gacccaagcc 6060 acggcagtca gattcttttt ctgcctttct ttctttcttt tctttttttt tttttttttt 6120 ttttgtcttt ttgccttttc taggtgcggc atatggaggt tcccaggcta ggtgtcgaat 6180 cagagctgta gacgccggcc taaaccacag ccacagcaac acaggatcca agccttgtct 6240 gtgacctaca ccacagctca cggcaacgct ggatccttaa cccgctgagc gaggccaggg 6300 attgaacccg caacctcatg gttcttagtt ggattcgtta accactgagc catgatggga 6360 actcctgcag tcagattctt aacccactat gccacagcag gaactcctag aagtgccctt 6420 tgaggctact ctgtagacag ctttgagcca gcgaggcaag acctgttttt ctggaggaag 6480 ataaatcctg ggtgagggat gggtgggctg tggtcttcct gggacccatc tctggagcct 6540 ctctccctca gcaaagccac cttggacaat aagagctgcc atctattttt tttttcttta 6600 aactaagatt tgatattttc cagagacctc cctcccaccg tttgatctga gtaattctga 6660 aatgacgaga gtcccgtgat atcatttttt cgatctcgaa ggtggaaacc tgggagtagc 6720 cacaacccag gctctcagct cagcctaggg tttcaatgat aatgattgca aaatagcttt 6780 tctctgcatt ccaagtaaca tgatatgttt ttatttccat ttgcttttag cccagaaggt 6840 tctttgttct ggctcgag 6858 <210> 13 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Forward for cloning neo <400> 13 ctaggaattc cttcgcgatg tacgggcc 28 <210> 14 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Reverse for cloning neo <400> 14 cagtgatatc cttatcgcta tcgattcaca c 31 <210> 15 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Forward for validation of homologous recombination of GT left arm <400> 15 ggtctgccta catctctctg atgaac 26 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse for validation of homologous recombination of GT left arm <400> 16 ggcatcagag cagccgattg 20 <210> 17 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Forward for validation of homologous recombination of GT right arm <400> 17 ccttctatcg ccttcttgac gag 23 <210> 18 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Reverse for validation of homologous recombination of GT right arm <400> 18 ctagagatat ttggaattca aagcacttac 30 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward for cloning IRES <400> 19 gatatccgcg tcgagcatgc 20 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Reverse for cloning IRES <400> 20 ctcgagtcgc cattcaggc 19 <210> 21 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Forward for cloning CD59 <400> 21 ctgagtcgac atgggaatcc aaggagggtc tg 32 <210> 22 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Reverse for cloning CD59 <400> 22 ctgagcggcc gcttagggat gaaggctcca gg 32 <210> 23 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Forward for CMAH partial intron 3 and partial exon 4 <400> 23 gagctccatg acaggaactc ctgagatgaa tatc 34 <210> 24 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Reverse for CMAH partial intron 3 and partial exon 4 <400> 24 gcggccgctc gttgcctctc tccaggtatt aag 33 <210> 25 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Forward for CMAH partial exon 6 and partial intron 6 <400> 25 gatatcacca tcaatactga tcaatgtttt ctg 33 <210> 26 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Reverse for CMAH partial exon 6 and partial intron 6 <400> 26 gtcgacagca ggaaaaactg agtctgcagt tc 32 <210> 27 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Forward for cloning CD39 <400> 27 tgcaatttcg cctcttggc 19 <210> 28 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Reverse for cloning CD39 <400> 28 gaggatccct ataccatatc tttccagaaa tatgaagg 38 <210> 29 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Forward for cloning CD39 + polyA <400> 29 gactcgagca tggaagatac aaaggagtct aacg 34 <210> 30 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Reverse for cloning CD39 + polyA <400> 30 gcattaatta agatttaaca aaaatttaac gcg 33 <210> 31 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Forward for validation of homologous recombination of CMAH <400> 31 accccagctc acaatgagca acaccagat 29 <210> 32 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Reverse for validation of homologous recombination of CMAH <400> 32 agagggaccc caatgacagt aacagcaga 29
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Cited By (6)
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| KR101048426B1 (en) * | 2009-05-27 | 2011-07-11 | 한국생명공학연구원 | Method for producing somatic cells obtained by adding the DAF gene to the position of the alpha 1,3-galactosyltransferase gene |
| WO2012134241A2 (en) * | 2011-03-30 | 2012-10-04 | 주식회사 툴젠 | Zinc finger nuclease targeting the cmah gene, and a use therefor |
| KR101236724B1 (en) * | 2010-11-23 | 2013-02-26 | 대한민국 | Gene targeting knock-in vector containing overexpression cassette, the method for constructing the same and transgenic cloned animal carrying the vector for xenotransplantation |
| WO2014178485A1 (en) | 2013-04-30 | 2014-11-06 | 건국대학교 산학협력단 | Cmp-acetylneuraminic acid hydroxylase targeting vector, vector-transduced transgenic animal for xenotransplantation, and method for producing same |
| KR101479671B1 (en) * | 2011-11-02 | 2015-01-08 | 건국대학교 산학협력단 | CMP-N-acetylneuraminic acid hydroxylase targeting vector and use of the same |
| KR20180121748A (en) * | 2017-04-28 | 2018-11-08 | 서울대학교산학협력단 | Transgenic pigs expressing both shTNFRI-Fc and HA-HO-1 without Gal epitope and N-glycolylneuraminic acid epitope and the Use of thereof |
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| CN102224245B (en) * | 2008-09-30 | 2016-01-13 | 诺维信股份有限公司 | Method that is positive and negative selectability gene is used in filamentous fungal cells |
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| DE69535752D1 (en) | 1994-04-13 | 2008-06-26 | Biotransplant Inc | ALPHA (1,3) GALACTOSYL TRANSFERASE NEGATIVE PIG |
| US6166288A (en) | 1995-09-27 | 2000-12-26 | Nextran Inc. | Method of producing transgenic animals for xenotransplantation expressing both an enzyme masking or reducing the level of the gal epitope and a complement inhibitor |
| CA2445945A1 (en) | 2001-04-30 | 2002-11-07 | Rbc Biotechnology, Inc. | Modified organs and cells for xenotransplantation |
| JP2006129736A (en) | 2004-11-02 | 2006-05-25 | Nippon Dobutsu Kogaku Kenkyusho:Kk | Pig cell for xenotransplantation, method for selecting the same, and pig for the xenotransplantation |
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Cited By (7)
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| KR101048426B1 (en) * | 2009-05-27 | 2011-07-11 | 한국생명공학연구원 | Method for producing somatic cells obtained by adding the DAF gene to the position of the alpha 1,3-galactosyltransferase gene |
| KR101236724B1 (en) * | 2010-11-23 | 2013-02-26 | 대한민국 | Gene targeting knock-in vector containing overexpression cassette, the method for constructing the same and transgenic cloned animal carrying the vector for xenotransplantation |
| WO2012134241A2 (en) * | 2011-03-30 | 2012-10-04 | 주식회사 툴젠 | Zinc finger nuclease targeting the cmah gene, and a use therefor |
| WO2012134241A3 (en) * | 2011-03-30 | 2013-01-10 | 주식회사 툴젠 | Zinc finger nuclease targeting the cmah gene, and a use therefor |
| KR101479671B1 (en) * | 2011-11-02 | 2015-01-08 | 건국대학교 산학협력단 | CMP-N-acetylneuraminic acid hydroxylase targeting vector and use of the same |
| WO2014178485A1 (en) | 2013-04-30 | 2014-11-06 | 건국대학교 산학협력단 | Cmp-acetylneuraminic acid hydroxylase targeting vector, vector-transduced transgenic animal for xenotransplantation, and method for producing same |
| KR20180121748A (en) * | 2017-04-28 | 2018-11-08 | 서울대학교산학협력단 | Transgenic pigs expressing both shTNFRI-Fc and HA-HO-1 without Gal epitope and N-glycolylneuraminic acid epitope and the Use of thereof |
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| WO2009069986A2 (en) | 2009-06-04 |
| WO2009069986A3 (en) | 2009-07-16 |
| KR101149475B1 (en) | 2012-05-25 |
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