KR100807644B1 - Piglet production method by simultaneous transfer of embryos formed by in vitro fertilization and nuclear transfer - Google Patents

Piglet production method by simultaneous transfer of embryos formed by in vitro fertilization and nuclear transfer Download PDF

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KR100807644B1
KR100807644B1 KR1020070026707A KR20070026707A KR100807644B1 KR 100807644 B1 KR100807644 B1 KR 100807644B1 KR 1020070026707 A KR1020070026707 A KR 1020070026707A KR 20070026707 A KR20070026707 A KR 20070026707A KR 100807644 B1 KR100807644 B1 KR 100807644B1
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vitro fertilization
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박창식
김명철
진동일
이영주
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충남대학교산학협력단
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract

A method for producing piglet is provided to be able to increase the normal birth rate of cloned pig by co-transplantation of in vitro fertilization and somatic cell cloned fertilized eggs, significantly shorten the culturing period of the fertilized egg and improve the normal birth rate from a small number of ova. A method for producing piglet comprises the steps of: (a) culturing collected immature ova of slaughtered pig; (b) after preparing (i) a fertilized egg obtained by in vitro fertilization of the mature ova prepared by the step(a) and liquid semen, and (ii) a somatic cell transplanted fertilized egg obtained by transplanting a donor somatic cell into an enucleated prepared mature ova prepared by enucleating the mature ova of the step(a), respectively culturing (i) and (ii) for 16-36 hours; and (c) transplanting 150-250 fertilized eggs prepared by the in vitro fertilization and the somatic cell transplantation into one surrogate mother. Further, a culture ground of the immature ova contains 2-6 follicle films having 4-6 mm of size.

Description

체외수정 수정란 및 체세포 복제 수정란의 동시 이식에 의한 돼지 생산 방법{Piglet production method by simultaneous transfer of embryos formed by in vitro fertilization and nuclear transfer} Piglet production method by simultaneous transfer of embryos formed by in vitro fertilization and nuclear transfer}

도 1은 돼지의 난소 중 미성숙 난자의 체외성숙 시 사용된 난포막을 보여주는 사진.1 is a photograph showing the follicle membrane used during in vitro maturation of immature eggs in pig ovaries.

도 2는 본 발명의 방법에 의해 생성된 체외수정 유래 돼지(A 및 B)와 체세포 복제 유래 돼지(C)의 사진.Figure 2 is a photograph of in vitro fertilized pigs (A and B) and somatic cloning-derived pigs (C) produced by the method of the present invention.

본 발명은 수정란 이식에 의한 돼지의 생산에 관한 것으로, 보다 상세하게는 돼지 미성숙 난자를 체외 성숙한 후 체외수정한 수정란과 체세포의 핵을 이식한 수정란을 동시에 이식하는 것에 의한 돼지의 생산 방법에 관한 것이다.The present invention relates to the production of pigs by fertilized egg transplantation, and more particularly, to a method of producing pigs by simultaneously implanting fertilized eggs fertilized in vitro after fertilization of immature pigs in vitro and fertilized eggs implanted with nuclei of somatic cells. .

오늘날까지 돼지의 번식학적 측면에서의 개량은 주로 우수한 수퇘지를 이용한 인공수정에 의존하여 왔으나, 인공수정만으로는 돼지 개량의 한계점에 와있다. 따라서 우수한 암퇘지를 이용한 돼지 개량에 눈을 돌리게 되었고, 이러한 필요성에 의하여 개발된 기술이 수정란 이식(embryo transfer)이다. 수정란 이식은 돼지의 유전적 개량뿐만 아니라, 성비조절, 일란성 단태생산, 핵이식, 외래유전자의 도입, 배아의 초기발생과 유전자 발현의 기전 구명, 질병의 조절 등의 연구에도 크게 기여할 수 있다는 측면에서 중요시 되고 있다.To date, improvements in reproductive aspects of pigs have been reliant on artificial insemination using good boars, but artificial insemination alone is at the limit of pig improvement. Therefore, the focus was on improving pigs using excellent sows, and the technology developed by this necessity is embryo transfer. Embryo transplantation can contribute not only to genetic improvement in pigs, but also to contribute to studies on sex ratio control, identical production of embryos, nuclear transfer, introduction of exogenous genes, early development of embryos and the mechanism of gene expression, and regulation of diseases. It becomes important.

포유동물의 수정란 이식을 최초로 성공한 사람은 영국의 Heape(1890, Proc. Roy. Soc. 48: 457)로서 그는 순종 앙고라 토끼의 수정란을 벨지안종 암토끼에 이식하여 앙고라종 자토를 생산하는데 성공하였다. 돼지의 수정란 이식은 Kvansnickii(1951, Interbreed ova transplantation. Sovetsk, Zootech. 1: 36)에 의하여 최초로 성공하였으며, 오늘날 대부분의 실험실에서 많이 사용되고 있는 수정란의 회수와 이식방법이 1960년대 Hancock과 Hovell (1962, J. Reprod. Fert. 4: 195), Dziuk 등 (1964, J. Anim. Sci. 23: 37), 그리고 Vincent 등 (1964, J. Anim. Sci. 23: 1084)에 의하여 개발된 이래 주로 실험실 내에서의 연구에 한정되어 왔다. Heape (1890, Proc. Roy. Soc. 48: 457) of England was the first to successfully implant a fertilized egg in a mammal, and he succeeded in producing an Angora bovine by implanting a fertilized angora rabbit into a Belgian female rabbit. The first successful transplantation of fertilized eggs in pigs was carried out by Kvansnickii (1951, Interbreed ova transplantation. Sovetsk, Zootech. 1: 36), and the recovery and transplantation of fertilized eggs, which are widely used in most laboratories today, was carried out by Hancock and Hovell (1962, J. Reprod.Fert. 4: 195), Dziuk et al. (1964, J. Anim. Sci. 23: 37), and Vincent et al. (1964, J. Anim. Sci. 23: 1084). It has been limited to research within.

그러나 최근 생체내 (in vivo)에서 성숙된 난자를 체외수정시켜 정상자돈을 생산한 결과가 Cheng(1985, In vitro fertilization of farm animal oocytes. Ph. D. Thesis. Council for Nation Academic Awards), Yoshida(1987, Jpn. J. Vet. Sci. 49: 711; 1989, Jpn. J. Anim. Reprod. 35: 34), Nagai 등(1988, J. Reprod. Fert. 84: 585)에 의하여, 생체외 (in vitro)에서 성숙시킨 난자를 체외수정시켜 정상자돈을 생산한 결과가 Mattioli 등(1989, Theriogenology 31: 1201)에 의해서 보고됨으로써 돼지 수정란 이식 기술의 실용화 가능성을 나타내었다. 우리나라에서는 이 등(1988, 한국축산학회지 30(7): 403)이 5두의 공란돈으로부터 67개의 수정란을 회수한 후 5두의 수란돈에 이식하여 18두의 자돈을 생산함으로써 돼지 수정란 이식의 역사가 시작되었다.However, recently, in vitro fertilization of mature eggs in vitro to produce normal piglets resulted in Cheng (1985, In vitro fertilization of farm animal oocytes.Ph. D. Thesis.Council for Nation Academic Awards), Yoshida ( 1987, Jpn. J. Vet. Sci. 49: 711; 1989, Jpn. J. Anim. Reprod. 35: 34), by Nagai et al. (1988, J. Reprod. Fert. 84: 585), ex vivo ( The results of in vitro fertilization of in vitro fertilized eggs to produce normal piglets were reported by Mattioli et al. (1989, Theriogenology 31: 1201), suggesting the possibility of the practical application of a pig fertilized egg transplantation technique. In Korea, Lee et al. (1988, Journal of the Korean Society for Animal Science 30 (7): 403) recovered 67 fertilized eggs from 5 blank pigs and transplanted them into 5 pig eggs to produce 18 pigs. History began.

Kikuchi 등(2002, Biology of Reproduction 66:1033)은 체외에서 성숙시킨 돼지 난자를 체외수정 시킨 후 5일과 6일동안 체외배양 시키고 배양 후 발달한 양질의 배반포만을 선별하여 3마리의 대리모에 각각 50개의 배반포를 이식한 결과 총 19마리의 자돈을 생산하였다. 또한 Suzuki 등(2006, Theriogenology 65:374)은 우태아혈청을 첨가한 배지에서 체외성숙시킨 난자를 체외수정시켜 6일간 배양시킨 후 생산된 배반포를 두 마리의 대리모에 이식하여 7마리의 자돈을 생산하였다. 그러나, 기존의 보고들은 많은 수의 배반포를 얻기 위하여 많은 난자를 준비해야 하고, 수정란을 5-6일간 배양한 후 이식하여야 하는 번거로움이 있다.Kikuchi et al. (2002, Biology of Reproduction 66: 1033) in vitro fertilized in vitro fertilized pig eggs and in vitro cultured for 5 and 6 days, and selected only high-quality blastocysts developed after incubation. Implantation of the blastocyst produced a total of 19 piglets. In addition, Suzuki et al. (2006, Theriogenology 65: 374) produced in vitro fertilized eggs in vitro fertilized with fetal bovine serum and incubated them for 6 days before transplanting blastocysts into two surrogate mothers to produce 7 piglets. It was. However, the existing reports have to prepare a large number of eggs in order to obtain a large number of blastocysts, and it is cumbersome to incubate the fertilized egg after 5-6 days.

핵이식 기법(nuclear transfer)은 우수한 유전형질 또는 목적하는 형질전환 개체의 세포핵을 탈핵(enucleation)된 난자에 이식하여 유전적으로 동일한 개체를 생산하는 방법으로 체세포 복제라고도 한다. 실험동물을 이용한 기초연구를 바탕으로 근래에는 산업동물을 중심으로 한 우량가축의 대량생산과 의약학 분야에의 이용을 목표로 활발하게 진행되고 있다. 이러한 노력의 결과로 면양의 복제생산(Wilmut 등, 1997, Nature 385:810)을 처음으로 성공한 이래, 마우스 및 소 등(Wakayama 등, 1998, Nature 394:369; Kato 등, 1998, Science 282:2095)에서도 성공예가 속출하고 있으며, 최근에는 가장 까다롭기로 알려져 있던 돼지에서의 체세포 복제동물 생산에 성공 (PPL Therapeutics, 2000)하는 성과를 달성하게 되었다. Nuclear transfer, also known as somatic cell cloning, is a method of transplanting the cell nucleus of a good genetic trait or the desired transgenic individual into an enucleated egg to produce genetically identical individuals.  Based on the basic research using experimental animals, it is actively progressed in recent years with the aim of mass production of high quality livestock and industrial medicine. As a result of this effort, since the first successful reproduction of sheep (Wilmut et al., 1997, Nature 385: 810), mice and cattle (Wakayama et al., 1998, Nature 394: 369; Kato et al., 1998, Science 282: 2095) ), And success in recent years has been achieved in the production of somatic clones (PPL Therapeutics, 2000).

최근 이식외과술의 발전으로 심장, 신장, 간 등 장기이식에 의한 생명 연장이 확대되고 있으나, 그 특성 상 장기의 공급원이 인간에 한정되어 있기 때문에 수요에 비해 공급이 절대적으로 부족한 상태이다. 따라서, 장기 공급원의 확보를 위한 동물의 장기의 이용 가능성이 제기되면서 그 중요성이 부각되고 있으며, 그 중에서도 생리 구조 및 형태학적 측면에서 인간과 가장 유사한 동물인 돼지가 장기이식 대체동물이 될 수 있음이 시사 되어왔다. 이에 장기 제공용 대체동물의 생산과 함께, 알츠하이머, 파킨슨씨병, 당뇨병 등의 만성질병의 치료수단으로 해당 장기나 조직의 특정 세포를 이식하는 세포 이식치료법(cell theraphy)이 주목을 받고 있다. 여기에, 공급이 용이한 돼지 등 가축의 난자를 수핵란으로 이용하여 인간의 체세포를 이식하는 핵이식기술(cross-species nuclear transplantation)을 적용하여 복제수정란을 작성, 이식 전 단계까지 발육 후 배아세포와 같은 기간세포를 수립, 분화시키게 되면 목적으로 하는 세포를 직접 대량 생산할 수 있다.Recently, due to the development of transplant surgery, life extension by organ transplantation such as heart, kidney, liver, etc. has been extended, but the supply of organs is limited to humans. Therefore, as the availability of organs of animals for the supply of organs has been raised, its importance is highlighted. Among them, pigs, which are the most similar to humans in terms of physiology and morphology, can be organ transplant alternatives. It has been suggested. Along with the production of alternative animals for organ supply, cell therapies for transplanting specific cells of organs or tissues as a means of treating chronic diseases such as Alzheimer's, Parkinson's disease, and diabetes are drawing attention. In addition, by using a cross-species nuclear transplantation technique that uses human eggs, such as pigs, which are easy to supply, as a nucleus for oocytes, a cloned fertilized egg is prepared, and embryos and embryos are developed after development. By establishing and differentiating cells in the same period, the target cells can be mass produced directly.

이와 같이 가축번식학적 측면이나 의학적 활용면에서 매우 유용한 핵이식 기법을 이용하여 복제돼지를 생산한 보고가 있다 (Park 등, 2001, Animal Biotechnology 12:173; Lai 등 2002, Science 295:1089). 돼지의 임신은 임신 12일경 4개 이상의 수정란이 착상하였을 시 감지되는데(Polge 등, 1966, Reproduction & Fertility 12:395), 핵이식 기법 유래의 수정란은 정상 배반포 발 생율이 낮으므로 수정란 이식 시 다수의 수정란을 이식하여야 하는 문제가 있다. 또한 핵이식 후 정상적인 배반포 발생율이 낮고 그로 인한 임신율이 매우 낮아 자돈 생산율이 극히 떨어지는 실정이다 (Lai 등, 2003, Reproductive Biology and Endocrinology 1:82). As such, there have been reports that cloned pigs have been produced using nuclear transfer techniques which are very useful in animal breeding and medical applications (Park et al., 2001, Animal Biotechnology 12: 173; Lai et al. 2002, Science 295: 1089). Pregnancy in pigs is detected when 4 or more fertilized eggs are implanted around 12 days of gestation (Polge et al., 1966, Reproduction & Fertility 12: 395). Since embryos derived from nuclear transfer technique have a low incidence of normal blastocysts, There is a problem in that a fertilized egg should be transplanted. In addition, post-nuclear transplantation has a low rate of normal blastocysts and a very low pregnancy rate, resulting in extremely low piglet production (Lai et al., 2003, Reproductive Biology and Endocrinology 1:82).

본 발명은 상기와 같은 종래기술의 문제점을 해결하기 위한 것으로, 체외수정 및 체세포 복제된 수정란의 동시 이식에 의해 복제 돼지의 정상 분만율을 높일 수 있는 돼지의 생산 방법을 제공하고자 하는 것을 목적으로 한다.The present invention is to solve the problems of the prior art as described above, it is an object of the present invention to provide a pig production method that can increase the normal delivery rate of cloned pigs by simultaneous transplantation of in vitro fertilization and somatic cloned fertilized egg.

본 발명의 다른 목적은 체외수정에 의한 돼지의 생산에 있어서, 수정란의 배양 기간을 대폭 단축하고 적은 수의 난자로부터 정상 분만율을 향상시킨 돼지의 생산 방법을 제공하는 것이다.Another object of the present invention is to provide a method for producing pigs, in which the production period of fertilized eggs is significantly shortened in the production of pigs by in vitro fertilization and the normal rate of fertilization is improved from a small number of eggs.

전술한 목적을 달성하기 위한 본 발명의 돼지 생산방법은 (A) 도축 돼지의 미성숙 난자를 채취하여 배양하는 단계; (B) a) (A)단계에서 준비된 성숙난자와 액상정액의 체외수정에 의한 수정란 및 b) (A)단계에서 준비된 성숙난자를 탈핵하고 공여 체세포를 탈핵 난자에 이식하는 것에 의한 체세포 이식 수정란을 준비하여 각각 배양하는 단계; (C) (B) 단계에서 준비된 체외수정에 의한 수정란과 체세포 이식에 의한 수정란을 동시에 한 마리의 대리모에 이식하는 단계;를 포함하는 것을 특징으로 한다.Pig production method of the present invention for achieving the above object comprises the steps of (A) harvesting the immature eggs of the slaughtered pigs and culturing; (B) a fertilized egg by in vitro fertilization of mature egg and liquid sperm prepared in step (A) and b) somatic cell transplant fertilized egg by denuclearizing mature egg prepared in step (A) and transplanting donor somatic cells into denuclear egg Preparing and culturing each; (C) transplanting the fertilized egg by in vitro fertilization and somatic cell transplantation prepared in step (B) to one surrogate mother at the same time; characterized in that it comprises a.

상기 미성숙 난자의 체외 배양은 체외수정 및 체세포 복제의 성공에 가장 기본 적인 단계로서, 당분야에서 공지의 방법에 의해 이루어 질 수 있다. 그러나, 체외 성숙의 효율을 높이기 위해서는 난포막(follicle shells)을 첨가한 배지에서 36~48시간 이루어 지는 것이 바람직하다. 돼지 미성숙 난자는 보통 체외배양 후 36시간 이후에는 핵 성숙이 이루어져 난자의 핵은 제 2감수분열 중기(metaphase II)에 이르러서 정자의 자극 (체외수정)전까지 휴지하게 되므로 최소 36시간이상 배양하여야 한다. 그러나, 난자의 성숙이 완전해 지기 위해서는 세포질 성숙도 필요하므로 일반적으로 체외성숙 배양은 44~48시간 정도인 것이 가장 바람직하다. 48시간 이후의 휴지기 성숙 난자는 정자의 자극이 없으면, 스스로 퇴화되어 정상적인 수정이 이루어지지 않는다. In vitro culturing of immature eggs is the most basic step for the success of in vitro fertilization and somatic replication, and can be accomplished by methods known in the art. However, in order to increase the efficiency of in vitro maturation, it is preferable to make 36 to 48 hours in a medium containing follicle shells. Porcine immature oocytes usually undergo nuclear maturation 36 hours after in vitro culture and the nucleus of the oocytes reaches metaphase II (metaphase II), which is at rest until sperm stimulation (in vitro fertilization). However, in order to complete the maturation of the oocytes, cytoplasmic maturation is required, so in vitro maturation culture is most preferably about 44-48 hours. Resting mature eggs after 48 hrs degenerate themselves without stimulation of sperm, resulting in normal fertilization.

난포막은 돼지 미성숙 난자의 세포질 성숙을 향상시키고(Mattioli 등, 1989, Theriogenology 31:1201), 미성숙 난자내 cyclic adenosine monophosphate (cAMP) 농도와 glutathione 농도를 증가시켜 체외수정 후 초기 배아 발달을 촉진시킨다(Abeydeera 등, 1998, Biol Reprod 58:213)고 알려져 있다. 따라서 Abeydeera 등(Biol Reprod 58:213, 1998)은 난포로부터 주사기로 난포액을 채취하고, 현미경하에서 난포액에 포함되어 있는 난포막 조각 (follicle shell piece)을 실험자가 주관적으로 선택한 후 세척하여 미성숙 난자의 체외 배양 시 첨가하여 이용하였다. 그러나 이는 처리과정의 번거로움으로 인하여 실용화 되지 못하였다. 본 발명에서는 도축장에서 수집된 난소에서 4-6 mm 직경의 난포막을 수술용 가위로 잘라, 성숙배지에 넣고 돼지 미성숙 난자와 배양시켰다. 실시예에서 구체적인 데이터를 도시하지는 않았으나, 난포막이 첨가된 성숙배지에서 체외성숙시킨 난자를 체외수정시키고, CO2 배양기에서 7일간 배양한 후 그 발달율을 조사한 결과 난포막을 첨가하지 않았을 때보다 최고 약 175%의 증가율을 나타내어 난포막의 첨가가 수정란의 배반포 발달에 현저한 효과가 있음을 알 수 있었다.Follicle membranes enhance cellular maturation of immature pigs (Mattioli et al., 1989, Theriogenology 31: 1201), and promote early embryonic development after in vitro fertilization by increasing cyclic adenosine monophosphate (cAMP) and glutathione levels in immature eggs (Abeydeera). Et al., 1998, Biol Reprod 58: 213). Therefore, Abeydeera et al. (Biol Reprod 58: 213, 1998) used a syringe to collect follicular fluid from a follicle, and under the microscope, the subject's subjective selection of the follicle shell piece contained in the follicular fluid was performed by the experimenter, followed by washing in vitro. It was added to the culture used. However, this has not been put to practical use due to the cumbersome process. In the present invention, the follicle membrane of 4-6 mm diameter from the ovaries collected in the slaughterhouse was cut with surgical scissors, put into mature medium and incubated with immature pigs. Although the specific data are not shown in the examples, in vitro fertilization of the oocytes matured in the mature medium with the follicle membranes was performed in vitro, and after 7 days of incubation in a CO 2 incubator, the development rate was examined. It was found that the addition of follicular membrane had a significant effect on the development of blastocyst of fertilized egg.

상기 (A)단계에서 체외 성숙된 난자를 이용하여 체외수정 및 체세포 이식에 의한 수정란을 각각 준비하였다.In step (A), fertilized eggs by in vitro fertilization and somatic cell transplantation were prepared using in vitro mature eggs.

체외수정은 액상정액을 사용하여 통상의 방법에 의해 이루어 질 수 있으며, 보다 바람직하게는 본 발명의 실시예에 사용된 4℃ 보존 액상 정액을 사용하는 것이 좋다. 본 발명의 4℃ 보존 액상 정액은 조성물 100ml 수용액 당 락토오스 하이드레이트(lactose hydrate) 10~15g, 난황 15~25ml 및 N-아세틸-D-글루코사민(N-acetyl-D-glucosamine) 0.01~0.1g을 포함하는 4℃ 보존용 인공수정용 돼지정액 희석액을 사용하여 제조된 것으로서, 본 실시예에는 자세한 데이터를 기재하지 않았으나 4℃에서 5일간 보관한 후에도 정자의 운동성, 정상침체율 및 생존능이 우수하여 돼지의 수태율 및 산자수를 높일 수 있을 것으로 기대된다.In vitro fertilization can be made by a conventional method using a liquid sperm, more preferably it is preferred to use the 4 ℃ stored liquid semen used in the embodiment of the present invention. The 4 ° C. preservation liquid semen of the present invention comprises 10-15 g of lactose hydrate, 15-25 ml of egg yolk and 0.01-0.1 g of N-acetyl-D-glucosamine per 100 ml aqueous solution of the composition. It was prepared using a diluent solution for artificial insemination for 4 ℃ preservation, this example did not describe the detailed data, but after storage for 5 days at 4 ℃ excellent motility, normal stagnation rate and viability of the sperm It is expected to increase the conception rate and the number of litters.

체세포 이식에 의한 수정란 생산 역시 당분야에서 공지의 기술을 이용하여 용이하게 실시될 수 있으며, 특정 방법에 한정되는 것은 아니다.Fertilized egg production by somatic cell transplantation can also be easily carried out using techniques known in the art, and is not limited to specific methods.

체외수정 수정란 및 체세포 이식 수정란은 16~36시간 각각 체외배양한 후 대리모에 이식한다. 통상 체외수정에 의해 돼지를 생산하는 경우, 체외수정 수정란을 체외에서 5~6일간 배양한 후 양질의 배반포만을 선별하여 수정란을 이식하는 데 반하여, 본 발명에서는 체외수정된 수정란의 경우에도 16~36시간만 체외배양한 후 수정란을 이식하여도 되는 이점이 있다. In Vitro Fertilized Embryos and Somatic Cell Transplantation Embryos are transplanted in surrogate mothers after 16 to 36 hours of in vitro culture. In general, in the case of producing pigs by in vitro fertilization, in vitro fertilization of in vitro fertilized eggs for 5-6 days after culturing only the high quality blastocysts and transplanting fertilized eggs, whereas in the present invention 16-36 There is an advantage that you can transplant the fertilized egg after in vitro culture only.

체외에서 16~36시간 각각 배양된 체외수정에 의한 수정란과 체세포 이식에 의한 수정란을 개복수술에 의해 1두의 대리모에 동시에 이식한다. 이식에 사용되는 수정란의 총 개수는 150~250개인 것이 바람직하다. Fertilized eggs by in vitro fertilization and fertilized eggs by somatic cell transplantation incubated for 16 to 36 hours in vitro are transplanted to one surrogate mother simultaneously by laparotomy. The total number of fertilized eggs used for transplantation is preferably 150-250.

일반적으로 체외에서 성숙된 난자를 이용하여 체외수정과 체세포 복제를 한 후 그 수정란을 7일간 배양하였을 시 배반포 발생율은 약 20% 전후이다. 그러나 실제 수정란 이식 시 착상되는 배반포의 착상율이 2% 내외로 극히 낮은 것을 고려한다면, 수정란을 1일 배양한 후 이식할 경우 성공적인 수태를 위해서는 이론적으로 최소한 250개의 수정란을 이식하여야 한다. 한편 배반포를 이식할 경우에는 수정란을 5-7일간 배양한 후 양질의 배반포만을 선별하여 50개 이상을 주입하여야 한다. In general, after in vitro fertilization and somatic cell cloning using in vitro mature eggs, the embryonated blastocyst development rate is about 20%. However, considering that the implantation rate of blastocysts implanted during fertilized egg implantation is extremely low, about 2%, theoretically at least 250 fertilized eggs should be transplanted for successful conception when fertilizing embryos for 1 day. On the other hand, when transplanting blastocysts, fertilized eggs should be cultured for 5-7 days, and only 50 blastocysts should be selected and injected with 50 or more blastocysts.

본 발명의 실시예에서 생산된 체세포 복제 수정란만을 역시 실시예와 동일한 수술 방법에 의해 이식하여 복제 돼지의 생산을 시도하였을 경우, 아래 표 1과 같 이 11번의 시도에도 불구하고 임신이 되지 않았다. 상기 시험은 2005년 7월 15일부터 2006년 6월 2일에 걸쳐 실시되었다. 이는 통상 체세포 복제 수정란의 임신율이 극히 낮은 것과 일치되는 결과이다. 그러나 체세포 복제 유래 수정란 100개를 1일 배양된 체외수정 유래 수정란 100개와 동시에 이식한 결과 단 한번의 시도로 임신이 되어 3마리의 자돈이 정상적으로 생산되었다.When only the somatic cell cloned embryos produced in Examples of the present invention were transplanted by the same surgical method as in Example, and attempted to produce cloned pigs, despite 11 trials as shown in Table 1 below, pregnancy did not occur. The test was conducted from July 15, 2005 to June 2, 2006. This is usually consistent with an extremely low fertility rate of somatic replication embryos. However, when 100 embryos derived from somatic cloning were implanted at the same time with 100 embryos derived from in vitro fertilization, three piglets were normally produced in a single trial.

Figure 112007021811633-pat00001
Figure 112007021811633-pat00001

체외수정에 사용된 정액 및 체세포 이식에 이용된 체세포의 DNA와 비교한 결과 2마리는 체외수정 수정란 유래의 자돈이며, 1마리는 체세포 복제 수정란 유래의 자돈임을 확인할 수 있었다. 자돈의 생시 체중은 각각 1.2, 2.0 (체외수정 유래), 1.2 (체세포 복제 유래) Kg의 정상체중이었다.As a result of comparison with the DNA of semen used for in vitro fertilization and somatic cell transplantation, it was confirmed that two were piglets derived from in vitro fertilized eggs and one were piglets derived from somatic cloned fertilized eggs. The live weights of piglets were normal weights of 1.2, 2.0 (from in vitro fertilization) and 1.2 (from somatic cloning), respectively.

체세포 복제에 의한 돼지의 정상 분만율이 매우 낮음에도 불구하고 단 한번의 시도에 의해 2두의 체외수정 유래의 자돈과 1두의 체세포 복제 자돈이 정상분만된 것은 체외수정 유래의 수정란이 일반적으로 착상이 잘 되지 않는 체세포 복제 수정란의 착상에 유리한 효과를 준 것으로 사료된다. 또한, 체외수정에 의한 수정란을 배반포 형성 전에 단지 100개만 이식하였음에도 체외수정 유래의 자돈이 2마리가 생산 된 것 역시 체세포 이식 수정란의 동시 이식이 체외 수정 유래의 수정란에 대해서 역시 수태율 및 정상분만율을 높이는 데 상호 시너지 효과를 준 것을 의미한다. Despite the very low rate of normal birth of pigs by somatic cloning, two piglets derived from in vitro fertilization and one somatic cloning piglet were normally delivered in a single trial. It is thought that it gave a favorable effect on the implantation of poor somatic cell cloned embryos. In addition, even though only 100 embryos were transplanted before blastocyst formation, two piglets from in vitro fertilization were produced. Simultaneous transplantation of somatic cell embryos also improved fertility and normal delivery rate for fertilized eggs derived from in vitro fertilization. That means having a mutual synergy.

이하 실시예를 통하여 본 발명을 상세하게 설명한다. 그러나, 이들 실시예는 예시적인 목적일 뿐 본 발명이 이에 한정되는 것은 아니다.The present invention will be described in detail through the following examples. However, these examples are for illustrative purposes only and the present invention is not limited thereto.

실시예Example

1. 돼지 미성숙 난자의 수집 및 체외성숙1. Collection and in vitro maturation of pig immature eggs

도축장에서 도살 직후 미경산돈의 난소들을 적출하고 30-35oC의 0.9% 생리식염수에 담아 실험실로 운반하였다. 난포액과 난구세포(cumulus cell)가 부착된 미성숙 난자를 10 ml 주사기에 18게이지 바늘을 장착하여 2-6 mm 직경의 난포로부터 흡입하여 채취하였다. Immediately after slaughter in the slaughterhouse, the ovaries of unpolished pigs were extracted and transported to the laboratory in 0.9% saline at 30-35 o C. Immature eggs attached with follicular fluid and cumulus cells were collected by suctioning from a 2-6 mm diameter follicle with an 18-gauge needle attached to a 10 ml syringe.

흡입된 난포액을 37oC 수조에 정치시키고, 가라앉은 침전물만을 0.1% polyvinyl alcohol(PVA)이 포함된 TL-HEPES-PVA(114 mM NaCl, 3.2 mM KCl, 2 mM NaHCO3, 0.4 mM NaH2PO4, 0.27 mM glucose, 10 mM sodium lactate, 2 mM CaCl2·2H2O, 0.5 mM MgCl2·6H2O, 10 mM HEPES, 0.03 mM phenol red, 0.25 mM sodium pyruvate, 0.3% bovine serum albumin (BSA), 75 ㎍/ml penicillin G and 25 ㎍/ml gentamycin sulfate)에 취하여 미성숙 난자만을 골랐다. 미성숙 난자는 난구세포가 치밀하게 부착된 것만을 선택하여 TL-HEPES-PVA로 두 번 세척하고 하기 성숙배지로 다시 두 번 세척하였다. 체외성숙에는 tissue culture medium (TCM) 199 배양배지(Sigma, St. Louis, MO, USA)를 기본 배지로 사용하였고, 여기에 26.19 mM NaHCO3, 3.05 mM glucose, 0.91 mM sodium pyruvate, 75 ㎍/ml sodium penicillin G, 50 ㎍/ml streptomycin sulfate 그리고 0.1% PVA를 첨가하여 사용하였다. 미성숙 난자는 2 ml 체외성숙배지에 0.5 ㎍/ml luteinizing hormone (LH), 0.5 ㎍/ml follicular stimulating hormone (FSH), 10 ng/ml epidermal growth factor (EGF), 10% porcine follicular fluids (pFF), 0.57 mM cysteine, 2개의 난포막을 첨가한 것에 옮겨 38.5oC, 5% CO2의 항온기(incubator)에서 22시간 배양하고, 그 후 FSH와 LH를 제외한 성숙배지에서 추가로 22시간 배양하였다. 상기 난포막은 도축장에서 수집된 난소에서 도 1에 표시된 → 부분의 볼록한 부분을 수술용 가위로 4-6 mm 직경으로 잘라 성숙배지에 담가 3번 세척한 후 사용하였다.The inhaled follicle solution was allowed to stand in a 37 ° C. water bath and TL-HEPES-PVA (114 mM NaCl, 3.2 mM KCl, 2 mM NaHCO 3 , 0.4 mM NaH 2 PO) containing only 0.1% polyvinyl alcohol (PVA). 4 , 0.27 mM glucose, 10 mM sodium lactate, 2 mM CaCl 2 · 2H 2 O, 0.5 mM MgCl 2 · 6H 2 O, 10 mM HEPES, 0.03 mM phenol red, 0.25 mM sodium pyruvate, 0.3% bovine serum albumin (BSA ), And 75 g / ml penicillin G and 25 μg / ml gentamycin sulfate) were used to select only immature eggs. Immature oocytes were selected only with the densely attached cumulus cells and washed twice with TL-HEPES-PVA and again with the following maturation medium. For in vitro maturation, tissue culture medium (TCM) 199 culture medium (Sigma, St. Louis, MO, USA) was used as the basal medium, and 26.19 mM NaHCO 3 , 3.05 mM glucose, 0.91 mM sodium pyruvate, 75 ㎍ / ml Sodium penicillin G, 50 ㎍ / ml streptomycin sulfate and 0.1% PVA were used. Immature oocytes in 0.5 ml / ml luteinizing hormone (LH), 0.5 µg / ml follicular stimulating hormone (FSH), 10 ng / ml epidermal growth factor (EGF), 10% porcine follicular fluids (pFF) 0.57 mM cysteine, 2 follicle membranes were added, and then incubated for 22 hours in an incubator at 38.5 ° C and 5% CO 2 , followed by an additional 22 hours in mature medium excluding FSH and LH. The follicular membrane was used after washing three times by immersing the convex part of the part indicated in FIG.

2. 종모돈 원정액 채취와 액상정액의 제조2. Sampling of sow seed and preparation of liquid sperm

원정액(fresh semen)은 일주일에 1회 듀록 (Duroc) 종모돈으로부터 채취하였다. 사출된 농후정액(sperm rich fraction)은 85% 이상의 운동성과 정상첨체율 (normal acrosome)을 나타내는 것만을 이용하였다. 채취 후 2시간에 걸쳐 원정액의 온도를 서서히 실온으로 내렸다. 원정액을 15 ml 시험관(BD Falcon, USA)에 옮기고 실온에서 800×g, 10분간 원심분리하였다. 상층액은 깨끗이 버리고 남은 정자 (sperm)에 실온의 10 ml LEN(100 ml distilled water에 11.0 g lactose hydrate, 20 ml egg yolk, 0.05 g N-acetyl-D-glucosamine이 포함된 것) 희석액을 넣어 잘 섞고 최종 정자농도가 1.0×109 sperm/ml가 되도록 하였다. 액상정액은 4oC 냉장고에 넣어 5일간 보존하며 사용하였다.Fresh semen were collected from Duroc sows once a week. The injected spum rich fraction was used only for those exhibiting more than 85% of motility and normal acrosome. The temperature of the stock solution was gradually lowered to room temperature over 2 hours after the collection. The crude solution was transferred to a 15 ml test tube (BD Falcon, USA) and centrifuged at 800 x g for 10 minutes at room temperature. Discard the supernatant and add 10 ml LEN (11.0 g lactose hydrate, 20 ml egg yolk, 0.05 g N-acetyl-D-glucosamine in 100 ml distilled water) at room temperature to the remaining sperm. The final sperm concentration was 1.0 × 10 9 sperm / ml. The liquid sperm was stored in a 4 o C refrigerator for 5 days and used.

3. 돼지 난자의 체외수정3. In vitro fertilization of pig eggs

1에서 44시간 성숙된 난자를 0.1% hyaluronidase가 첨가된 TL-HEPES-PVA에 옮겨 피펫팅(pipetting)하여 확장된 난구세포를 제거하였다. 난구세포가 제거된 난자를 TL-HEPES-PVA에 세척한 후, 실체 현미경하에서 난자내에 극체(polar body)가 방출된 것만을 선별하였다. 다시 TL-HEPES-PVA와 체외수정 배지로 각각 두 번 세척한 후 500 μl 체외수정 배지에 30-40개의 난자를 넣었다. 체외수정 배지로는 Tris-buffered medium(mTBM; 113.1 mM NaCl, 3.0 mM KCl, 10.0 mM CaCl2, 20.0 mM Tris (hydroxymethyl aminomethane), 11.0 mM glucose, 5.0 mM sodium pyruvate, 1.0 mM caffeine, 75 μg/ml penicillin G, 50 μg/ml streptomycin, 0.57 mM cysteine and 1 mg/ml BSA 포함)을 사용하였으며 4-well multi dish(Nunc, Denmark)에 well당 500 μl씩 분주하고 mineral oil로 피복하여 38.5oC, 5% CO2의 항온기에서 4시간 이상 평형 시켜둔 것을 사용하였다. Extended oocytes were removed by transferring the mature eggs from 1 to 44 hours to TL-HEPES-PVA added with 0.1% hyaluronidase. After the oocytes from which the oocytes had been removed were washed with TL-HEPES-PVA, only those in which the polar body was released into the egg under a stereoscopic microscope were selected. After washing twice with TL-HEPES-PVA and in vitro fertilization medium, 30-40 eggs were added to 500 μl in vitro fertilization medium. In vitro fertilization medium included Tris-buffered medium (mTBM; 113.1 mM NaCl, 3.0 mM KCl, 10.0 mM CaCl 2 , 20.0 mM Tris (hydroxymethyl aminomethane), 11.0 mM glucose, 5.0 mM sodium pyruvate, 1.0 mM caffeine, 75 μg / ml penicillin G, 50 μg / ml streptomycin, 0.57 mM cysteine and 1 mg / ml BSA) was used. Dispense 500 μl per well in a 4-well multi dish (Nunc, Denmark) and coat with mineral oil to cover 38.5 o C, An equilibrium of at least 4 hours in a 5% CO 2 thermostat was used.

체외수정을 위하여, 2에서 제조한 500 μl 액상정액을 취하여 실온의 TL-HEPES-PVA 5ml를 넣고 잘 섞은 후 800xg에서 3분간 원심분리하였다. 상층액만을 버린 뒤 TL-HEPES-PVA를 넣고 섞은 뒤 다시 원심분리하고 상층액을 버린 뒤 실온의 체외수정 배지 mTBM로 희석하여 잘 섞었다. 위에서 제조한 체외수정배지에 있는 난자에 정자의 농도가 1×106sperm/ml가 되도록 넣은 뒤 38.5oC, 5% CO2의 항온기에서 6시간 동안 공배양하였다.For in vitro fertilization, the 500 μl liquid solution prepared in 2 was taken, 5 ml of TL-HEPES-PVA at room temperature was added, mixed well, and centrifuged at 800xg for 3 minutes. After discarding only the supernatant, mixed with TL-HEPES-PVA, centrifuged again, discarded the supernatant, diluted with mTBM at room temperature in vitro fertilization medium and mixed well. The sperm concentration was 1 × 10 6 sperm / ml in the oocytes in the in vitro fertilization medium prepared above and co-cultured for 6 hours in a thermostat of 38.5 o C, 5% CO 2 .

6시간의 체외수정 후, 난자는 체외배양 배지인 NCSU-23 (108.73 mM NaCl, 4.78 mM KCl, 1.19 mM KH2PO4, 1.19 mM MgSO4·7H2O, 5.55 mM D-glucose, 1.00 mM glutamine, 7.00 mM Taurine, 5.00 mM hypotaurine, 25.07 mM NaHCO3, 1.70 mM CaCl2·2H2O, 75 μg/ml penicillin G, 50 μg/ml streptomycin)에 배지에 50 μM 2-mercatoethanol, 0.17 mM sodium pyruvate, 2.73 mM sodium lactate를 첨가한 것에 옮기고, 16-36 h 배양시킨 뒤 수정란 이식을 하였다.After 6 hours of in vitro fertilization, the egg was in vitro culture medium NCSU-23 (108.73 mM NaCl, 4.78 mM KCl, 1.19 mM KH 2 PO 4 , 1.19 mM MgSO 4 .7H 2 O, 5.55 mM D-glucose, 1.00 mM glutamine , 7.00 mM Taurine, 5.00 mM hypotaurine, 25.07 mM NaHCO 3 , 1.70 mM CaCl 2 · 2H 2 O, 75 μg / ml penicillin G, 50 μg / ml streptomycin) in 50 μM 2-mercatoethanol, 0.17 mM sodium pyruvate, Transfer to the addition of 2.73 mM sodium lactate, incubated for 16-36 h and then fertilized egg transplant.

4. 체세포 복제4. Somatic Replication

1에서 44시간 성숙된 난자를 0.1% hyaluronidase를 첨가한 TL-HEPES-PVA에 옮겨 피펫팅(pipetting)하여 확장된 난구세포를 제거하였다. 돼지 난자는 micromanipulator (Narishige, Japan)가 장착된 inverted microscope (Nikon, Japan)하에서 미세조작에 의하여 핵을 제거하고, 임신 35일의 돼지에서 적출해낸 태아에서 분리하여 배양된 태아섬유아세포 (fetal fibroblast)를 난자에 주입하였다. Extended oocytes were removed by transferring the matured eggs from 1 to 44 hours to TL-HEPES-PVA with 0.1% hyaluronidase. Pig eggs were removed by micromanipulation under an inverted microscope (Nikon, Japan) equipped with a micromanipulator (Narishige, Japan) and isolated from the embryos extracted from pigs at 35 days of gestation and cultured with fetal fibroblasts. Was injected into the egg.

서로 다른 세포에 대한 융합을 위하여 ECM 2001 electrocell manipulator (BTX Inc., San Diego, CA, USA)를 이용하여 1.2 kV/cm, 30 μs, 1 DC pulse로 전기적 자극을 주었다. 융합된 난자는 TCM-199배지에서 3시간 동안 배양하고 다시 NCSU23에 옮겨 16-36시간 배양한 후 수정란 이식에 사용하였다.For fusion to different cells, ECM 2001 electrocell manipulator (BTX Inc., San Diego, Calif., USA) was used for electrical stimulation with 1.2 kV / cm, 30 μs, 1 DC pulse. The fused eggs were incubated for 3 hours in TCM-199 medium and transferred to NCSU23 for 16-36 hours before being used for fertilized egg transplant.

5. 수정란 이식 및 자돈 생산5. Embryo Transfer and Piglet Production

돼지의 발정주기는 대개 20-22일(평균 21일)이며 발정지속시간, 즉 수퇘지 허용시간은 평균 48시간이다. 따라서 하루에 두 번 발정조사를 하여 발정 발견 시작부터 12-24시간 후 수정란 이식을 시술하였다.The estrous cycle of pigs is usually 20-22 days (average 21 days) and the estrous duration, or boar tolerance, is 48 hours on average. Therefore, estrus irradiation was performed twice a day, and fertilized egg transplantation was performed 12-24 hours after the onset of estrus discovery.

수정란 이식을 위하여 대리모 암퇘지에 말레인산 아세프로마진(acepromazine maleate) 35 mg과 황산아트로핀(atropine sulfate) 2.5 mg을 이근부 피하주사하여 진정시키고, 30분 후 말레인산 아세프로마진 20 mg과 염산 케타민(ketamine hydrochloride) 1,200 mg을 귀정맥에 주사하여 전신마취하였다. 전신마취시킨 돼지를 앙와자세로 수술대에 보정하고, 수술부위를 세척, 제모, 소독한 후 최종유두에서 다음번 유두 사이를 정중선을 따라 15 cm 절개하였다. 피하지방을 절개부위에서 좌우로 둔성분리한 후 근육과 복막을 절개하고 자궁, 난관, 난소를 노출시킨 후 수정란 이식을 실시하였다. Subcutaneous subcutaneous injection of 35 mg of maleic acid acepromazine maleate and 2.5 mg of atropine sulfate in the surrogate mother sow was used to sub- fertilize the fertilized egg sow. After 30 minutes, 20 mg of maleic acid acepromazine and ketamine hydrochloride General anesthesia was performed by injecting 1,200 mg into the ear vein. The general anesthetized pig was calibrated on the operating table with the supine position, and the surgical site was washed, depilated, and disinfected, and a 15 cm incision was made between the final papilla and the next papilla along the midline. Subcutaneous fat was separated from left and right at the incision site, muscles and peritoneum were cut, exposed the uterus, fallopian tube and ovary, and fertilized egg transplantation was performed.

1-4세포기의 수정란(체외수정된 수정란 100개와 체세포 복제된 수정란 100개, 즉 총 200개의 수정란을 함께 대리모 1두에 이식)을 내부직경 0.86 mm, 외부직경 1.27 mm의 비독성 수술용 폴리에틸렌 튜브(Intramedic, Clay Adams, USA)를 이용하여 난관심부에 이식하였다. 이식이 끝난 후 자궁, 난관, 난소를 원위치 시키고 복막, 근육, 피부의 순으로 봉합하였다.Non-toxic surgical polyethylene with 1-6 cell stages (100 in vitro fertilized eggs and 100 somatic cloned embryos, ie 200 total fertilized eggs implanted in one surrogate mother) with 0.86 mm inner diameter and 1.27 mm outer diameter A tube (Intramedic, Clay Adams, USA) was used to implant in the fallopian tube. After transplantation, the uterus, fallopian tubes, and ovaries were replaced and sutured in order of peritoneum, muscle and skin.

수정란 이식 후 115일만인 2006년 9월 25일 정상상태의 자돈 3마리를 생산하였다.Three healthy piglets were produced on September 25, 2006, 115 days after fertilization.

6. DNA 친자감별6. DNA paternity

자돈, 대리모돈, 액상정액 및 공여세포의 DNA를 분석하여 체외수정 수정란 유래의 자돈인지 체세포 복제 수정란 유래의 자돈인지를 감별하였다. DNA 감별은 축산연구소 동물유전체과(National Livestock Research Institute, 경기도 수원시 권선구 오목천동 564)에 의뢰하여, 돼지 액상정액 DNA 시료, 3마리 돼지의 각 DNA 시료 및 공여세포의 DNA 시료에 대해 하기의 방법으로 13개의 microsatellite marker (MS)를 분석하였다.DNA from piglets, surrogate sows, liquid sperm and donor cells was analyzed to discriminate whether piglets derived from in vitro fertilized eggs or piglets derived from somatic cloning embryos. DNA discrimination was commissioned by the Animal Livestock Research Institute (National Livestock Research Institute, 564, Omokcheon-dong, Suwon-si, Gyeonggi-do, Korea). For the liquid sample of swine, each of three pigs, and the DNA of donor cells, Microsatellite markers (MS) were analyzed.

1) DNA 추출1) DNA extraction

자돈 3마리, 대리모 1마리 각각의 귀를 1-2 cm 크기로 잘라 1.5 ml tube에 넣었다. 체외수정에 이용된 정액은 1 ml를 1.5 ml tube에 넣고 microcentrifuger (Bio-spin, Hanil, Korea)에서 10,000 rpm, 20 분간 원심분리하고 남아있는 pellet만을 준비하였다. 체세포 복제 시 이용한 체세포는 배양 중이던 체세포를 0.05% trypsin-EDTA(ethylenediamine tetra acetic acid)로 분리한 뒤 phosphate buffered saline (PBS)로 두 번 세척하여 준비하였다.Three piglets and one surrogate mother were cut into 1-2 cm ears and placed in a 1.5 ml tube. The semen used for in vitro fertilization was placed in a 1.5 ml tube and centrifuged at 10,000 rpm for 20 minutes in a microcentrifuger (Bio-spin, Hanil, Korea) and only the remaining pellets were prepared. Somatic cells used for cloning were prepared by separating the somatic cells in culture with 0.05% trypsin-EDTA (ethylenediamine tetra acetic acid) and washing them twice with phosphate buffered saline (PBS).

상기 준비된 샘플들에 대하여 500 μl DNA extraction buffer(50 nM Tris, 100 mM EDTA, 10% sodium dodecyl sulfate (SDS)) 및 10 mg/ml proteinase K(U302B, Promega, Madison, WI, USA) 50 μl를 각각 첨가하고 위 아래로 섞어 주었다. 55oC의 수조에서 상기의 각 샘플들을 8-24시간 정도 배양하여 샘플들이 부옇게 되었는지 확인하고, 500 μl PCI(phenol : chloroform : isoamylalcohol = 25 : 24: 1, Cat. C9017, Bioneer, Korea)를 넣어 위아래로 섞어준 후 10,000 rpm, 10 분간 원심분리하였다. 원심분리된 상층액만을 취하여 tube에 넣고 동량의 CI(chloroform : isoamylalcohol = 24: 1 (v/v))를 넣고 위아래로 섞어준 후 10,000 rpm, 10 분간 원심분리하고 다시 상층액만 새 tube에 넣었다. 3 M sodium acetate(pH 6.0)를 상기 상등액의 1/10 만큼 넣고, 이 혼합의 두 배에 해당하는 100% 에탄올을 넣고 잘 섞어 주었다. 상기 혼합물을 실온에 5시간 이상 그대로 두어 침전물을 완전히 가라앉게 한 후 10,000 rpm, 10 분간 원심분리하였다. Clean bench에서 상층액은 버리고 pellet만 취하여 염을 제거하기 위하여 100 μl 70% ethanol을 넣고 잘 흔든 후 버리기 두 번, 100 μl 100% ethanol을 넣고 잘 흔든 후 버리기 한 번 실시하였다. 세척된 pellet을 실온에서 5-20분간 두어 완벽하게 말린 후 고체상태가 된 pellet을 20-50 μl 증류수를 사용하여 녹였다. 상기 수용액의 UV 흡광도를 UV/Visible spectrophotometer(Ultrospec 2100 pro, Amersham Biosciences, Cambridge, UK)를 사용하여 260파장에서 측정하여 optical density(OD)를 얻은 후 DNA의 농도를 계산하여 DNA 최종농도가 1 μg/μl가 되도록 하고 -20oC에서 보관하여 사용하였다.50 μl of 500 μl DNA extraction buffer (50 nM Tris, 100 mM EDTA, 10% sodium dodecyl sulfate (SDS)) and 10 mg / ml proteinase K (U302B, Promega, Madison, WI, USA) were prepared for the prepared samples. Each was added and mixed up and down. Incubate each of the above samples for 8-24 hours in a 55 o C water bath to determine whether the samples were broken, and 500 μl PCI (phenol: chloroform: isoamylalcohol = 25: 24: 1, Cat. C9017, Bioneer, Korea) After mixing up and down, it was centrifuged for 10 minutes at 10,000 rpm. Only the centrifuged supernatant was taken into the tube, the same amount of CI (chloroform: isoamylalcohol = 24: 1 (v / v)) was added and mixed up and down, and then centrifuged at 10,000 rpm for 10 minutes and only the supernatant was added to a new tube. . 3 M sodium acetate (pH 6.0) was added by 1/10 of the supernatant, and 100% ethanol corresponding to twice the amount of the supernatant was added and mixed well. The mixture was left at room temperature for at least 5 hours to allow the precipitate to settle completely and then centrifuged at 10,000 rpm for 10 minutes. In the clean bench, the supernatant was discarded and pellet was removed. To remove the salt, 100 μl 70% ethanol was added, shaken well and discarded twice. 100 μl 100% ethanol was added, shaken well and discarded once. The washed pellet was allowed to dry for 5-20 minutes at room temperature, and then dissolved into 20-50 μl distilled water. The UV absorbance of the aqueous solution was measured at 260 wavelength using a UV / Visible spectrophotometer (Ultrospec 2100 pro, Amersham Biosciences, Cambridge, UK) to obtain an optical density (OD). / μl and stored at -20 o C was used.

2) Genomic DNA 분리 및 농도측정2) Genomic DNA Isolation and Concentration Measurement

Genomic DNA는 F2 354두의 기준집단으로부터 0.5 M EDTA(pH8.0)가 1 ml 첨가된 tube에 전혈 10 ml을 채취한 후 Wizard genomic DNA purification kit (Promega Co., USA)를 이용하여 추출하였다. 채취된 전혈 10 ml에 30 ml의 cell lysis solution을 첨가한 후 실온에서 10분간 적혈구를 용혈시켰다. 용혈 후, 2,000xg에서 10분간 원심분리하여 백혈구만을 모은 후 pellet을 잘 현탁시키고 10 ml의 nuclei lysis solution에 용해시켰다. 상기 용액을 15분간 37oC 진탕배양기에서 RNase A(20 μg/ml)로 처리한 뒤, 3.3 ml의 protein precipitation solution을 첨가하고 20초간 강하게 현탁시켰다. 상기 현탁액을 2,000xg에서 10분간 원심분리하여 정제하였고 isopropanol로 DNA를 침전시킨 다음 70% ethanol로 세척, 건조 후 약 50 μl의 Tirs-EDTA buffer (pH 8.0)에 녹여 PCR에 이용하였다.Genomic DNA was extracted using a wizard genomic DNA purification kit (Promega Co., USA) from 10 ml of whole blood in a tube containing 1 ml of 0.5 M EDTA (pH8.0) from 354 F 2 reference groups. . 30 ml of cell lysis solution was added to 10 ml of collected whole blood and red blood cells were hemolyzed at room temperature for 10 minutes. After hemolysis, centrifuged at 2,000xg for 10 minutes to collect only white blood cells, the pellet was well suspended and dissolved in 10 ml of nuclei lysis solution. The solution was treated with RNase A (20 μg / ml) in a 37 ° C. shaker incubator for 15 minutes, followed by the addition of 3.3 ml of protein precipitation solution and strong suspension for 20 seconds. The suspension was purified by centrifugation at 2,000xg for 10 minutes, and the DNA was precipitated with isopropanol, washed with 70% ethanol, dried and dissolved in about 50 μl of Tirs-EDTA buffer (pH 8.0) for use in PCR.

3) 초위성체 표지인자3) Supersatellite markers

유전자형 분석에 사용한 LEPR(Leptin receptor)내에 존재하는 초위성체 표지인자는 Genbank에 등록되어 있는 염기서열정보(Accession No. AF184172)를 근거로 선발하였으며, (CA)n의 nucleotide 반복서열 형태를 가지고 있었다. PCR을 위한 primer는 forward primer 5'-aatggaaactcttcccagct-3'(서열번호 1)와 reverse primer 5'-cattcgaactgttcattgccat-3'(서열번호 2)을 각각 선발 합성하여 사용하였다.The supersatellite markers present in the LEPR (Leptin receptor) used for genotyping were selected based on nucleotide sequence information (Accession No. AF184172) registered in the Genbank, and had a nucleotide repeat of (CA) n. The primers for PCR were selected by synthesizing forward primer 5'-aatggaaactcttcccagct-3 '(SEQ ID NO: 1) and reverse primer 5'-cattcgaactgttcattgccat-3' (SEQ ID NO: 2), respectively.

4) PCR에 의한 DNA 증폭4) DNA amplification by PCR

PCR 반응액 조성은 PCR reaction buffer(10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2)와 2.5 mM dNTPs, 3 pmol fluorescent dye labeling primer paris, 10 ng의 template DNA, 0.5 U Tag DNA polymerase(Takara Shuzo Co., Shiga, Japan)와 ddH2O를 사용하였으며 총 반응액은 10 μl로 하였다. PCR 반응에는 GeneAmp PCR System 9600(Perkin-Elmer Co., USA)를 사용하였고, PCR 조건은 94oC에서 5분간 pre-denaturation 한 후 94oC에서 30초(denaturation), 60oC에서 40초(annealing), 72oC에서 1분(extention)을 35 cycles 수행한 후 마지막으로 72oC에서 10분간 최종 extention 과정을 수행하였다. PCR 증폭 산물은 증폭된 단편의 크기가 예상된 allele size 범위내에 존재하는지, PCR 조건의 적정성 여부를 확인하기 위하여 EtBr(ethidium bromide)이 포함된 2% agarose gel에 전기영동하고 UV 상에서 관찰하였다.PCR reaction solution composition was PCR reaction buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl 2 ) and 2.5 mM dNTPs, 3 pmol fluorescent dye labeling primer paris, 10 ng template DNA, 0.5 U Tag DNA Polymerase (Takara Shuzo Co., Shiga, Japan) and ddH 2 O were used and the total reaction solution was 10 μl. PCR reaction was performed using GeneAmp PCR System 9600 (Perkin-Elmer Co., USA), PCR conditions were 40 seconds at 94 minutes at 5 o C and then pre-denaturation 30 sec at 94 o C (denaturation), 60 o C (annealing), 35 cycles of 1 min at 72 o C, and finally 10 min final extention at 72 o C. PCR amplification products were electrophoresed on 2% agarose gel containing EtBr (ethidium bromide) and observed on UV light to determine whether the size of the amplified fragment was within the expected allele size range and whether the PCR conditions were appropriate.

5) 유전자형 분석5) Genotyping

PCR 산물을 적정량의 DI(deionized)수로 희석하고 DNA : formamide : size standard(Genescan-350 TAMRA)를 1 μl : 12 μl : 0.5 μl 비율로 혼합하여 95oC이상에서 3분간 denaturation 한 후, ABI 310 Genetic Analyzer(Perkin-Elmer Co., USA)를 이용하여 분석하였다. GeneScan software version 2.1(Perkin-Elmer Co., USA)을 이용하여 PCR 산물인 DNA 절편의 양과 크기에 대한 자료를 모아 수집하였다. 전기영동시 Performance Optimized Polymer 4(POP4)(PE Applied Biosystems)와 10x Buffer(with EDTA)를 1x로 희석하여 사용하였고, run time은 22분으로 하였다. 유전자형은 Genotyper software version 2.5 (Perkin-Elmer Co., USA)를 이용하여 분석하였다. Dilute the PCR product with an appropriate amount of DI (deionized) water, mix DNA: formamide: size standard (Genescan-350 TAMRA) in 1 μl: 12 μl: 0.5 μl ratio, denaturate at 95 ° C for 3 minutes, and then ABI 310 Analysis was performed using a Genetic Analyzer (Perkin-Elmer Co., USA). GeneScan software version 2.1 (Perkin-Elmer Co., USA) was used to collect data on the amount and size of DNA fragments, PCR products. During electrophoresis, Performance Optimized Polymer 4 (POP4) (PE Applied Biosystems) and 10x Buffer (with EDTA) were diluted to 1x and run time was 22 minutes. Genotypes were analyzed using Genotyper software version 2.5 (Perkin-Elmer Co., USA).

6) DNA 분석결과6) DNA analysis result

자돈 3마리와 체외수정에 이용한 정자, 체세포 복제 시 공여세포로 사용한 체세포의 DNA를 분석하여 비교한 결과, 하기 표 1에 기재된 것과 같이 자돈 2마리의 경우에는 수정에 이용한 정자와 마이크로 새털라이트 (microsatellites) 마커 12종에 대하여 거의 모두 일치하였으며, 자돈 1마리는 마이크로 새털라이트 (microsatellites) 마커 12종이 모두 일치하였다.As a result of analyzing the DNA of three piglets, sperm used for in vitro fertilization, and somatic cell DNA used as donor cells for somatic cell replication, as shown in Table 1 below, sperm and microsatellites used for fertilization were used for two piglets. Almost all of the 12 markers were identical, and one pig was identical to all 12 microsatellites markers.

Figure 112007021811633-pat00002
Figure 112007021811633-pat00002

이로써, 돼지 1과 2(도 2의 A 및 B)는 체외수정에 의한 수정란 유래의 돼지임을 돼지 3(도 2의 C)은 체세포 복제에 의한 수정란 유래의 돼지임을 확인할 수 있었다.As a result, pigs 1 and 2 (A and B of FIG. 2) were pigs derived from fertilized eggs by in vitro fertilization, and pig 3 (C of FIG. 2) was confirmed to be pigs derived from fertilized eggs by somatic cell replication.

이상과 같이 본 발명에 의하면 체외수정된 수정란과 체세포 복제에 의한 수정란을 동시 이식하는 것에 의해, 정상 분만율이 극히 낮은 복제돼지의 정상 분만을 가능하게 한다. 뿐만 아니라 체외수정에 의한 수정란의 체외 배양 기간을 대폭 단축하고 적은 수의 수정란으로부터도 정상 분만이 가능하고, 체외수정된 수정란과 체세포 복제 수정란 유래의 자돈을 동시에 생산할 수 있다.As described above, according to the present invention, by simultaneously implanting in vitro fertilized embryos and fertilized eggs by somatic cell cloning, normal delivery of cloned pigs with a very low rate of normal delivery can be achieved. In addition, the in vitro culture period of fertilized eggs by in vitro fertilization can be significantly shortened and normal delivery from a small number of fertilized eggs is possible.

서열목록 전자파일 첨부 Attach sequence list electronic file  

Claims (5)

(A) 도축 돼지의 미성숙 난자를 채취하여 배양하는 단계; (A) harvesting and incubating immature eggs of slaughtered pigs; (B) a) (A)단계에서 준비된 성숙난자와 액상정액의 체외수정에 의한 수정란 및 b) (A)단계에서 준비된 성숙난자를 탈핵하고 공여 체세포를 탈핵 난자에 이식하는 것에 의한 체세포 이식 수정란을 준비하여 각각 배양하는 단계; 및(B) a fertilized egg by in vitro fertilization of mature egg and liquid sperm prepared in step (A) and b) somatic cell transplant fertilized egg by denuclearizing mature egg prepared in step (A) and transplanting donor somatic cells into denuclear egg Preparing and culturing each; And (C) (B) 단계에서 준비된 체외수정에 의한 수정란과 체세포 이식에 의한 수정란을 동시에 한 마리의 대리모에 이식하는 단계;(C) implanting the fertilized egg by in vitro fertilization and somatic cell transplantation prepared in step (B) into one surrogate mother at the same time; 를 포함하는 것을 특징으로 하는 돼지의 생산 방법.Piglet production method comprising a. 제 1 항에 있어서,The method of claim 1, (A) 단계의 미성숙 난자의 배양 배지는 4~6mm크기의 난포막을 2~6개 포함하는 것을 특징으로 하는 돼지의 생산 방법.The culture medium of the immature egg of step (A) is a pig production method characterized in that it comprises 2 to 6 follicle membrane of 4 ~ 6mm size. 제 1 항에 있어서,The method of claim 1, (B) 단계의 체외수정에 의한 수정란은 조성물 100ml 수용액 당 락토오스 하이드레이트(lactose hydrate) 10~15g, 난황 15~25ml 및 N-아세틸-D-글루코사민(N- acetyl-D-glucosamine) 0.01~0.1g을 포함하는 4℃ 보존용 인공수정용 돼지정액 희석액을 사용하여 제조된 액상정액을 이용하여 제조된 것임을 특징으로 하는 돼지의 생산 방법.The fertilized egg by in vitro fertilization of step (B) is 10-15 g of lactose hydrate per 15 ml aqueous solution, 15-25 ml of egg yolk and 0.01-0.1 g of N-acetyl-D-glucosamine Pigment production method characterized in that it was prepared using a liquid sperm prepared using a diluent solution for artificial insemination for 4 ℃ preservation. 제 1 항에 있어서, The method of claim 1, 체외수정 수정란 및 체세포 이식 수정란은 16~36시간 각각 체외배양한 후 대리모에 이식하는 것을 특징으로 하는 돼지의 생산 방법.In vitro fertilized fertilized egg and somatic cell transplant The fertilized egg is a production method of pig, characterized in that transplanted in surrogate mothers after in vitro culture for 16 to 36 hours each. 제 1 항에 있어서,The method of claim 1, (C)단계의 이식에 사용되는 수정란의 총 개수는 150~250개인 것을 특징으로 하는 돼지의 생산 방법.(C) The method of producing a pig, characterized in that the total number of fertilized eggs used for transplantation is 150-250.
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