KR20070081531A - Mammary gland-specific human erythropoietin expression vector, transgenic animal and method for producing human erythropoietin using same - Google Patents

Mammary gland-specific human erythropoietin expression vector, transgenic animal and method for producing human erythropoietin using same Download PDF

Info

Publication number
KR20070081531A
KR20070081531A KR1020060013485A KR20060013485A KR20070081531A KR 20070081531 A KR20070081531 A KR 20070081531A KR 1020060013485 A KR1020060013485 A KR 1020060013485A KR 20060013485 A KR20060013485 A KR 20060013485A KR 20070081531 A KR20070081531 A KR 20070081531A
Authority
KR
South Korea
Prior art keywords
hepo
vector
human erythropoietin
epo
pbc1
Prior art date
Application number
KR1020060013485A
Other languages
Korean (ko)
Other versions
KR100769291B1 (en
Inventor
김진회
Original Assignee
조아제약주식회사
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 조아제약주식회사 filed Critical 조아제약주식회사
Priority to KR1020060013485A priority Critical patent/KR100769291B1/en
Publication of KR20070081531A publication Critical patent/KR20070081531A/en
Application granted granted Critical
Publication of KR100769291B1 publication Critical patent/KR100769291B1/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/505—Erythropoietin [EPO]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/20—Animal model comprising regulated expression system
    • A01K2217/206—Animal model comprising tissue-specific expression system, e.g. tissue specific expression of transgene, of Cre recombinase
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/01—Animal expressing industrially exogenous proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/48—Vector systems having a special element relevant for transcription regulating transport or export of RNA, e.g. RRE, PRE, WPRE, CTE

Abstract

Mammary gland-specific human erythropoietin expression vectors, a transgenic animal and a method for producing human erythropoietin by using the same vector and animal are provided to express high concentration of erythropoietin in milk, and improve stability and physiologically activating effects of the produced human erythropoietin. The mammary gland-specific human erythropoietin expression vector, pBC1-hEPO-WPRE, contains a DNA sequence of beta-casein promoter of SEQ ID NO:2 as a mammary gland-specific promoter, a DNA sequence encoding human erythropoietin of SEQ ID NO:1 and WPRE(woodchuck hepatitis virus post-transcriptional regulatory element) DNA sequence of SEQ ID NO:3. The method for producing human erythropoietin comprises the steps of: transforming an animal selected from pig, mouse, cow, goat, sheet and chicken with the mammary gland-specific human erythropoietin expression vector; and isolating and purifying the human erythropoietin expressed in milk produced from the transgenic animal.

Description

유선 특이적 인간 에리트로포이에틴 발현 벡터, 이를 이용한 형질전환 동물 및 이를 이용한 인간 에리트로포이에틴의 생산 방법{Mammary gland-specific human erythropoietin expression vector, transgenic animal and method for producing human erythropoietin using same}Mammary gland-specific human erythropoietin expression vector, transgenic animal and method for producing human erythropoietin using same}

도 1은 본 발명의 pBC1/hEPO/NEO 발현벡터의 구조를 나타낸 도이다.1 is a diagram showing the structure of the pBC1 / hEPO / NEO expression vector of the present invention.

도 2a는 본 발명의 pBC1/hEPO/NEO 발현벡터 구조이고, 도 2b는 이를 확인한 PCR 결과도이다.Figure 2a is a pBC1 / hEPO / NEO expression vector structure of the present invention, Figure 2b is a PCR result confirmed this.

도 3은 실험용 생쥐의 유선, 간, CHO 세포에서 글리코실화에 관여하는 유전자의 발현양상을 분석한 결과이다.Figure 3 is a result of analyzing the expression patterns of genes involved in glycosylation in mammary gland, liver, CHO cells of the experimental mice.

도 4는 본 발명의 hEPO 발현벡터로 형질전환시킨 생쥐에서의 hEPO 발현여부를 관찰한 도이다.4 is a diagram observing the expression of hEPO in the mouse transformed with the hEPO expression vector of the present invention.

도 5는 본 발명의 형질전환 생쥐의 유선 조직에서 hEPO 단백질 발현을 관찰한 결과도이다.5 is a result of observing the expression of hEPO protein in the mammary gland tissue of the transgenic mouse of the present invention.

도 6은 본 발명의 형질전환 생쥐의 유즙에서 생산된 hEPO 단백질 발현을 관찰한 결과도이다.Figure 6 is a result of observing the expression of hEPO protein produced in the milk of the transgenic mice of the present invention.

도 7은 본 발명의 형질전환 생쥐의 유즙에서 생산된 hEPO와 환자의 혈청내 EPO의 2차원 전기영동 젤 사진이다.Figure 7 is a two-dimensional electrophoresis gel photograph of hEPO produced in the milk of the transgenic mice of the present invention and EPO in the serum of the patient.

도 8은 본 발명의 형질전환 생쥐의 유즙에서 생산된 hEPO와 이포에틴알파의 올리고당 분석결과이다.8 is a result of oligosaccharide analysis of hEPO and the epoetin alpha produced in the milk of the transgenic mice of the present invention.

도 9는 본 발명의 형질전환 생쥐의 유즙에서 생산된 hEPO의 in vitro 활성측정결과이다.9 is a result of in vitro activity measurement of hEPO produced in the milk of the transgenic mice of the present invention.

도 10은 본 발명의 형질전환 생쥐의 유즙에서 생산된 hEPO의 in vivo 활성측정결과이다.10 is a result of in vivo activity measurement of hEPO produced in the milk of the transgenic mice of the present invention.

본 발명은 유선 특이적 인간 에리트로포이에틴(hEPO) 발현 벡터, 이를 이용한 형질전환 동물 및 이를 이용한 EPO의 생산 방법에 관한 것이다.The present invention relates to a mammary gland specific human erythropoietin (hEPO) expression vector, a transgenic animal using the same, and a method for producing EPO using the same.

에리트로포이에틴(erythropoietin, EPO)은 태아의 간이나 성인의 신장에서 주로 생성되는 당단백질로 적혈구 생성(erythropoiesis)을 촉진하고, 적혈구 수를 유지하는 데에 중요한 역할을 하는 조혈 호르몬이다.Erythropoietin (erythropoietin, EPO) is a glycoprotein mainly produced in the liver of the fetus and the kidneys of adults is a hematopoietic hormone that plays an important role in promoting erythropoiesis and maintaining the number of red blood cells.

천연형 인간 EPO는 분자량이 약 34~38kd 이고, 24, 38, 83번 아미노산에 N-linked 당쇄첨가부위를 가지고 있으며, 또한 126번 아미노산에 O-linked 당쇄첨가결합부위를 가지고 있는데, 이 당쇄 부분은 EPO의 생체 내 활성에 필수적인 역할을 수행한다.Natural human EPO has a molecular weight of about 34 to 38 kd, has an N-linked sugar chain at amino acids 24, 38, and 83, and an O-linked sugar chain at the amino acid 126. Plays an essential role in the in vivo activity of EPO.

EPO는 적혈구 형성에 필수적이이 때문에 빈혈, 특히 신장성 빈혈 치료에 유효하며, HIV-감염환자의 AZT 치료와 관련된 빈혈, 화학치료중인 비-골수암 환자의 빈혈 등의 치료에 EPO를 이용할 수 있다.Because EPO is essential for red blood cell formation, it is effective for treating anemia, especially renal anemia, and EPO can be used for the treatment of anemia associated with AZT treatment of HIV-infected patients and anemia in non-myeloid cancer patients undergoing chemotherapy.

이러한 의약 분야에서 경제적 부가가치가 높은 EPO 등의 생산을 극대화하는 방법으로서, 세포배양법에 의한 대량생산방법이 주로 사용되어 왔다. 그러나, 이 방법은 동물의 혈액을 배양 배지로 이용하기 때문에 생산 비용이 높아지고, 배양기술에 있어서 전문적인 지식이 요구된다. 또한 배지 성분에 함유된 동물의 EPO와 새로 생산한 EPO를 완전히 분리하는 것이 불가능하기 때문에 최종적으로 얻는 EPO의 순도가 낮고, 현재까지 동물의 세포에서 생산한 재조합 인간 EPO의 경우는 상기한 천연형 EPO 단백질과 글리코실화(glycosylation)의 상태가 동일하지 않아 생리활성도 낮은 문제점을 가지고 있다.As a method of maximizing the production of EPO and the like having high economic added value in the pharmaceutical field, a mass production method by cell culture has been mainly used. However, since this method uses animal blood as a culture medium, production costs are high, and expertise in culture technology is required. In addition, since it is impossible to completely separate the animal EPO and the newly produced EPO contained in the medium component, the purity of the finally obtained EPO is low, and in the case of the recombinant human EPO produced from animal cells to date, the above-described natural type EPO Protein and glycosylation is not the same state has a low physiological activity.

반면 형질전환동물을 이용한 유용단백질 생산 방법은, 동물이 분비하는 체액 중에 목적단백질이 포함되므로 기존의 세포배양법에 비해 목적단백질의 분리ㆍ정제가 용이하며, 활성 또한 우수하게 유지되므로 이 분야에 대한 관심이 급증하고 있다. On the other hand, the useful protein production method using a transgenic animal contains the target protein in the body fluids secreted by the animal, which makes it easier to separate and purify the target protein and to maintain excellent activity compared to the conventional cell culture method. This is soaring.

현재까지의 형질전환동물 생산기술에서 목적 단백질을 생산하는 조직은 주로 단백질 발현율이 높은 것으로 알려진 유선 조직이었다. 그러나 지금까지 개발된 유선 특이적 프로모터를 이용하여 형질전환된 동물의 단백질 생산 효율은 실제로 매우 낮은 수준에 머무르고 있으며, 또한, 기존에 보고된 유선 특이적 벡터의 경우 정규 장소 밖의(ectopic) 발현을 보이는 등 여러 가지 문제점이 있다.In the transgenic animal production technology to date, the tissue producing the target protein was mainly a mammary tissue known to have high protein expression rate. However, the protein production efficiency of transgenic animals using the mammary gland specific promoters developed so far remains very low, and the previously reported mammary gland specific vector shows ectopic expression. There are several problems.

국내공개특허 특2001-81456호는 생쥐의 유선으로부터 분리한 WAP 프로모터 및 인간의 EPO 게놈유전자를 포함하는 EPO 형질전환 발현 벡터 및 그 형질전환 돼 지에 대하여 개시하고 있다.  Korean Patent Laid-Open No. 2001-81456 discloses an EPO transgenic expression vector comprising a WAP promoter and a human EPO genomic gene isolated from a mouse mammary gland and a transgenic pig.

또한, 국내공개특허 제10-2004-101793호는 소의 베타-카제인 프로모터를 사용하여 유선에서 사람 에리트로포이에틴(hEPO)을 발현하는 형질전환 복제소에 대하여 개시하고 있다. 그러나, hEPO의 유즙 내 농도 및 생산된 EPO의 활성에 대한 결과는 전혀 없으므로, 이러한 방법으로 생산된 hEPO가 실용화될 수 있는지 전혀 알 수 없다. In addition, Korean Patent Publication No. 10-2004-101793 discloses a transgenic clone that expresses human erythropoietin (hEPO) in the mammary gland using a bovine beta-casein promoter. However, since there is no result in the milk concentration of hEPO and the activity of the produced EPO, it is not known whether hEPO produced by this method can be put to practical use.

이에 본 발명자는 유선에서만 특이적으로 인간 EPO를 높은 효율로 생산하는 발현벡터를 제작하고, 이를 도입한 형질전환동물이 높은 생리활성을 갖는 인간 EPO를 생산함을 확인함으로써 본 발명을 완성하였다.In this regard, the present inventors completed the present invention by making an expression vector that produces human EPO with high efficiency only in the mammary gland, and confirming that the transgenic animal introduced therein produces human EPO having high physiological activity.

본 발명은 유선 특이적으로 인간 에리트로포이에틴(hEPO) 단백질을 발현하는 벡터를 제공하고자 한다.The present invention seeks to provide a vector expressing a human erythropoietin (hEPO) protein specifically mammary gland.

또한, 본 발명은 상기 벡터를 도입한 형질전환 동물을 제공하고자 한다.In addition, the present invention is to provide a transgenic animal introduced with the vector.

또한, 본 발명은 상기 벡터를 이용한 형질전환 동물 생산방법을 제공하고자 한다.In addition, the present invention is to provide a method for producing a transgenic animal using the vector.

또한, 본 발명은 상기 형질전환 동물을 이용한 인간 에리트로포이에틴을 생산하는 방법을 제공하고자 한다.In addition, the present invention is to provide a method for producing human erythropoietin using the transgenic animal.

본 발명은 유선 특이적으로 인간 에리트로포이에틴(hEPO) 단백질을 발현하는 발현벡터를 제공한다.The present invention provides an expression vector expressing a human erythropoietin (hEPO) protein specifically mammary gland.

본 발명은 유선 특이적 프로모터인 베타-카제인 프로모터 염기서열, 그 3' 쪽으로 인간 에리트로포이에틴(hEPO)을 코딩하는 DNA 서열 및 hEPO 유전자의 3'쪽으로 WPRE DNA 서열을 포함하는 pBC1-hEPO-WPRE 벡터를 제공한다.The present invention provides a pBC1-hEPO-WPRE vector comprising a beta-casein promoter sequence, a mammary gland specific promoter, a DNA sequence encoding human erythropoietin (hEPO) towards its 3 'and a WPRE DNA sequence toward the 3' of the hEPO gene. To provide.

본 발명의 pBC1-hEPO-WPRE 벡터에서, 상기 hEPO을 코딩하는 DNA 서열은 서열번호 1 에 기재된 것이 바람직하고, 상기 베타-카제인 프로모터 DNA 서열은 서열번호 2 에 기재된 것이 바람직하며, 상기 WPRE DNA 서열은 서열번호 3 에 기재된 것이 바람직하다.In the pBC1-hEPO-WPRE vector of the present invention, the DNA sequence encoding the hEPO is preferably described in SEQ ID NO: 1, the beta-casein promoter DNA sequence is preferably described in SEQ ID NO: 2, and the WPRE DNA sequence is Preference is given to SEQ ID NO: 3.

구체적으로, 본 발명에서는 염소의 베타-카제인 프로모터를 포함하는 pBC1 발현벡터에 서열번호 1의 인간 EPO 유전자를 삽입시켜, EPO의 유선 특이적 발현을 가능하게 하는 발현 벡터 pBC1-hEPO를 제공한다. Specifically, the present invention provides an expression vector pBC1-hEPO that enables the mammary gland specific expression of EPO by inserting the human EPO gene of SEQ ID NO: 1 into a pBC1 expression vector comprising a beta-casein promoter of goat.

본 발명의 발현 벡터 pBC1-hEPO는 필요에 따라 인슐레이터(insulator), 또는 WPRE(woodchuck hepatitus virus post-transcriptional regulatory element), 네오마이신 저항성 유전자(neomycin-resistant gene) 등을 추가로 포함함으로써, 형질전환 세포주의 구축을 용이하게 하며, 목적 단백질 발현량의 극대화 및 발현의 안정성을 도모할 수 있다. The expression vector pBC1-hEPO of the present invention further comprises an insulator, a woodchuck hepatitus virus post-transcriptional regulatory element (WPRE), a neomycin-resistant gene, and the like, as necessary, thereby transforming the cell line. It is possible to facilitate the construction of and to maximize the target protein expression amount and to stabilize the expression.

인슐레이터는 프로모터 부근에 존재하는 조절인자의 영향을 돕고, 위치 비의존적인 (position-independent) 발현을 도와주는 인자로, 베타-카제인 프로모터의 조절 하에 단백질을 안정적으로 발현할 수 있게 한다. Insulators help to influence the regulators present in the vicinity of the promoter and position-independent expression, which allows for stable expression of the protein under the control of the beta-casein promoter.

WPRE는 mRNA의 안정화에 기여하여 단백질 합성량을 증대시킬 수 있는 조절인자로, 베타-카제인 프로모터의 조절 하에 단백질을 대량으로 발현할 수 있게 한다. WPRE의 염기서열은 서열번호 3에 기재된 바와 같다.WPRE is a regulator that can contribute to the stabilization of mRNA to increase the amount of protein synthesis, and allows for the expression of large amounts of protein under the control of the beta-casein promoter. The base sequence of the WPRE is as described in SEQ ID NO: 3.

네오마이신 저항성 유전자는 세포주 구축시 사용되는 G418 시약에 대해 저항성을 나타내는 유전자로, 베타-카제인 프로모터의 조절 하에 단백질을 발현하는 동물세포주(animal cell line) 구축시 효율적인 선택적 표지 유전자로서 작용할 수 있다. 네오마이신 저항성 유전자의 염기서열은 서열번호 4에 기재된 바와 같다.Neomycin resistance gene is a gene that is resistant to the G418 reagent used in cell line construction, and can act as an efficient selective marker gene when constructing an animal cell line expressing a protein under the control of a beta-casein promoter. The base sequence of the neomycin resistance gene is as described in SEQ ID NO: 4.

본 발명은 상기 조절인자들을 추가로 포함하는 발현 벡터의 바람직한 예로, WPRE를 포함하는 pBC1-hEPO-WPRE 벡터 및 상기 pBC1-hEPO-WPRE 벡터에 네오마이신 저항성 유전자를 추가로 포함하는 pBC1/hEPO/NEO 벡터를 제공한다.The present invention is a preferred example of the expression vector further comprising the regulator, pBC1-hEPO-WPRE vector containing WPRE and pBC1 / hEPO / NEO further comprising a neomycin resistance gene in the pBC1-hEPO-WPRE vector Provide a vector.

상기 벡터들은 본 발명의 pBC1-hEPO 벡터 내 EPO 유전자의 3' 쪽에 WPRE를 삽입하거나, 여기에 다시 네오마이신 저항성 유전자를 삽입함으로써 제조된다.The vectors are prepared by inserting the WPRE into the 3 'side of the EPO gene in the pBC1-hEPO vector of the present invention, or by inserting the neomycin resistance gene again.

이러한 본 발명의 발현 벡터 pBC1/hEPO/NEO는 2005년 12월 2일 한국 농업미생물자원센터 유전자은행에 기탁번호 KACC 95043P로 기탁하였다.The expression vector pBC1 / hEPO / NEO of the present invention was deposited on Dec. 2, 2005 with the accession number KACC 95043P to the Genetic Bank of Korea Microbial Resources Center.

또한, 본 발명의 발현벡터는 필요에 따라 조절인자들, 즉 또 다른 프로모터, 인핸서(enhancer), 선택적 표지 유전자(selective marker), 5'-UTR(untranslated region), 3'-UTR, 폴리아데닐화 신호(polyadenylation signal), 리보솜 결합 서열, 게놈의 특정 부위로 삽입될 수 있는 염기서열, 또는 인트론을 적절한 위치에 추가로 포함할 수 있다.In addition, the expression vector of the present invention, if necessary, regulators, that is, another promoter, enhancer, selective marker, 5'-UTR (untranslated region), 3'-UTR, polyadenylation A polyadenylation signal, a ribosome binding sequence, a base sequence that can be inserted into a specific site of the genome, or an intron may be further included in an appropriate position.

본 발명은 또한, 상기 발현 벡터를 이용하여 형질전환시킨 체세포를 제공한다.The present invention also provides a somatic cell transformed using the expression vector.

본 발명은 또한, 상기 발현 벡터를 이용하여 형질전환시킨 체세포를 탈핵된 난자에 핵이식하여 만든 수정란을 제공한다.The present invention also provides a fertilized egg made by nuclear transfer of denuclearized egg cells somatic cells transformed using the expression vector.

본 발명은 또한, 상기 발현 벡터를 이용하여 형질전환시킨 동물을 제공한다.The present invention also provides an animal transformed using the expression vector.

본 발명의 발현 벡터로 형질전환될 수 있는 동물은 유즙을 분비하는 모든 동물, 즉 돼지, 생쥐, 소, 염소, 양 또는 닭 등을 포함한다.Animals that can be transformed with the expression vectors of the invention include all animals that secrete milk, ie pigs, mice, cows, goats, sheep or chickens.

본 발명의 발현 벡터를 이용한 형질전환동물의 생산 방법은 통상적인 방법에 의한다.The production method of the transgenic animal using the expression vector of the present invention is by a conventional method.

형질전환하고자 하는 동물이 생쥐일 경우, 건강한 개체로부터 수정란을 채취하고, 수정란에 본 발명의 발현 벡터를 도입한 후, 정관결찰 생쥐를 이용하여 위임신 생쥐를 얻고, 이를 대리모로 하여 난관 내에 수정란을 이식한 후, 대리모로부터 얻은 자손 중 형질전환된 개체를 선별하는 과정으로 이루어진다.When the animal to be transformed is a mouse, a fertilized egg is collected from a healthy individual, the expression vector of the present invention is introduced into the fertilized egg, and a delegation mouse is obtained using a vas defermented mouse, and the fertilized egg is placed in the fallopian tube as a surrogate mother. After transplantation, the process consists in selecting transformed individuals from offspring obtained from surrogate mothers.

형질전환하고자 하는 동물이 돼지일 경우, 먼저 건강한 개체로부터 난포란을 채취하고 체외성숙용 배양액에서 배양한다. 또한, 돼지 태아로부터 채취, 배양한 공여체세포로 본 발명의 발현 벡터를 도입한 후 벡터가 도입된 체세포를 선별, 배양한다. 상기 체외성숙된 난자에서 핵을 제거하고, 이 공간에 공여세포를 주입한 다음, 전기융합을 통해 핵이식이 완료된 난자의 공여세포와 세포질을 융합시킨 후 체외배양한다. 이 복제수정란을 과배란을 유도한 수란돈에 이식한 후, 수란돈으로부터 얻은 자손 중 형질전환된 개체를 선별하는 과정으로 이루어진다.When the animal to be transformed is a pig, first, follicular eggs are collected from healthy individuals and cultured in in vitro mature culture. In addition, after introducing the expression vector of the present invention into donor cells collected and cultured from pig embryos, somatic cells into which the vector is introduced are selected and cultured. The nucleus is removed from the in vitro mature egg, and the donor cells are injected into this space, followed by fusion of the donor cell and the cytoplasm of the nuclear transplantation completed through electrofusion, followed by in vitro culture. The cloned embryos are transplanted into over-ovulation-induced poached pigs, followed by screening for transformed individuals from offspring obtained from poached pigs.

이후 형질전환된 것으로 확인된 개체로부터 유즙을 수집한 후, 목적 단백질을 분리ㆍ정제함으로써 유용 단백질을 생산하게 된다(A. Gokana, J.J. Winchenn , A. Ben-Ghanem, A. Ahaded, J.P. Cartron, P. Lambin(1997) Chromatographic separation of recombinant human erythropoietin isoforms, Journal of chromatography, 791, 109-118).After the milk is collected from the individuals identified as transformed, useful proteins are produced by isolating and purifying the desired protein (A. Gokana, JJ Winchenn, A. Ben-Ghanem, A. Ahaded, JP Cartron, P). Lambin (1997) Chromatographic separation of recombinant human erythropoietin isoforms, Journal of chromatography, 791, 109-118).

또한, 본 발명은 형질전환동물로부터 생산된 유즙 중 발현된 에리트로포이에틴을 분리, 정제하는 방법으로 이루어진 인간 에리트로포이에틴의 생산방법을 제공한다.The present invention also provides a method for producing human erythropoietin consisting of a method for isolating and purifying erythropoietin expressed in milk produced from a transgenic animal.

본 발명의 인간 에리트로포이에틴 생산방법에서, 분리ㆍ정제 방법은 통상적으로 사용되는 방법을 사용할 수 있으며, 구체적으로는 여과법 또는 크로마토그래피법 등이 될 수 있다.In the method for producing human erythropoietin of the present invention, a separation and purification method may be a conventionally used method, and specifically, may be a filtration method or a chromatography method.

이렇게 하여 제조되는 본 발명의 형질전환동물은 유선 특이적으로 에리트로포이에틴을 발현하며, 기존의 방법에 비해 매우 높은 농도로 유즙 중에 에리트로포이에틴을 발현한다. 또한 특이적으로 수유기 동안의 알비올라 세포에서만 EPO 단백질을 발현한다.The transgenic animal of the present invention thus produced expresses erythropoietin specifically in mammary gland, and expresses erythropoietin in milk at a much higher concentration than conventional methods. It also specifically expresses EPO protein only in alviola cells during lactation.

그 예로, 본 발명의 발현 벡터 pBC1/hEPO/NEO로 형질전환된 생쥐의 경우, 200,000 IU~ 400,000 IU/㎖ 수준의 높은 EPO 발현율을 나타낸다. 원래 EPO는 태아의 조기 사망을 유발하기 때문에 발현시키기 어려운 단백질임에도 불구하고, 본원발명의 형질전환 동물에서는 기존의 유선 특이적 프로모터를 이용하여 제조된 형질전환동물의 유즙 내 단백질 발현율에 비해 1000배 이상의 높은 발현율을 나타낸다. For example, mice transformed with the expression vector pBC1 / hEPO / NEO of the present invention exhibit high EPO expression rates of 200,000 IU to 400,000 IU / ml. Although the original EPO is a protein that is difficult to express because it causes premature death of the fetus, the transgenic animal of the present invention is 1000 times higher than the protein expression rate in the milk of the transgenic animal prepared using the existing mammary gland specific promoter. High expression rate.

또한 본 발명의 형질전환동물로부터 생산된 EPO 단백질은 시판되는 동종 단 백질이 나타내는 것 이상의 안정성 및 우수한 생리활성을 나타낸다.In addition, the EPO protein produced from the transgenic animal of the present invention exhibits more stability and superior physiological activity than that of commercially available homologous proteins.

그 예로, 본 발명의 발현 벡터 pBC1/hEPO/NEO로 형질전환된 생쥐로부터 얻은 EPO는, 시알릭산(sialic acid)을 많이 함유하고 있어 RBC 전구세포에 작용하여 우수한 단백질 활성을 나타낸다. For example, EPO obtained from a mouse transformed with the expression vector pBC1 / hEPO / NEO of the present invention contains sialic acid, and thus acts on RBC progenitor cells and shows excellent protein activity.

또한, 본 발명의 형질전환동물로부터 생산된 EPO 단백질을 투여시 혈액 내 혈소판, 적혈구, 혈색소, 헤마토크릿(hematocrit)을 증가시키는 작용효과를 나타낸다. In addition, the administration of EPO protein produced from the transgenic animal of the present invention shows an effect of increasing the platelets, red blood cells, hemoglobin, hematocrit in the blood (hematocrit).

따라서 본 발명의 EPO 발현 벡터 및 형질전환동물은 기존의 EPO 보다 우수한 생리활성을 갖는 EPO 단백질을 생산하는데 유용하게 사용될 수 있다.Therefore, the EPO expression vector and the transgenic animal of the present invention can be usefully used to produce an EPO protein having superior physiological activity than conventional EPO.

이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the examples.

[실시예 1] 본 발명의 최적의 유선특이적 인간 에리트로포이에틴(hEPO) 발현 벡터 제작Example 1 Construction of Optimal Mammary Specific Human Erythropoietin (hEPO) Expression Vectors of the Present Invention

본 발명은 유선(mammary gland)에서 hEPO를 분비하는 최적 벡터를 구축하였다.The present invention constructed an optimal vector that secretes hEPO in the mammary gland.

1) pBC1-hEPO 벡터 구축1) pBC1-hEPO Vector Construction

본 발명의 유선 특이적 발현벡터를 제작하기 위해, 염소의 베타-카제인 프로 모터(goat beta-casein promoter)를 갖고 있는 Invitrogen사의 pBC1을 사용하여, 이의 제한효소 XhoI 위치에 인간 에리트로포이에틴 genomic DNA (서열번호 1)을 클로닝하였다.To prepare a mammary gland specific expression vector of the present invention, human erythropoietin genomic DNA at its restriction enzyme XhoI site using pBC1 of Invitrogen having a goat beta-casein promoter SEQ ID NO: 1) was cloned.

2) pBC1-hEPO-WPRE 벡터 구축2) pBC1-hEPO-WPRE Vector Construction

hEPO의 발현량을 증가시키기 위하여, 상기 pBC1-hEPO 벡터에 WPRE 유전자를 삽입하였다.In order to increase the expression level of hEPO, the WPRE gene was inserted into the pBC1-hEPO vector.

WPRE(woodchuck hepatitis virus post-transciptional regulatory element)는 mRNA의 안정화에 기여하여, 최종 단백질 합성을 증대시키는데 관여한다고 보고된 조절인자이다.WPRE (woodchuck hepatitis virus post-transciptional regulatory element) is a regulator that has been reported to contribute to the stabilization of mRNA and to enhance final protein synthesis.

이 WPRE 조절인자를 hEPO 바로 뒤에 연결하여 EPO가 전사될 때 함께 mRNA가 만들어지도록 하기 위하여, 상기 1)의 pBC1-hEPO 벡터에서 XhoI 제한효소 위치에 삽입하였다. This WPRE modulator was inserted at the XhoI restriction site in the pBC1-hEPO vector of 1) above, in order to connect immediately after hEPO to produce mRNA together when EPO is transcribed.

WPRE는 Hepatitis virus 로부터 정방향 프라이머(forward primer) 5'-ACCAGGTTCTGTTCCTGTTAATCAACCTC-3'(서열번호 5)과 역방향 프라이머(reverse primer) 5'-CTCGAGGAGCCCGAGGCGAAACAGGCG-3' (서열번호 6)을 이용하여 0.6 kb 길이의 PCR 산물을 증폭하였고 이를 pGEM T-easy 벡터에 클로닝하였다. T-벡터에 클로닝된 0.6 kb WPRE를 SalI 및 XhoI 제한효소로 절단하여 삽입조각(insert) 부위를 준비하였고, pBC1-hEPO을 Xho1 제한효소로 절단하여 준비한 벡터와 라이게이션(ligation) 하여 23975 bp인 pBC1-hEPO-WPRE 벡터를 구축하였다. WPRE was 0.6 kb in length from Hepatitis virus using forward primer 5'-ACCAGGTTCTGTTCCTGTTAATCAACCTC-3 '(SEQ ID NO: 5) and reverse primer 5'-CTCGAGGAGCCCGAGGCGAAACAGGCG-3' (SEQ ID NO: 6). The product was amplified and cloned into pGEM T-easy vector. 0.6 kb WPRE cloned into T-vector was digested with SalI and XhoI restriction enzyme to prepare an insertion site, and ligation was performed with the vector prepared by cleaving pBC1-hEPO with Xho1 restriction enzyme to 23975 bp. pBC1-hEPO-WPRE vectors were constructed.

3) pBC1/hEPO/NEO 벡터 구축3) pBC1 / hEPO / NEO Vector Construction

유선 특이적인 베타 카제인 프로모터의 조절 하에 hEPO 생산을 극대화할 수 있는 벡터만을 가진 세포들을 효과적으로 선택하기 위해, neo 유전자(neomycin-resistance gene)를 클로닝하였다. In order to effectively select cells with only vectors capable of maximizing hEPO production under the control of the mammary gland specific beta casein promoter, the neogene (neomycin-resistance gene) was cloned.

G418 drug에 저항할 수 있는 neo' 유전자는 pEGFP-N1 벡터(Clontech, Catalog #6085-1)로부터 정방향 프라이머 5'-GCGGCCGCGCGCGTCAGGTGGCAC-3'(서열번호 7)와 역방향 프라이머인 5'-CGATCGGACGCTCAGTGGAACGAAAACTC-3'(서열번호 8)을 이용하여 1.9 kb PCR 산물을 증폭하여 수득하였으며, 이 후 pGEM T-easy 벡터에 클로닝하였다. Neo 'genes that can resist G418 drug are forward primer 5'-GCGGCCGCGCGCGTCAGGTGGCAC-3' (SEQ ID NO: 7) from the pEGFP-N1 vector (Clontech, Catalog # 6085-1) and reverse primer 5'-CGATCGGACGCTCAGTGGAACGAAAACTC-3 ' (SEQ ID NO: 8) was used to amplify the 1.9 kb PCR product, which was then cloned into the pGEM T-easy vector.

T-벡터에 클로닝된 1.9 kb neo 유전자는 NotI과 PvuI 제한효소로 절단하여 삽입 부위를 준비하였다. 또한, 상기 2)의 pBC1-hEPO-WPRE 벡터의 amp' 유전자 (ampicillin-resistance gene) 부위를 NotI과 PvuI 제한효소로 절단하여 제거한 후 벡터 부위를 준비하였다. The 1.9 kb neo gene cloned into the T-vector was digested with NotI and PvuI restriction enzymes to prepare the insertion site. In addition, the amp 'gene (ampicillin-resistance gene) region of pBC1-hEPO-WPRE vector of 2) was removed by cleavage with NotI and PvuI restriction enzymes, and then a vector region was prepared.

이렇게 준비된 두 삽입조각 및 벡터 부위를 라이게이션을 통해, 기존에 구축된 pBC1-hEPO 벡터에 WPRE 조절인자와 neo 유전자(neomycin-resistance gene)가 삽입된 pBC1/hEPO/NEO 벡터를 구축하였다. The two fragments and vector sites thus prepared were ligated to construct a pBC1 / hEPO / NEO vector in which a WPRE regulator and neo gene (neomycin-resistance gene) were inserted into the previously constructed pBC1-hEPO vector.

이 결과 얻은 본 발명의 발현벡터의 구조는 도 1에 나타나 있으며, 유선 특이적 프로모터인 베타-카제인 프로모터의 조절 하에서 hEPO를 발현하게 된다. As a result, the structure of the expression vector of the present invention is shown in Figure 1, and expresses hEPO under the control of the beta-casein promoter, a mammary gland specific promoter.

이를 pBC1-5' + WPRE-R 프라이머쌍 및 hEPO3+WPRE-R 프라이머쌍을 이용한 PCR을 통해 3kb 및 1.5 kb의 PCR 산물을 얻음으로써 pBC1/hEPO/NEO 벡터 제작이 제대로 되었음을 확인하였다(도 2a 및 2b).The PCR product of pBC1-5 '+ WPRE-R primer pair and hEPO3 + WPRE-R primer pair was obtained to obtain 3 kb and 1.5 kb PCR products. 2b).

이러한 본 발명의 발현 벡터 pBC1/hEPO/NEO는 2005년 12월 2일 농업생명공학연구원에 기탁번호 KACC 95043P로 기탁하였다.The expression vector pBC1 / hEPO / NEO of the present invention was deposited on December 2, 2005, Agricultural Research Institute of Agriculture and Biotechnology under the accession number KACC 95043P.

pBC1/hEPO/NEO는 WPRE 조절인자의 도입으로 EPO 단백질 합성이 매우 증가되고, 또한 neo' 유전자의 삽입은 세포주 구축에 매우 효율적인 선택적 표시 유전자로 활용된다. pBC1 / hEPO / NEO significantly increases EPO protein synthesis by the introduction of WPRE modulators, and neo 'gene insertion is used as a selective marker gene that is highly efficient for cell line construction.

[실시예 2] hEPO 발현 형질 전환 동물 제작 및 발현된 hEPO 단백질 분석Example 2 Preparation of hEPO Expressed Transgenic Animal and Analysis of Expressed hEPO Protein

본 발명의 유선 특이적 hEPO 발현 형질전환 생쥐의 생산에 의한 생리활성 확인 및 안정성 평가를 위해 하기와 같은 실험을 수행하였다.The following experiments were performed to confirm the physiological activity and stability evaluation by the production of mammary gland specific hEPO expressing transgenic mice of the present invention.

1) 생쥐의 유선에서 글리코실화에 관여하는 유전자의 발현양상 분석 1) Analysis of expression patterns of genes involved in glycosylation in the mammary gland of mice

단백질의 글리코실화(glycosylation)는 단백질의 번역 후 변형(post-translational modification)의 한 과정으로, 단백질의 기능을 강화하고 결정하는데 중요한 역할을 하는 것으로 알려져 있다. 이러한 글리코실화는 대장균을 포함한 원핵생물, 효모, 동물세포 또는 생쥐 등의 실험동물에서 단백질 발현시 각각 다른 양상으로 나타난다. 이 중 인간과 진화단계가 비슷한 실험동물에게서 생산된 단백질의 글리코실화정도가 인간 생체 내에서 생산된 단백질의 글리코실화와 유사하다고 알려져 있다.Glycosylation of proteins is a process of post-translational modification of proteins and is known to play an important role in enhancing and determining the function of proteins. Such glycosylation appears in different ways when protein expression in experimental animals such as prokaryotes, yeasts, animal cells or mice, including E. coli. Among them, glycosylation of proteins produced in experimental animals with similar evolutionary stages to humans is known to be similar to glycosylation of proteins produced in humans.

따라서 In vivo에서 재조합된 인간 EPO 단백질의 글리코실화 관여하는 글리코실 트랜스퍼라제(glycosyltransferase)의 발현을 생쥐의 유선, 간 및 EPO의 생산에 유용하게 이용되고 있는 CHO 세포주에서 확인하였다.Therefore, expression of glycosyltransferase involved in glycosylation of recombinant human EPO protein in vivo was confirmed in CHO cell line which is useful for the production of mammary gland, liver and EPO.

먼저, 생쥐의 유선과 간 그리고 CHO 세포주로부터 전체 RNA를 분리한 후, 3종류의 글리코실 트랜스퍼라제(GnT-V, GnT-III 와 Fuc-TIV)의 프라이머쌍(표 1)을 이용하여 RT-PCR을 수행하였다.First, the whole RNA was isolated from mouse mammary gland, liver and CHO cell line, and then RT- using three primer pairs of glycosyl transferase (GnT-V, GnT-III and Fuc-TIV) (Table 1). PCR was performed.

프라이머 서열Primer sequence 서열번호SEQ ID NO: mouse GnT-V Forward primermouse GnT-V Forward primer 5'-CACTGTTAATTCGCCCACCT-3'5'-CACTGTTAATTCGCCCACCT-3 ' 99 mouse GnT-V Reverse primermouse GnT-V Reverse primer 5'-GCTTGGTCCTCCTGACTCTG-3'5'-GCTTGGTCCTCCTGACTCTG-3 ' 1010 CHO GnT-V Forward primer CHO GnT-V Forward primer 5'-GTACAGAGTGACCTGCCAAA-3'5'-GTACAGAGTGACCTGCCAAA-3 ' 1111 CHO GnT-V Reverse primer CHO GnT-V Reverse primer 5'-GTCTTTGCATAGGGCCACTT-3' 5'-GTCTTTGCATAGGGCCACTT-3 ' 1212 mouse GnT-III Forward primer mouse GnT-III Forward primer 5'-GCTCAGGCCTCTAGTAATCT-3'5'-GCTCAGGCCTCTAGTAATCT-3 ' 1313 mouse GnT-III Reverse primer mouse GnT-III Reverse primer 5'-TCCTGACCCCTAACCTACTC-3' 5'-TCCTGACCCCTAACCTACTC-3 ' 1414 CHO GnT-III Forward primer CHO GnT-III Forward primer 5'-GCATCTACTTCAARCTCGTG-3' 5'-GCATCTACTTCAARCTCGTG-3 ' 1515 CHO GnT-III Reverse primer CHO GnT-III Reverse primer 5'-GTGCTCRTGGGCTCCCGGTA-3'5'-GTGCTCRTGGGCTCCCGGTA-3 ' 1616 mouse & CHO Fuc-TIV Forward primer mouse & CHO Fuc-TIV Forward primer 5'-CACACDGTGGCCCGCTACAA-3'5'-CACACDGTGGCCCGCTACAA-3 ' 1717 mouse & CHO Fuc-TIV Reverase primermouse & CHO Fuc-TIV Reverase primer 5'-TCCCAGAARGARGTGATGTG-3'5'-TCCCAGAARGARGTGATGTG-3 ' 1818

그 결과, 도 3에서 보는 바와 같이, 세 종류의 글리코실 트랜스퍼라제(GnT-III, GnT-V, Fuc-TIV)는 간이나 CHO 세포주보다 유선(M.G.)에서 상대적으로 높게 발현됨을 알 수 있었다.As a result, as shown in Figure 3, three types of glycosyl transferase (GnT-III, GnT-V, Fuc-TIV) was found to be expressed higher in the mammary gland (M.G.) than the liver or CHO cell line.

따라서 유선 유래 재조합된 인간 EPO는 현재 CHO 세포주에서 생산되어 시판되고 있는 EPO와 비교시 단백질의 글리코실화가 탁월하여, 단백질의 생리활성 뿐만 아니라 질이 구별됨을 확인할 수 있다.Therefore, mammary gland-derived recombinant human EPO is superior in glycosylation of proteins as compared to EPO currently produced and marketed in CHO cell lines, and it can be confirmed that the quality as well as the physiological activity of proteins are distinguished.

2) 형질 전환 생쥐의 생산2) Production of Transgenic Mice

상기 실시예 1에서 구축한 유선 특이적인 베타-카제인 프로모터의 조절 하에 EPO를 발현하는 23kb pBC1/hEPO/NEO 발현벡터(도 1)를 사용하여 현미미세주입에 의하여 형질전환 생쥐를 제작하였다(마크로젠에서 제작). Transgenic mice were prepared by microscopic injection using a 23 kb pBC1 / hEPO / NEO expression vector (FIG. 1) expressing EPO under control of the mammary gland specific beta-casein promoter constructed in Example 1 (in macrogen). making).

형질전환 생쥐는 BDF1(C587BL/6 DBA)을 사용하였고, 738개의 수정란에 pBC1/hEPO/NEO를 주입하고, 이 중 700 개의 수정란을 30마리의 대리모에게 이식하였다. 몇 주 후 태어난 총 85마리의 산자 중 9 마리가 형질전환되었음을 PCR과 Genomic Southern blot 결과를 통해 확인하였다(표 2 및 도 4B). The transgenic mice used BDF1 (C587BL / 6 DBA), and 738 fertilized eggs were injected with pBC1 / hEPO / NEO, 700 of which were implanted into 30 surrogate mothers. Nine out of a total of 85 live births after several weeks were confirmed by PCR and Genomic Southern blot results (Table 2 and Figure 4B).

PCR에 사용된 프라이머는 TG check용 정방향 프라이머 5'-CTCCTTGGCAGAAGGAAGCC-3'(서열번호 19)과 TG check용 역방향 프라이머 5'-CAGCCATGGAAAGGACGTCA-3'(서열번호 20)이며 이의 PCR 예상 산물 크기는 600 bp 이었다. The primers used for PCR were forward primer 5'-CTCCTTGGCAGAAGGAAGCC-3 '(SEQ ID NO: 19) for TG check and reverse primer 5'-CAGCCATGGAAAGGACGTCA-3' (SEQ ID NO: 20) for TG check, and its expected PCR product size was 600 bp. It was.

서던 블롯에 사용한 프로브는 hEPO genomic DNA로 서열번호 1의 서열을 포함하는 2.3kb 전체 유전자를 사용하였다.The probe used in the Southern blot used hEPO genomic DNA, which used the entire 2.3 kb gene containing the sequence of SEQ ID NO: 1.

도 4b는 라인 6 및 라인 37에서 확인된 EPO 유전자 PCR 및 서던블롯 결과이다. 도 4b는 유선 특이적으로 EPO를 발현하는 형질전환 생쥐 중 대표적인 두 라인을 선택하여 분석한 결과 염색체내 10-30 copy 정도 삽입되었으며 생식세포를 통해 다음 세대까지 EPO 유전자가 전달됨을 확인하였다.4B is EPO gene PCR and Southern blot results identified at lines 6 and 37. FIG. 4b shows that two representative lines of the mammary gland specific EPO-expressing transgenic mice were selected and inserted into the chromosome 10-30 copies and confirmed that the EPO gene was transferred to the next generation through germ cells.

Mouse strainMouse strain No. of embryos injectedNo. of embryos injected No. of embryos transferredNo. of embryos transferred No. of recipientsNo. of recipients No. of mice born No. of mice born No. of transgenic mice (%)No. of transgenic mice (%) BDF1 (C57BL/6xDBA)BDF1 (C57BL / 6xDBA) 738738 700700 3030 8585 9 (10.6)9 (10.6)

3) 형질전환 생쥐에서 EPO의 유선 특이적 발현 확인3) Confirmation of Mammary Specific Expression of EPO in Transgenic Mice

상기 2)에서 제작한 형질전환 생쥐에서 EPO 유전자가 유선 특이적으로 발현되는지를 확인하였다. In the transgenic mice prepared in 2) it was confirmed whether the EPO gene is specifically expressed in the mammary gland.

먼저, 상기 형질전환된 생쥐로부터 수립한 라인 6 및 라인 37 생쥐의 여러 조직(유선, 뇌, 신장, 심장, 비장, 간, 자궁, 폐)에서 각각 RNA를 추출한 후 당업계에 통상적인 방법으로 RT-PCR과 노던 블롯 (Northern blot) 분석을 하였다.First, RNA is extracted from various tissues (wired, brain, kidney, heart, spleen, liver, uterus, lung) of Line 6 and Line 37 mice established from the transformed mice, followed by RT in a conventional manner. PCR and Northern blot analysis.

EPO cDNA를 탐지하기 위하여, RT-PCR에서는 hEPO 특이적 프라이머로 정방향 프라이머 5'-GTAGAAGTCTGGCAGGGCCT-3'(서열번호 21)과 역방향 프라이머 5'-TCATCTGTCCCCTGTCCTGC-3'(서열번호 22)을 사용하였다. In order to detect EPO cDNA, forward primer 5′-GTAGAAGTCTGGCAGGGCCT-3 ′ (SEQ ID NO: 21) and reverse primer 5′-TCATCTGTCCCCTGTCCTGC-3 ′ (SEQ ID NO: 22) were used as hEPO specific primers in RT-PCR.

노던 블롯 분석에서 사용한 프로브는 EPO full genomic DNA로 서던 블롯 분석에서 사용한 2.3kb 동일한 프로브를 사용하였다.The probe used in Northern blot analysis was the same 2.3kb probe used in Southern blot analysis with EPO full genomic DNA.

도 4C를 보면, RT-PCR 결과에서는 유선(mammary gland: M.G.)에서는 강하게 발현되는 반면, 신장(kidney:K)에서 아주 약하게 발현이 되는 것으로 확인되었다. 또한, 도 4D의 Northern blot 분석결과에서는 EPO 유전자가 다른 조직에서는 전혀 발현되지 않고 오직 유선(M.G.)에서만 특이적으로 발현되는 것을 확인하였다.Referring to FIG. 4C, it was confirmed that the RT-PCR result was strongly expressed in the mammary gland (M.G.), but very weakly in the kidney (kidney: K). In addition, the Northern blot analysis of FIG. 4D confirmed that the EPO gene was not specifically expressed in other tissues but specifically expressed only in the mammary gland (M.G.).

(도면 중 B: brain, 뇌; H: heart, 심장; S: spleen, 비장; L: liver, 간; U: uterus, 자궁; Lu: lung, 폐를 의미함)(B: brain, brain; H: heart, heart; S: spleen, spleen; L: liver, liver; U: uterus, uterus; Lu: lung, meaning lung)

또한, 형질전환 생쥐에서 EPO 단백질 발현 여부 및 발현하는 세포종류를 알아보기 위해 조직에 대하여 면역조직화학법(immunohistochemistry)을 수행하였고, 이 때 EPO 항체는 Anti-human antibody (AB-286-NA, R&D system)를 사용하였다.In addition, immunohistochemistry was performed on tissues to determine whether the EPO protein was expressed in transgenic mice, and the EPO antibody was an anti-human antibody (AB-286-NA, R & D). system).

그 결과를 도 5에 나타내었다. The results are shown in FIG.

도 5A의 대조군 생쥐의 임신 16일째 유선에서는 EPO의 발현을 확인할 수 없었다. 반면, 형질전환 생쥐의 임신 16일(도 5B)과 분만 후 5일(도 5C)에서는 유선폐포 세포(alveolar cell)에서 EPO가 발현하는 것을 확인할 수 있었으며, 분만 후 16일(도 5D)에서는 유선폐포 세포가 퇴행하여 EPO의 발현이 없어지는 것을 확인하였다. Expression of EPO could not be confirmed in the mammary gland on day 16 of gestation of the control mice of FIG. 5A. On the other hand, EPO was expressed in mammary alveolar cells on the 16th day of pregnancy (FIG. 5B) and 5 days after delivery (FIG. 5C) of the transgenic mice, and on day 16 (FIG. 5D) after delivery. It was confirmed that alveolar cells regressed and the expression of EPO disappeared.

도 5와 같이 본 발명의 hEPO 형질전환 생쥐에서는 수유기 동안의 유선 폐포 세포에서만 EPO 단백질이 특이적인 발현을 보이는 것을 확인할 수 있었다.As shown in Figure 5 in the hEPO transgenic mice of the present invention it was confirmed that the EPO protein specific expression only in mammary alveolar cells during lactation.

4) 발현되는 EPO 단백질 분석 및 안정성 확인 4) Analysis of the expressed EPO protein and confirmation of stability

상기 2)에서 제작한 형질전환 생쥐의 유즙에 발현되는 EPO 단백질 및 그 농도를 당업계의 통상적인 웨스턴 블롯 분석법(Anti-human antibody 사용(AB-286-NA, R&D system))으로 확인하였다. EPO protein and its concentration expressed in the milk of the transgenic mice prepared in 2) was confirmed by the conventional Western blot analysis (anti-human antibody use (AB-286-NA, R & D system)).

그 결과를 도 6에 나타내었다. The results are shown in FIG.

도 6A는 형질전환 생쥐의 유즙을 이용한 웨스턴 블롯 분석결과로, 레인 1과 2 는 양성 대조군으로써 GST가 결합(tagging)되어 있는 EPO 항원 5ng, 10ng을 나타낸 것이며, 레인 3은 대조군 생쥐의 유즙, 레인 4와 5는 각각 형질전환 생쥐 라인 6과 37의 유즙 내 EPO을 나타낸 것이다.6A is a result of Western blot analysis using milk of transgenic mice. Lanes 1 and 2 show 5ng and 10ng of EPO antigen tagged with GST as a positive control, and lane 3 is milk and lane of control mice. 4 and 5 show EPO in milk of transgenic mice lines 6 and 37, respectively.

도 6A의 결과 본 발명의 형질전환 생쥐 유즙에서 34 KDa 분자량을 가진 단백질이 발현됨을 확인했으며, 전체 단백질 중 0.7-1.4%를 차지하였다.As a result of Figure 6A it was confirmed that the protein having a molecular weight of 34 KDa in the transgenic mouse milk of the present invention, accounting for 0.7-1.4% of the total protein.

또한, 형질전환 생쥐의 유즙유래 EPO의 글리코실화 패턴을 확인하기 위하여 N-글리코시다제-F(N-glycosidase F) 또는 N-글리코시다제-F와 O-글리코시다제로 자른 후 웨스턴 블롯을 실시하였다. In addition, to confirm the glycosylation pattern of milk-derived EPO of transgenic mice, western blot was performed after cutting with N-glycosidase F or N-glycosidase-F and O-glycosidase. It was.

도 6B에서 레인 1은 유즙유래 EPO, 레인 2는 N-글리코시다제-F로 자른 EPO, 레인 3은 N-글리코시다제-F 와 O-글리코시다제로 자른 EPO를 나타낸 것이다. In FIG. 6B, lane 1 is milk-derived EPO, lane 2 is EPO cut with N-glycosidase-F, and lane 3 shows EPO cut with N-glycosidase-F and O-glycosidase.

도 6B 결과를 통해 N-글리코실화 부위 3개와 O-글리코실화 부위가 잘려 최종적으로 18KDa의 EPO를 확인함으로써 후변형(post-modification)이 제대로 일어났음을 확인하였다.6B, the N-glycosylation site and O-glycosylation site were cut and finally confirmed that the post-modification occurred properly by confirming EPO of 18 KDa.

또한, 본 발명의 hEPO 형질전환 생쥐의 유즙 내 EPO의 농도를 확인하기 위하여 ELISA 키트(Human Erythropoietin ELISA, #01630, stem cell technology사)를 사용하여 정량분석한 결과, 수유기 1-5일 사이 약 200,000-400,000 IU/㎖ 이 발현됨을 확인하였다(표 3).In addition, in order to confirm the concentration of EPO in the milk of the hEPO transgenic mice of the present invention, as a result of quantitative analysis using an ELISA kit (Human Erythropoietin ELISA, # 01630, stem cell technology), about 200,000 between lactation period 1-5 days It was confirmed that -400,000 IU / ml was expressed (Table 3).

형질전환 생쥐의 유즙 내 EPO의 정량적인 분석Quantitative Analysis of EPO in Milk of Transgenic Mice micemice ELISA Kit (Stem Cell Technology)ELISA Kit (Stem Cell Technology) LineLine Days of lactationDays of lactation 66 1One 456 ± 33 IU/㎕456 ± 33 IU / μl 66 33 383 ± 28 IU/㎕ Transgenic383 ± 28 IU / μl Transgenic 66 55 233 ± 24 IU/㎕233 ± 24 IU / μl 3737 33 384 IU/㎕384 IU / μl * rhEPO concentration was measured after complete N- and O- digestion (18 kDa).rhEPO concentration was measured after complete N- and O-digestion (18 kDa).

또한, EPO의 안정성을 확인하기 위해 형질 전환된 생쥐의 유즙에서 생산된 인간 EPO와 신부전증 환자의 혈액을 이용해 2D 분석을 수행하였다.In addition, 2D analysis was performed using human EPO produced from the milk of transformed mice and blood of renal failure patients to confirm the stability of EPO.

도 7에 나타난 2D 분석결과를 통해 형전전환 생쥐 유래 EPO는 전하(charge)와 크기(size)가 heterogeneous 나타남을 알 수 있다. 일반적으로 단백 말단에 시알릭산과 같은 당사슬(sugar chain)이 많을수록 PI값이 낮게 되는데, 도 7에서 형질 전환된 라인 6과 37의 생쥐의 유즙에서 재조합된 인간 EPO는 대조군과 신부전증 환자 혈청보다 acidic 상태로 나타나, 시알릭산을 많이 함유하고 있음을 확인할 수 있다.The results of 2D analysis shown in FIG. 7 indicate that the EPO derived from transgenic mice is heterogeneous in charge and size. In general, the more sugar chains such as sialic acid at the protein terminus, the lower the PI value. The human EPO recombined in the milk of the mice in the transformed lines 6 and 37 in FIG. 7 is more acidic than the control and serum of renal failure patients. It can be seen that it contains a lot of sialic acid.

반면, 대조군과 신부전증 환자의 혈액은 asialic 상태임을 확인하였다. On the other hand, the blood of the control group and renal failure patients was confirmed to be asialic state.

시알릭산과 같은 당사슬은 단백질의 구조와 기능에 영향을 미치며 sialic 상태는 RBC 전구세포에 작용해 단백질 활성을 가지며 asialic 상태는 liver receptor와 결합해 오줌으로 배출되어 활성을 가지지 못한다고 보고되어 있다(Parekh RB, et al. (1989) N-glycosylation and in vitro enzymatic activity of human recombinant tissue plasminogen activator expressed in Chinese hamster ovary cells and a murine cell line. Biochemistry 28: 7670-7679; Tam RC, et al. (1991) Comparisons of human, rat and mouse erythropoietins by isoelectric focusing: differences between serum and urinary erythropoietins. Br J Haematol 79: 504-511).It is reported that oligosaccharides such as sialic acid affect protein structure and function, sialic states act on RBC progenitor cells and have protein activity, while asialic states bind to liver receptors and are excreted in the urine and are not active (Parekh RB). (1989) N-glycosylation and in vitro enzymatic activity of human recombinant tissue plasminogen activator expressed in Chinese hamster ovary cells and a murine cell line.Biochemistry 28: 7670-7679; Tam RC, et al. (1991) Comparisons of human, rat and mouse erythropoietins by isoelectric focusing: differences between serum and urinary erythropoietins.Br J Haematol 79: 504-511).

따라서 본원발명의 EPO 발현 형질전환 생쥐로부터 생산된 hEPO는 RBC 전구세포에 작용해 hEPO 단백질 활성을 가질 것으로 사료된다.Therefore, it is considered that hEPO produced from EPO expressing transgenic mice of the present invention acts on RBC progenitor cells and has hEPO protein activity.

5) 형질전환 생쥐의 유즙으로부터의 hEPO 분리5) hEPO isolation from milk of transgenic mice

먼저 본 발명의 형질전환 생쥐의 유즙 3 ㎖을 10mM 트리스 완충액 (pH 6.8) 20 ㎖에 현탁하여 막필터(membrane filter)에 담아 4 ℃, 10mM 트리스 완충액 (pH 6.8) 1,000 ㎖로 2회 밤새 투석하였다. 투석을 마친 현탁액을 원심분리관에 옮겨 담아 4,000rpm으로 30분간 냉장원심분리하여 침전물을 제거하였다. First, 3 ml of the milk of the transgenic mice of the present invention was suspended in 20 ml of 10 mM Tris buffer (pH 6.8), and placed in a membrane filter, and dialyzed twice overnight with 1,000 ml of 10 mM Tris buffer (pH 6.8) at 4 ° C. . After the dialysis suspension was transferred to a centrifuge tube, the precipitate was removed by refrigeration centrifugation at 4,000 rpm for 30 minutes.

상등액 약 20 ㎖을 pM 100막 (Amicon) 필터로 여과하고 그 여액을 10mM 트리스 완충액 (pH 6.8) 300 ㎖에 현탁한 다음 미리 10mM 트리스 완충액 (pH 6.8)로 충분히 평형화시킨 DEAE sephadex column(2Φ× 15cm, bed volume 40 ㎖)에 3 ㎖/min 유속으로 주입하였다. 그 다음 10mM 트리스 완충액 (pH 6.8) 500 ㎖로 칼럼을 충분히 세척한 후 0 mM에서 325mM NaCl을 함유한 10mM 트리스 완충액으로 각 분획의 사이즈는 3 ㎖, 유속은 21 ㎖/h로 gradient 용출시켰다. Approximately 20 ml of the supernatant was filtered through a pM 100 membrane filter and the filtrate was suspended in 300 ml of 10 mM Tris buffer (pH 6.8) and then sufficiently equilibrated with 10 mM Tris buffer (pH 6.8) in advance to a DEAE sephadex column (2Φ x 15 cm). , bed volume 40 ml) was injected at a flow rate of 3 ml / min. Then, the column was sufficiently washed with 500 ml of 10 mM Tris buffer (pH 6.8), and then gradient eluted with 10 ml Tris buffer containing 325 mM NaCl at 0 mM at a size of 3 ml and a flow rate of 21 ml / h.

상기 용출된 각 분획 중 EPO를 함유한 분획을 모은 후, 이를 pM 10막에 넣고 0.15M NaCl을 함유한 10mM 트리스 완충액 (pH 7.2)으로 3배 세척하여 3 ㎖로 농축하였다. 이 액을 앞서와 동일한 완충액으로 미리 평형화시킨 Sephadex G-100(2Φ× 100cm)에 주입한 후 유속 24 ㎖/h, Fraction size 3m로 용출시켰다. After collecting the fractions containing EPO in each of the eluted fractions, it was put into a pM 10 membrane and washed three times with 10 mM Tris buffer (pH 7.2) containing 0.15M NaCl and concentrated to 3 ml. The solution was injected into Sephadex G-100 (2Φ × 100 cm) previously equilibrated with the same buffer solution, and eluted at a flow rate of 24 ml / h and fraction size of 3 m.

상기 용출된 각 분획 중 EPO를 함유한 분획을 다시 모아 pM 10막으로 농축한 후 0.15M NaCl을 함유하는 10mM 트리스 완충액(pH 7.5)에 희석하여 아래와 같이 면역정제법(Immunopurification)을 실시하였다. EPO-containing fractions of the eluted fractions were collected again, concentrated to a pM 10 membrane, and diluted in 10 mM Tris buffer (pH 7.5) containing 0.15M NaCl, and subjected to immunopurification as follows.

① IgG의 분리하기 위해서, 채취한 복수액을 Protein-G agarose affinity system을 이용하여 정제하였다. ① In order to isolate IgG, the obtained ascites liquid was purified using the Protein-G agarose affinity system.

먼저 복수액을 5배의 결합 완충액(0.1M NaH2PO4, 0.15M NaCl, 5mM EDTA, pH 7,0)에 희석한 다음 Protein-A agarose 컬럼에 천천히 흘러 보낸 다음, 같은 완충액으로 세척하였다. 충분히 세척이 끝난 후 용출완충액(0.1M glycine/HCl, 0.01% sodium azide, pH 2.7)으로 용출하였다. 용출물은 동량의 중성화 완충액(1M Tris, 0.01% sodium azide, pH 9)이 들어있는 튜브에 용출하였다. 이 일련의 과정은 12% SDS-PAGE로 수행하여, 쿠마시블루 염색으로 확인하였다. 이 항체용액은 3L의 PBS 용액에서 투석을 3일 동안 6회 반복한 다음 항체량을 정량한 다음 실험에 사용하였다.First, the ascites solution was diluted in 5-fold binding buffer (0.1 M NaH 2 PO 4, 0.15 M NaCl, 5 mM EDTA, pH 7,0) and then slowly flowed into a Protein-A agarose column and washed with the same buffer. After sufficient washing, the solution was eluted with elution buffer (0.1M glycine / HCl, 0.01% sodium azide, pH 2.7). The eluate was eluted in a tube containing the same amount of neutralization buffer (1M Tris, 0.01% sodium azide, pH 9). This series of procedures was performed by 12% SDS-PAGE and confirmed by Coomassie Blue staining. This antibody solution was repeated 6 times for 3 days in 3L PBS solution, and then the amount of antibody was quantified and used in the experiment.

② 면역친화컬럼(Immunoaffinity column)의 제조② Preparation of immunoaffinity column

상기에서 정제된 IgG 50mg를 수소카보네이트 완충용액 (0.1M NaHCO3, 0.5M NaCl, pH 8.3)으로 4 ℃에서 하룻밤 투석하였다. CNBr로 활성화된 3g의 Sepharose 4B (Pharmacia)를 1mM 염산으로 여러 번 씻었다. 대략 12 ㎖ 정도의 젤을 컬럼에 부어 수소카보네이트 완충액으로 씻어 평형상태로 만든 후 IgG를 넣고 하룻밤 천천히 저어주어 반응하도록 하였다. 그 다음 1M 에탄올아민 (pH 8.8) 용액으로 상온에서 3시간 동안 흔들어 주어 반응하지 않고 남아 있는 활성화 잔기를 중화시켰다. 최종적으로 30 ㎖의 카보네이트 완충용액으로 3번, 그리고 30 ㎖의 0.1M 글리신-염산 (pH 2.5)용액으로 한번 씻어주었다. 0.2% NaN3를 함유하는 PBS 완충용액으로 바꿔주고 사용할 때까지 4 ℃에서 보관하였다.The purified IgG 50 mg was dialyzed overnight at 4 ° C. in hydrogen carbonate buffer (0.1 M NaHCO 3 , 0.5 M NaCl, pH 8.3). 3 g of Sepharose 4B (Pharmacia) activated with CNBr were washed several times with 1 mM hydrochloric acid. About 12 ml of gel was poured into the column, washed with hydrogen carbonate buffer, equilibrated, and then IgG was added and stirred slowly overnight. It was then shaken for 3 hours at room temperature with a 1M ethanolamine (pH 8.8) solution to neutralize the remaining activating residue. Finally washed three times with 30 ml of carbonate buffer and once with 30 ml of 0.1 M glycine-hydrochloric acid (pH 2.5) solution. Changed to PBS buffer containing 0.2% NaN 3 and stored at 4 ° C. until use.

③ Immunopurification③ Immunopurification

준비된 컬럼을 0.15M NaCl을 함유하는 10mM 트리스 완충용액(pH 7.5)으로 전처리하여 평형을 유지시켰다. 희석한 샘플용액 20 ㎖을 4 ℃에서 하룻밤동안 부드럽게 교반시키며 젤에 흡착시키고, 젤을 칼럼에 부은 후, 전처리 완충용액으로 충분히 씻고, 0.1M 아세테이트 완충용액(pH 4.5)을 60 ㎖/h의 속도로 흘려 씻어주었다. 젤에 결합한 EPO를 30 ㎖의 0.1M 글리신-염산(pH 2.5) 용액으로 용출시켰다. 용출된 분획은 바로 1M 트리스 용액(pH 9)을 가하여 빠른 시간 내에 pH 7.5까지 적정하였다. 그 후 pM 10막 (Amicon)을 가지고 용출된 분획을 농축하여 hEPO 단백질을 수득하였다.The prepared column was pretreated with 10 mM Tris buffer (pH 7.5) containing 0.15 M NaCl to maintain equilibrium. 20 ml of diluted sample solution was adsorbed onto the gel with gentle stirring overnight at 4 ° C., the gel was poured into a column, washed well with pretreatment buffer, and 0.1M acetate buffer (pH 4.5) was added at a rate of 60 ml / h. Rinse and wash. EPO bound to the gel was eluted with 30 ml of 0.1 M glycine-hydrochloric acid (pH 2.5) solution. The eluted fraction was immediately titrated to pH 7.5 by adding 1M Tris solution (pH 9). The eluted fractions were then concentrated with pM 10 membrane (Amicon) to obtain hEPO protein.

6) EPO 분자의 올리고당(oligosaccharide) 분석6) Oligosaccharide Analysis of EPO Molecules

본 발명의 형질전환 생쥐의 유즙 내 hEPO의 올리고당 구조(oligosaccharide structure)를 분석하기 위하여, 글리코단백질의 대조군으로 소 페투인(bovine serum fetuin, Glycosciences), 상품화된 에리트로포이에틴인 이포에틴 알파(epoetin α, LG생명과학), 그리고 형질전환 생쥐의 유즙 내 hEPO을 HPLC를 이용하여 분석하였다. In order to analyze the oligosaccharide structure of hEPO in milk of transgenic mice of the present invention, bovine serum fetuin (Glycosciences), a commercialized erythropoietin, epoetin alpha as a control of glycoproteins , LG Life Sciences), and hEPO in milk of transgenic mice were analyzed using HPLC.

도 8의 결과에서 A는 소 페투인, B는 형질전환 생쥐 유래 hEPO, C는 epoetin α를 나타낸다. In the results of FIG. 8, A represents bovine fetuin, B represents transformed mouse-derived hEPO, and C represents epoetin α.

도 8에서와 같이, 형질전환 생쥐의 유즙 유래 EPO는 CHO 세포주 유래 epoetin α와 비교시 비슷한 mono-, di-, tri-acidic oligosaccharide를 가지지만, 첨가된 tetra-acidic oligosaccharide 구조를 갖는 것을 확인하였다. 이는 글리코실화에 관여하는 효소에 의해 형질전환동물에서 EPO 발현시 번역 후 변형(post-translational modification)이 정상적으로 이루어졌으며, 따라서 유선에서 생산된 EPO는 의약용으로 사용하는데 용이하다라는 것을 의미한다.As shown in FIG. 8, the milk-derived EPO of the transgenic mice had similar mono-, di-, tri-acidic oligosaccharides compared to the epoetin α derived from the CHO cell line, but had an added tetra-acidic oligosaccharide structure. This means that post-translational modification of EPO in transgenic animals was normally performed by enzymes involved in glycosylation, so that EPO produced in the mammary gland is easy to use for medical purposes.

그리고 그 수치를 표 4에 나타내었다.And the numerical value is shown in Table 4.

사람의 혈청 내 EPO와 형질전환 생쥐의 유즙 내 EPO의 N-linked charge 분석N-linked charge analysis of EPO in human serum and EPO in milk of transgenic mice SourceSource Natural glycan %Natural glycan% Mono-acidic glycan %Mono-acidic glycan% Di-acidic glycan %Di-acidic glycan% Tri-acidic glycan %% Tri-acidic glycan Tetra-acidic glycan %Tetra-acidic glycan% CommentsComments Human serum EPOHuman serum EPO 2020 99 4848 2323 00 Venke kibeli, et al. (2001), BLOOD, 98(13), 3626-3634Venke kibeli, et al. (2001), BLOOD, 98 (13), 3626-3634 Milk-derived rhEPOMilk-derived rhEPO ndnd 3.82±0.23.82 ± 0.2 26.75±1.026.75 ± 1.0 40.05±1.940.05 ± 1.9 29.38±3.12a 29.38 ± 3.12 a Epoetin αEpoetin α ndnd 2.73±2.73 ± 36.79 ±0.436.79 ± 0.4 40.23±0.740.23 ± 0.7 20.60±0.17b 20.60 ± 0.17 b -Quantitative data were obtained from experiments performed in triplicate, and presented as means ㅁ S.D. Rows of different superscripts within a column are significantly different (P<0.05). -nd represents 'not detectable'Quantitative data were obtained from experiments performed in triplicate, and presented as means in S.D. Rows of different superscripts within a column are significantly different (P <0.05). -nd represents 'not detectable'

7) 형질전환 생쥐의 유즙 내 hEPO의 활성 확인7) Confirmation of hEPO activity in milk of transgenic mice

7-가) 형질전환 생쥐의 유즙 내 hEPO의 in vitro 활성 확인7-A) Confirmation of in vitro activity of hEPO in milk of transgenic mice

본 발명의 형질전환 생쥐의 유즙 내 hEPO의 in vitro 활성을 확인하기 위하여 하기와 같은 실험을 수행하였다.In order to confirm the in vitro activity of hEPO in milk of the transgenic mice of the present invention, the following experiment was performed.

EPO는 EPO 수용체(EPO receptor, EPOR)와 결합하여 전사인자인 STAT5를 활성화시키는 것으로 알려져 있다. 그래서 STAT5가 포함되어 있는 BCL-XL 루시퍼라제(lucifease) 발현 벡터를 구축한 후, EPOR가 발현되는 MCF-7 세포주(인간 유방암 세포주)에 안정하게 발현될 수 있도록 BCL-XL 루시퍼라제 발현 벡터를 도입하였다. 이 세포(2 ×105/35 mm plate)에 본 발명의 형질전환 생쥐의 rhEPO를 포함한 유즙 단백질을 각각 0 ng, 10 ng, 100 ng, 1 ㎍을 16 시간 동안 처리하였다.EPO is known to bind to the EPO receptor (EPOR) to activate the transcription factor STAT5. Therefore, after constructing the BCL-XL luciferase expression vector containing STAT5, the BCL-XL luciferase expression vector was introduced to be stably expressed in the MCF-7 cell line (human breast cancer cell line) expressing EPOR. It was. The cells (2 × 10 5/35 mm plate) 0 ng each of the milk protein containing rhEPO of transgenic mice of the present invention to, 10 ng, 100 ng, 1 ㎍ was treated for 16 hours.

음성대조군으로는 일반 유즙단백질 1 ㎍을 세포(2 ×105/35 mm plate)에 처리하고, 양성대조군으로 이포에틴 알파는 10 IU, 100 IU로 처리하였다. 처리 16 시간 후, 상대적인 루시퍼라제 활성을 Microplate Luminometer (Perkin Elmer)를 이용하여 측정하였다. 그 결과를 도 9에 나타내었다.The negative control was treated the common milk protein 1 ㎍ the cells (2 × 10 5/35 mm plate) , and alpha-ethynyl epoch as positive control were treated with 10 IU, 100 IU. After 16 hours of treatment, relative luciferase activity was measured using a Microplate Luminometer (Perkin Elmer). The results are shown in FIG.

도 9의 결과에서, MCF-7 세포에 농도별로 형질전환 생쥐의 유즙 유래의 EPO를 첨가한 결과 용량 의존적(dose-dependent) 방식으로 루시퍼라제의 활성이 증가하는 것을 확인하였다. 이러한 증가 정도는 이포에틴 알파 처리시 루시퍼라제 활성도 증가와 유사하므로, 본원발명의 형질전환 생쥐의 유즙 유래의 hEPO가 우수한 in vitro 활성을 나타냄을 알 수 있었다.In the results of FIG. 9, it was confirmed that the luciferase activity was increased in a dose-dependent manner by adding EPO derived from milk of transgenic mice to the MCF-7 cells by concentration. This increase was similar to that of luciferase activity when treated with Ipoietin alpha, and thus it was found that the milk-derived hEPO of the transgenic mice of the present invention showed excellent in vitro activity.

7-나) 형질전환 생쥐의 유즙 내 EPO의 in vivo의 생리활성 확인7-b) Confirmation of in vivo physiological activity of EPO in milk of transgenic mice

또한, 본 발명의 형질전환 생쥐의 유즙 내 EPO의 in vivo의 생리활성을 확인하기 위하여 형질전환 생쥐의 유즙 유래 EPO를 대조군 마우스에 주입한 후(200 ng/Kg, 1회 정맥주사) 시간별로 혈액을 채취하여 성분을 분석하였다. In addition, in order to confirm the in vivo physiological activity of EPO in the milk of the transgenic mice of the present invention, after injection of milk-derived EPO of the transgenic mice into the control mouse (200 ng / Kg, once intravenous injection) blood by time The components were collected and analyzed.

그 결과를 도 10에 나타내었다.The results are shown in FIG.

도 10에서 보는 바와 같이 형질전환 생쥐의 유즙 유래의 EPO를 첨가한 결과 시간 의존적(time-dependent) 방식으로 혈소판, 적혈구, 혈색소, 헤마토크릿(Hematocrit)이 증가하는 것을 확인하였다. 이러한 증가 정도는 이포에틴 알파 처리시의 증가 정도와 유사한 것을 알 수 있어, 본원발명의 형질전환 생쥐의 유즙 유래의 EPO가 우수한 in vivo 활성을 나타냄을 알 수 있었다.As shown in FIG. 10, it was confirmed that platelet, erythrocyte, hemoglobin, and hematocrit were increased in a time-dependent manner when the EPO-derived EPO was added. The increase was similar to the increase in the treatment with Ipoietin alpha, it can be seen that the EPO derived from milk of the transgenic mice of the present invention shows excellent in vivo activity.

[실시예 3] 본 발명의 EPO 발현벡터를 도입한 체세포를 이용한 형질전환 복제돼지의 생산 제조 Example 3 Production and Production of Transgenic Cloned Pigs Using Somatic Cells Incorporated with EPO Expression Vector of the Present Invention

상기 실시예 1에서 제조한 본 발명의 발현벡터 pBC1/hEPO/NEO를 도입하여 체세포를 이용한 형질전환 복제돼지를 다음과 같이 제조하였다.The transgenic cloned pig using somatic cells was prepared by introducing the expression vector pBC1 / hEPO / NEO of the present invention prepared in Example 1 as follows.

가) 배양액 준비A) Preparation of culture solution

난포란의 체외성숙에 사용한 배양액은 NCSU 23 배양액을 기본배양액으로 하여 Baxter (Baxter Healthcare Co., U.S.A.)의 물 1리터로 제조하여 0.2 ㎛ filter (Gelman Sci., U.S.A.)로 여과한 후 pH 7.2~7.3으로 조정하여 50 ㎖ tissue culture flask (Falcon, U.S.A.)에 45 ㎖씩 분주하여 4 ℃의 냉장고에 보관하면서 약 2 주간 사용하였다. The culture medium used for in vitro maturation of follicles was prepared with 1 liter of water from Baxter (Baxter Healthcare Co., USA) using NCSU 23 culture as a basic culture medium and filtered with a 0.2 μm filter (Gelman Sci., USA), followed by pH 7.2 ~ 7.3 45 ml of each was injected into a 50 ml tissue culture flask (Falcon, USA) and stored in a 4 ° C. refrigerator for about 2 weeks.

체외성숙용 배양액은 NCSU 23 배양액에 10% 난포액, 0.1 ㎎/㎖ cysteine, 0.01 ㎍/㎖ EGF, 10 IU/㎖ eCG 그리고 10 IU/㎖ hCG를 첨가하여 제조하였다. 체외배양액은 NCSU 23 배양액에 0.4% BSA를 첨가하여 사용하였다. In vitro maturation broth was prepared by adding 10% follicles, 0.1 mg / ml cysteine, 0.01 μg / ml EGF, 10 IU / ml eCG and 10 IU / ml hCG to NCSU 23 culture. In vitro culture was used by adding 0.4% BSA to the NCSU 23 culture.

돼지 난포액은 2~7 ㎜ 크기의 난포에서 난포액을 채취하여 1,900 × g로 3회 원심분리하고, 최종 0.2 ㎛ 필터로 거른 후 -20℃ 냉장고에 보관하여 사용하였다.Porcine follicle solution was collected from the follicle of 2 ~ 7 mm size follicles, centrifuged three times 1,900 × g, filtered with a final 0.2 ㎛ filter and stored in -20 ℃ refrigerator used.

나) 난포란의 채란B) egg follicles

본 실시예에서 사용한 난소는 도축장에서 도살 직후 난소를 적출하여 페니실린 G (100 units/㎖)와 스트렙토마이신 (100 ㎍/㎖)이 함유된 생리식염수(30~35℃)가 들어있는 보온병에 담아 3~4시간 내에 실험실로 운반하였고, 미성숙 난포란을 채란하기 전 난소주위의 지방과 결합조직을 제거하고, 생리식염수로 3~4회 세척한 후, 18-G needle이 부착된 20 ㎖ 주사기를 이용하여 2~7 ㎜의 가시난포를 흡입하여 난포란을 채란하였다. 난포란의 채취시 사용된 배양액은 0.1 ㎎/㎖ PVA가 첨가된 TALP-HEPES (Prather, R. S., et al. 1995. In vitro development of embryos from sinclair miniature pigs: A preliminary report. Theriogenology, 43:1001-1007)를 사용하였다. The ovaries used in this example were extracted from the ovary immediately after slaughter and placed in a thermos containing physiological saline (30-35 ° C.) containing penicillin G (100 units / mL) and streptomycin (100 μg / mL). It was transported to the laboratory within 4 hours, the fat and connective tissues around the ovary were removed before the immature follicular egg was egged, washed 3-4 times with physiological saline, and then using a 20 ml syringe with an 18-G needle. 2 to 7 mm of visible follicles were inhaled and the oocytes were ovulated. The culture medium used for oocyte collection was TALP-HEPES (Prather, RS, et al. 1995. In vitro development of embryos from sinclair miniature pigs: A preliminary report.Theriogenology, 43: 1001-1007 ) Was used.

흡입된 난포액은 5~10 분간 정치시킨 후 침전된 하부액을 5 ㎖의 피펫으로 흡입하여 직경 60 ㎜ 배양접시에 옮겨 40×배율의 도립현미경(Olympus Co., Japan)에서 난포란을 수집한 후 체외성숙용 기본배양액 NCSU 23으로 4~5회 세척하면서 선발하였다. 난포란의 선발은 난구세포의 부착 정도와 세포질의 충실도에 따라 실시하였으며, 최소한 2층 이상의 난구세포층으로 되어 있고, 세포질이 균일하고 충실한 것을 선발하여 하기 실시예에 사용하였다. The inhaled follicle solution was allowed to stand for 5 to 10 minutes, and the sedimented lower solution was sucked with a 5 ml pipette and transferred to a culture dish of 60 mm in diameter. Selected by washing 4-5 times with the basic culture medium NCSU 23 for maturation. The selection of follicular eggs was carried out according to the degree of adhesion of the cumulus cells and the fidelity of the cytoplasm. The follicle cells were composed of at least two layers of cumulus cells.

다) 난포란의 체외성숙C) In vitro maturation of follicular eggs

난포란의 체외성숙은 체외성숙용 배양액을 4-well dish (Nunc, Denmark)에 500 ㎕씩 분주하여 18 시간 이상 전 배양을 실시하여 평형을 유도한 다음, 체외성숙용 배양액에 난구세포가 2층 이상이고 세포질이 충실한 100~150 개의 난포란을 넣고 5％ CO2, 98~99％ 습도, 39℃ CO2 incubator에서 20~22시간 동안 호르몬이 첨가된 체외성숙용 배양액에서 배양하였다. 이어서, 다음 20~22시간은 호르몬이 되지 않은 배양액에서 배양하여 총 40~44시간 동안 배양하였다. In vitro maturation of follicles was performed by incubating 500 µl of IVF culture in 4-well dish (Nunc, Denmark) and incubating for 18 hours or more to induce equilibrium. 100 to 150 follicles with high cytoplasm were added and cultured in vitro culture medium containing hormones for 20-22 hours in a 5% CO 2 , 98-99% humidity, 39 ℃ CO 2 incubator. Subsequently, the next 20 to 22 hours were incubated in a non-hormonal culture medium and incubated for a total of 40 to 44 hours.

라) 인간 에리트로포이에틴(hEPO)을 발현하는 유전자의 도입 및 적중을 위한 벡터의 제조D) Preparation of a vector for introduction and targeting of genes expressing human erythropoietin (hEPO)

상기 실시예 1에 기재된 방법을 따라 인간 에리트로포이에틴(hEPO)을 발현하는 pBC1/hEPO/NEO 발현벡터를 제작하였다.A pBC1 / hEPO / NEO expression vector expressing human erythropoietin (hEPO) was prepared according to the method described in Example 1 above.

마) 공여체세포의 채취, 배양 및 세포라인 확립E) collecting, culturing, and establishing cell lines for donor cells;

본 실험에 사용된 공여체세포는 임신 30일령 된 돼지 태아의 조직을 채취하여 이용하였다. Donor cells used in this experiment were collected from the tissues of pig fetuses 30 days old pregnant.

채취한 태아의 머리, 사지 및 내장을 제외한 나머지 전체조직을 미세하게 세절하여 0.05% 트립신(trypsin, Gibco, USA)과 EDTA (Sigma, USA)가 첨가된 D-PBS로 3분간 처리한 후, D-PBS로 원심분리를 실시하여 트립신과 EDTA를 제거하였다. The whole tissue except for the collected fetus's head, limbs and intestines was finely divided and treated with D-PBS added with 0.05% trypsin (Gibco, USA) and EDTA (Sigma, USA) for 3 minutes. -Trypsin and EDTA were removed by centrifugation with PBS.

분리된 세포는 10% FBS가 첨가된 DMEM을 가하고, 25 ㎤ 플라스크 (Falcon, USA)에 분주하여 CO2 인큐베이터에서 배양하였으며, 배양 12시간 후 바닥에 붙지 않은 세포는 제거하고, 10% FBS가 첨가된 신선한 DMEM으로 교체하면서 3~5일간 배양하였다. 계대배양은 공여세포가 플라스크에 80% 이상 자랐을 때, 0.05% 트립신과 EDTA를 처리하여 부유시킨 다음, 1/3~1/4씩 나누어 10회 이상 계대해서 배양하였다. The isolated cells were added DMEM with 10% FBS, aliquoted in a 25 cm 3 flask (Falcon, USA) and cultured in a CO 2 incubator. After 12 hours of culture, non-bottom cells were removed and 10% FBS was added. Incubated for 3-5 days while replacing with fresh DMEM. Subcultures were cultured by donor cells grown at 80% or more in the flask, treated with 0.05% trypsin and EDTA, and then divided into 1/3 to 1/4 passages at least 10 times.

계대배양한 공여세포는 10% DMSO가 첨가된 DMEM 배양액으로 동결 보존해 두고, 핵이식에 사용할 때는 38~39℃ 온수에 융해하여 동결보호제를 제거한 다음 10% FBS가 첨가된 신선한 DMEM 배양액으로 1회 계대배양한 후 세포가 배양디쉬(culture dish)에 단층(monolayer)을 충분히 형성하여 confluency 상태로 2~3일 정도 배양함으로써 세포주기상의 G0나 G1단계로 유도한 다음 공여세포로 사용하였다. Donor cells that were passaged were cryopreserved in DMEM medium with 10% DMSO, and when used for nuclear transfer, they were thawed in hot water at 38-39 ° C to remove cryoprotectant and then added once with fresh DMEM medium with 10% FBS. After subculture, the cells were sufficiently formed with a monolayer in a culture dish and cultured in a confluency state for 2 to 3 days to induce G0 or G1 phase in the cell cycle, and then used as donor cells.

바) 확립된 체세포로의 외래 유전자 도입F) introduction of foreign genes into established somatic cells;

상기 실시예 1에서 제조하여 -20℃에서 보관한 pBC1/hEPO/NEO DNA를 꺼내 녹인 후, 쓰기 직전에 냉장 보관한 이펙틴(Effectene, Qiagen사)을 사용하여 각각 섞이지 않도록 주의하여 e-tube에 DNA 농도가 2 ㎍/㎖가 되도록 담아 표시한 후, Qiagen kit 내의 Enhancer 8 ㎕을 각각 튜브에 천천히 넣어 주었다. Take out and melt the pBC1 / hEPO / NEO DNA prepared in Example 1 and stored at -20 ° C, and carefully mix them in an e-tube so as not to mix with each other using an effectine (Effectene, Qiagen) stored in the refrigerator immediately before writing. After the DNA concentration of 2 ㎍ / ㎖ was indicated, 8 μl of Enhancer in the Qiagen kit was slowly added to each tube.

DNA+Enhancer+Buffer EC 양이 Total 150 ㎕이 되도록 계산하여 Buffer EC를 넣어 주고, 이것을 1초간 볼텍싱한 다음 RT에서 2~5분간 두었다. 여기에 이펙틴 25㎕을 각각 넣어 준 후 파이펫팅(pipetting)하여 RT에서 5~10분간 두었다. Calculate the amount of DNA + Enhancer + Buffer EC to be 150 μl in total, add Buffer EC, vortex this for 1 second and leave for 2 to 5 minutes at RT. 25 μl of each of the effectin was added thereto, followed by pipetting (pipetting) and placed at RT for 5-10 minutes.

또한, 상기 마)에서 준비된, 배양 디쉬에 50~80% 깔린 섬유아세포(Fibroblast)는 D-PBS로 2~3번 가량 10분 동안 세척하여 준 다음, FBS가 첨가되지 않은 DMEM 배지를 4 ㎖씩 각각 넣어준 후 인큐베이터에 넣어두었다. In addition, fibroblasts 50-80% crushed in a culture dish prepared in the above) were washed with D-PBS for 2 to 3 times for 10 minutes, and then 4 ml of DMEM medium without FBS was added thereto. After each put in the incubator.

10분 후 각각 E-tube에 FBS가 첨가되지 않은 DMEM을 1 ㎖까지 채워 파이펫팅하여 잘 섞어준 후, 섬유아세포 및 DMEM이 4 ㎖ 들어있는 디쉬에 넣어 유전자 세포내 도입(transfection)시킨 후, 인큐베이터에서 다시 12~18시간 방치하였다. 12~18시간 후 D-PBS로 세척하여 준 후 다시 FBS가 10% 첨가된 DMEM으로 교체해 주었다.After 10 minutes, pipette and mix well with 1 ml of DMEM without FBS added to the E-tube, and put it in a dish containing 4 ml of fibroblasts and DMEM, and then transfect the gene into an incubator. Was left for another 12 to 18 hours. After 12 to 18 hours, the cells were washed with D-PBS and replaced with DMEM with 10% FBS.

사) EPO가 도입된 체세포 라인의 선별, 계대배양 및 동결보존G) Screening, passage and cryopreservation of somatic cell lines incorporating EPO

상기에서 바)에서 유전자 세포내 도입시킨 날로부터 72시간 후부터 G418 농도 600~800 ㎍/㎖ 정도를 사용하여 약 2주간 네오마이신 저항성을 갖는 세포를 선별(selection)하였다. 이렇게 선별된 섬유아세포는 보통 3~5일 정도면 깔리게 하여 계대배양 해주었다.In the above bar, cells having neomycin resistance were selected for about 2 weeks using the G418 concentration of about 600-800 µg / ml from 72 hours after the gene was introduced into the cell. Fibroblasts thus screened were usually subcultured for 3 to 5 days.

자세하게는, 분리할 세포의 배양용기(bottle)에서 배양배지를 제거하고, 그 배양용기에 Trypsin+EDTA 0.25%를 1.5 ㎖ 가량 넣어 인큐베이터에 약 3~5분가량 넣어 두었다. 현미경으로 관찰하여 약 70% 가량 세포들이 떨어져 있는 상태일 때 인큐베이터에서 꺼내어, Transfer pipette를 이용해 미리 준비해 놓은 D-PBS가 10 ㎖정도 들어 있는 15 ㎖ 튜브에 넣어 1500 RPM에서 3분가량 원심분리하였다. 상층액 제거 후 밑의 펠렛에 10% FBS+DMEM+G418을 3 ㎖정도 넣고 충분히 펠렛을 풀어준 후, 배양용기(bottle)에 넣어 인큐베이터에서 배양하였다.In detail, the culture medium was removed from the culture vessel of the cells to be separated, and about 1.5 ml of Trypsin + EDTA 0.25% was added to the culture vessel and placed in the incubator for about 3-5 minutes. Observed by microscope, the cells were removed from the incubator when the cells were about 70% apart and placed in a 15 ml tube containing 10 ml of D-PBS prepared in advance using a transfer pipette and centrifuged at 1500 RPM for 3 minutes. After removing the supernatant, about 3 ml of 10% FBS + DMEM + G418 was added to the lower pellet, and the pellet was fully released. The pellet was placed in a culture vessel and cultured in an incubator.

세포의 동결보존을 위해서는 상기 원심분리 후 수득한 펠렛에 DMSO를 1 ㎖정도 넣고 충분히 펠렛을 풀어 준 후, Cryotube에 넣어 -70 ℃ Deep Freezer에 24시간 보관 후 -196℃ 액체 질소 탱크에 넣어 보관하였다. For cryopreservation of cells, 1 mL of DMSO was added to the pellet obtained after centrifugation, and the pellet was fully released. The pellet was placed in a Cryotube and stored in a -70 ° C Deep Freezer for 24 hours, and then stored in a -196 ° C liquid nitrogen tank. .

아) 핵이식에 공여될 동결 체세포의 융해 및 준비H) fusion and preparation of frozen somatic cells for donation to nuclear transfer;

1. 융해: 해동(Thawing)할 세포를 저장고에서 꺼내어 37℃ 수조(water bath)에서 해동시켰다. 미리 준비해놓은 D-PBS가 10 ㎖정도 들어 있는 15 ㎖ 튜브에 넣어 1500 RPM에 3분가량 원심분리하였다. 상층액 제거 후 펠렛에 10% FBS+DMEM+G418을 3 ㎖정도 넣고 충분히 펠렛을 풀어 준 후 bottle에 넣어 인큐베이터에 넣어 배양하였다.1. Thawing: The cells to be thawed were removed from the reservoir and thawed in a 37 ° C. water bath. The D-PBS prepared in advance was placed in a 15 ml tube containing about 10 ml and centrifuged at 1500 RPM for about 3 minutes. After removing the supernatant, 3 ml of 10% FBS + DMEM + G418 was added to the pellet, and the pellet was fully released. The pellet was placed in a bottle and incubated.

2. 공여핵 준비: 보통 공여세포(Donor cell) 준비는 4-well Dish에 하는데 분리할 세포의 배양배지를 제거하고, Dish에 Trypsin+EDTA 200 ㎕ 가량 넣어 인큐베이터에 약 3~5분가량 넣어 두었다. 현미경으로 관찰하여 약 70% 가량 세포가 떨어져 있는 상태일 때 인큐베이터에서 꺼내어 200 ㎕ 파이펫으로 세포를 분리하여 공여세포 준비용 배양배지가 약 3 ㎖ 정도 들어있는 dish에 넣어 세포를 풀어 준 후 쓰기 전까지 인큐베이터에 넣어 두었다.2. Donor Nuclear Preparation: Donor cell preparation is usually carried out in 4-well dish, and the culture medium of the cells to be separated is removed, and 200 μl of Trypsin + EDTA is added to the dish and placed in the incubator for about 3-5 minutes. . Observed under a microscope, the cells were removed from the incubator when the cells were about 70% apart and separated into 200 μl pipettes and placed in a dish containing about 3 ml of the donor cell preparation medium. Placed in the incubator.

자) 핵이식Nuclear transfer

핵이식에 사용된 파이펫은 직경이 1 mm인 capillary tube (Narishige, Japan)를 사용하여 보정용(holding), 탈핵용(enucleation) 및 주입용(injection) 파이펫을 각각 제작하였다. Holding 파이펫의 외경은 150~180 ㎛, 탈핵과 주입용 파이펫은 외경이 30~40 ㎛로 조절하였다. 제작이 완료된 파이펫은 PVP로 코팅처리한 후 사용하였다. 체외성숙된 수핵난자를 0.1% 히알우로니다제(hyaluronidase, Sigma, USA)가 첨가된 D-PBS에 넣어 난구세포를 제거하고, 3회~4회 PVA-TALP-HEPES로 세척한 다음, 0.05 M 슈크로즈 (sucrose, Sigma, USA) 및 0.4% BSA가 첨가된 배양액에서 세포질이 양호하고 제1극체가 뚜렷하게 보이는 난자만을 선별하여 사용하였다. Pipettes used for nuclear transfer were prepared using a capillary tube (Narishige, Japan) having a diameter of 1 mm for holding, enucleation, and injection pipettes, respectively. The outer diameter of the holding pipette was adjusted to 150 ~ 180 ㎛, the denuclearization and injection pipette was adjusted to 30 ~ 40 ㎛. The pipettes were fabricated and used after coating with PVP. In vitro matured nucleated oocytes were added to D-PBS with 0.1% hyaluronidase (Sigma, USA) to remove cumulus cells, washed 3-4 times with PVA-TALP-HEPES, and then 0.05 M. In the culture medium added with sucrose (sucrose, Sigma, USA) and 0.4% BSA, only eggs that had a good cytoplasm and a clear first polar body were selected and used.

핵이식은 NCSU-23에 0.4% BSA가 첨가된 배양액 소적에 7.5 ㎍/㎖의 사이토칼라신 B (cytochalasin B, Sigma, USA)와 0.05 M 슈크로즈를 첨가하여 30% 정도의 세포질을 흡입함으로써 핵을 제거하였다. Nuclear transplantation was performed by inhaling about 30% of the cytoplasm by adding 7.5 µg / ml cytochalasin B (Sigma, USA) and 0.05 M sucrose to a culture droplet containing 0.4% BSA added to NCSU-23. Was removed.

상기에서 탈핵한 난자는 세포질이 제거된 공간에 G0기나 G1기로 유도한 공여세포를 loading하여 세포질과 부착되게 주입하였다. 공여세포가 주입된 난자는 전기융합을 하기 전까지 NCSU-23에 0.4% BSA가 첨가된 IVC 배양액에 처리하였다. The denucleated oocytes were loaded with donor cells derived from G0 or G1 group into the cytoplasm-removed space and attached to the cytoplasm. The donated cells were treated with IVC culture containing 0.4% BSA in NCSU-23 before electrofusion.

차) 핵이식 수정란의 융합 및 활성화F) Fusion and activation of nuclear transfer embryos

핵이식이 완료된 난자의 공여세포와 세포질의 융합은 전기세포융합장치(BTX, USA)로 실시하였다. 이때 전기융합 배양액은 0.1 mM CaCl2 (Sigma, USA) 및 0.1 mM MgCl2 (Sigma, USA)가 첨가된 0.28 M Mannitol (Sigma, USA) 용액을 사용하고, 여기에서 2~3분간 평형을 실시한 다음, 핵이식란을 전기 융합용 chamber로 옮겨 양 전극 사이에 일렬로 주입하여 핵은 전극의 양극(+)쪽으로 향하게 하고 세포질은 음극(-)쪽으로 향하게 하여 전기자극을 가하였다. 전기융합 및 활성화는 150 V, 50 μsec, 2 pulse를 주어, 수정란의 융합과 전기활성화를 동시처리하였다. Fusion of donor cells and cytoplasm of oocytes after nuclear transfer was performed using an electric cell fusion device (BTX, USA). At this time, the electrofusion culture solution was prepared using 0.28 M Mannitol (Sigma, USA) solution with 0.1 mM CaCl 2 (Sigma, USA) and 0.1 mM MgCl 2 (Sigma, USA), and then equilibrated for 2-3 minutes. In addition, the nuclear transfer embryos were transferred to the chamber for electrofusion and injected in a row between the two electrodes so that the nucleus was directed toward the anode (+) of the electrode and the cytoplasm toward the cathode (-). Electrofusion and activation were given 150 V, 50 μsec, 2 pulses, and the fusion and electroactivation of the fertilized egg was co-processed.

핵이식 수정란의 융합과 활성화를 유도한 다음 사이토칼라신 B에 4시간 처리 후, 0.4% BSA가 첨가된 배양액에서 체외배양을 실시하였다. After inducing the fusion and activation of the nuclear transfer embryos, and then treated with cytocalin B for 4 hours, in vitro culture was performed in a culture medium containing 0.4% BSA.

카) 수란돈 준비K) Sorlandon preparation

복제 수정란을 이식하기 위하여 임신 30~40일된 수란돈에 PGF2α를 근육주사하여 유산을 유도한 후 24시간이 경과되었을 때 PGF2α와 PMSG를 동시에 주사하였다. 그 후 72시간이 경과되었을 때 hCG를 근육 주사하여 과배란을 실시하였다. 48시간 후에 복제수정란을 이식하였다. PGF2α and PMSG were injected at the same time 24 hours after abortion was induced by intramuscular injection of PGF2α to 30-40 days of gestation. After 72 hours, hCG was injected intramuscularly to over-ovulate. After 48 hours, the embryos were implanted.

타) 수정란 이식 및 임신진단5) Fertilized egg transplant and pregnancy diagnosis

돼지의 복제 수정란을 이식하여 산자를 생산할 목적으로 외과적인 방법에 의해서 수정란 이식을 하였다. 먼저 호르몬 처리된 수란돈은 케타민과 럼푼을 귀 정맥에 주입하여 전신 마취를 실시하였다. 마취된 수란돈의 정중선을 중심으로 광범위하게 면도기를 이용하여 털을 제거하고 소독을 한 다음 정중선을 따라 약 10~15cm를 절개하여 자궁을 노출시킨 후 난소 표면의 출혈체 혹은 난포상태를 확인한 후, 복제된 수정란을 난관 팽대부에 주입하였다. 일부 수란돈은 복제 수정란의 체내 발달 여부를 확인하기 위하여 이식 후 7일째에 외과적 방법으로 개복하여 이식된 수정란의 발달률을 조사하였다. 또한 수란돈의 임신 여부는 수정란 이식 후 27~30일째에 초음파진단기를 이용하여 확인하였다.A fertilized egg was implanted by a surgical method to produce a litter by transplanting a cloned fertilized egg of a pig. First, hormone-treated suranthones were subjected to general anesthesia by injecting ketamine and lumps into the ear veins. After removing the hair with a razor and disinfecting it with a wide range around the midline of the anesthetized lanthanide, excision about 10-15 cm along the midline to expose the uterus, and then check the bleeding or follicle on the surface of the ovary. The cloned fertilized egg was injected into the tubal bulge. In order to confirm the development of cloned fertilized eggs in the body, 7 days after transplantation, the surgical rate of the implanted embryos was examined. Also, whether or not pregnancy was successful was confirmed using an ultrasound diagnostic device at 27-30 days after fertilization.

생산된 자돈에서 PCR과 genomic Southern blot으로 확인하여, 유선 특이적으로 EPO를 발현하는 형질전환돼지가 생산됨을 확인하였다.PCR and genomic Southern blot were confirmed in the produced piglets to confirm the production of transgenic pigs expressing EPO specifically in mammary glands.

본 발명의 hEPO 발현 형질전환동물은 유선 특이적으로 에리트로포이에틴을 발현하며, 기존의 방법에 비해 매우 높은 농도로 유즙 중에 에리트로포이에틴을 발현한다.The hEPO expressing transgenic animals of the present invention express erythropoietin specifically in mammary gland, and express erythropoietin in milk at a much higher concentration than conventional methods.

또한, 본 발명의 형질전환동물로부터 생산된 hEPO 단백질은 시판되는 동종 단백질이 나타내는 것 이상의 안정성 및 우수한 생리활성을 나타낸다.In addition, the hEPO protein produced from the transgenic animal of the present invention exhibits more stability and superior physiological activity than that of commercially available homologous proteins.

또한, 본 발명의 형질전환동물로부터 생산된 hEPO 단백질을 투여시 혈액 내 혈소판, 적혈구, 혈색소, 헤마토크릿(hematocrit)을 증가시키는 작용효과를 나타낸다. 따라서 본 발명의 hEPO 발현 벡터 및 형질전환동물은 기존의 hEPO 보다 우수한 생리활성을 갖는 hEPO 단백질을 생산하는데 유용하게 사용될 수 있다.In addition, the administration of the hEPO protein produced from the transgenic animal of the present invention shows an effect of increasing platelets, red blood cells, hemoglobin, hematocrit in the blood (hematocrit). Therefore, the hEPO expression vector and the transgenic animal of the present invention can be usefully used to produce hEPO protein having superior physiological activity than conventional hEPO.

<110> CHO-A PHARM Co., LTD. <120> Mammary gland-specific human erythropoietin expression vector, transgenic animal and method for producing human erythropoietin using same <130> 05p-295 <160> 22 <170> KopatentIn 1.71 <210> 1 <211> 2148 <212> DNA <213> Homo sapiens <220> <221> gene <222> (1)..(2148) <223> human erythropoietin <400> 1 atgggggtgc acggtgagta ctcgcgggct gggcgctccc gccgcccggg tccctgtttg 60 agcggggatt tagcgccccg gctattggcc aggaggtggc tgggttcaag gaccggcgac 120 ttgtcaagga ccccggaagg gggagggggg tggggcagcc tccacgtgcc agcggggact 180 tgggggagtc cttggggatg gcaaaaacct gacctgtgaa ggggacacag tttgggggtt 240 gaggggaaga aggtttgggg gttctgctgt gccagtggag aggaagctga taagctgata 300 acctgggcgc tggagccacc acttatctgc cagaggggaa gcctctgtca caccaggatt 360 gaagtttggc cggagaagtg gatgctggta gctgggggtg gggtgtgcac acggcagcag 420 gattgaatga aggccaggga ggcagcacct gagtgcttgc atggttgggg acaggaagga 480 cgagctgggg cagagacgtg gggatgaagg aagctgtcct tccacagcca cccttctccc 540 tccccgcctg actctcagcc tggctatctg ttctagaatg tcctgcctgg ctgtggcttc 600 tcctgtccct gctgtcgctc cctctgggcc tcccagtcct gggcgcccca ccacgcctca 660 tctgtgacag ccgagtcctg gagaggtacc tcttggaggc caaggaggcc gagaatatca 720 cggtgagacc ccttccccag cacattccac agaactcacg ctcagggctt cagggaactc 780 ctcccagatc caggaacctg gcacttggtt tggggtggag ttgggaagct agacactgcc 840 cccctacata agaataagtc tggtggcccc aaaccatacc tggaaactag gcaaggagca 900 aagccagcag atcctacggc ctgtgggcca gggccagagc cttcagggac ccttgactcc 960 ccgggctgtg tgcatttcag acgggctgtg ctgaacactg cagcttgaat gagaatatca 1020 ctgtcccaga caccaaagtt aatttctatg cctggaagag gatggaggtg agttcctttt 1080 tttttttttt tcctttcttt tggagaatct catttgcgag cctgattttg gatgaaaggg 1140 agaatgatcg ggggaaaggt aaaatggagc agcagagatg aggctgcctg ggcgcagagg 1200 ctcacgtcta taatcccagg ctgagatggc cgagatggga gaattgcttg agccctggag 1260 tttcagacca acctaggcag catagtgaga tcccccatct ctacaaacat ttaaaaaaat 1320 tagtcaggtg aagtggtgca tggtggtagt cccagatatt tggaaggctg aggcgggagg 1380 atcgcttgag cccaggaatt tgaggctgca gtgagctgtg atcacaccac tgcactccag 1440 cctcagtgac agagtgaggc cctgtctcaa aaaagaaaag aaaaaagaaa aataatgagg 1500 gctgtatgga atacattcat tattcattca ctcactcact cactcattca ttcattcatt 1560 cattcaacaa gtcttattgc ataccttctg tttgctcagc ttggtgcttg gggctgctga 1620 ggggcaggag ggagagggtg acatgggtca gctgactccc agagtccact ccctgtaggt 1680 cgggcagcag gccgtagaag tctggcaggg cctggccctg ctgtcggaag ctgtcctgcg 1740 gggccaggcc ctgttggtca actcttccca gccgtgggag cccctgcagc tgcatgtgga 1800 taaagccgtc agtggccttc gcagcctcac cactctgctt cgggctctgg gagcccaggt 1860 gagtaggagc ggacacttct gcttgccctt tctgtaagaa ggggagaagg gtcttgctaa 1920 ggagtacagg aactgtccgt attccttccc tttctgtggc actgcagcga cctcctgttt 1980 tctccttggc agaaggaagc catctcccct ccagatgcgg cctcagctgc tccactccga 2040 acaatcactg ctgacacttt ccgcaaactc ttccgagtct actccaattt cctccgggga 2100 aagctgaagc tgtacacagg ggaggcctgc aggacagggg acagatga 2148 <210> 2 <211> 4101 <212> DNA <213> goat <220> <221> promoter <222> (1)..(4101) <223> beta-casein promoter <400> 2 ggatccctcg acctgcaggt caacggatca caacaaactg gaaaattctt caagagaaga 60 ataccagacc accctacctg cttcctgaga aatctgtttg ctgctcagaa gcaacagtta 120 gaaccagaca tggaacaaca gactggttcc aaatcaggaa aggagtatgt caaggctgta 180 tatcgtcacc ctgattattt aacttatatg catagtacat aatacaaaat gccaggctgg 240 atgaatcgca agctggaatc aagatttctg ggagaaatat caataaacga gatacaaaga 300 tacaccacac ttatggcaga aaactaagaa gaactaaaga gcctcttgat gaaagtgaaa 360 gaggagagtg aaaaagccag cttaaaaccc aacattcaaa atcaagatca tcatttcatg 420 gcaaataaat ggggaaacaa tggaaacagt gagagacttt attttcttgg gctccaaaat 480 cactgcagat tgtgactaca gccatgatta aaagatgctt gctccttgga agagaagcta 540 ttaccaaact agaaagcata ttaaaaagca gagacgttac tttgctgact aagttctgtc 600 tagtcaaacc tatggttttt ccagtagtca tatatggatg tgagttgaac tataaagaaa 660 gctgagcacc aaagaattga tgcttttgaa atttggtgtt ggagaagtct cttgagagtc 720 ccttgaacct gcaaggagat ccaaccagtc catcctaaag gaaatcagtc ctgaatattc 780 attggaagga ctgatgctga aattgaagat taacgttttg gactcaccta atgcagaaga 840 gccaactcac tagaaaagac cccatgttgg caaaaattga agccaggaag agaagtgaat 900 gacagaggat gagatggttg gatggcatcg ttgactgaat ggacatgagt ctgatcaagt 960 tccgggagac agcaaaggac agggctgcct ggtctgctgc agtccatggg gttgcaaaga 1020 gtcggtctca aatgagtaac taaacaacaa ccaagcagta gaaaaataaa taaaatttgt 1080 ctctgagatc tcagtacctc tttctgtgca tatccgtctc ctgttattgt actttgtctt 1140 ctgcttgtaa taaagctgtc ctgttagtaa aatctgtttg ggtcctctga attcttttag 1200 ctatcaaaaa tggaaggtga ttattgtgca atgtccacct ctgagtaata tacagagaat 1260 aaaagaaggg agaaattatg tgcaagttct ctctcatctc ctgcttctca tttaaaagat 1320 tctacctcag tgggggctaa aactccacat ttaacagtag caaaaaccaa tattccatag 1380 cttcttagga aaccattttt tatactcttg tatgtaatta cattcaagct caaaagcaaa 1440 gaagtgattc tgcgttggtg aaggcccaac catagaaaag aggaagaaaa taggccacat 1500 actgtgcttc ccccatagct cagttggtaa agaatctacc tacaatgcag gaggcctggg 1560 cttgatccct gggtaaggga gatcccctgg agaaggaaat ggtaacccac tccagtactc 1620 ttgcctgtaa atcccatgga cggaggagcc tggcagctac agccttgggg tggcaagagt 1680 tggacatgat taacaactaa accactgcca ccactccaca tactgagtgc tccccagtgg 1740 cactagtggt aaagaaccac ctgccggtgc agaagacatt aaagacactg gctctatccc 1800 tgcttgggaa gtagggaaga tcccctagag agggaaatag caacccactc cagaattctt 1860 gcctggaaaa tcccatgaat gaagactggc gggctgtagt aactggggtc acaaagagtt 1920 aaacatgatt tagcaactaa acatcaccac attaaaaaaa ttaccaccaa aatagtcata 1980 ttccaggcta aggggaataa tagcactagt acctgagaga actttctcag attctctgtc 2040 aagttcttcc ttctctcata taaccagtag tctagtttac ctcatcagat attaactact 2100 catcgattct aaattatcta attatggggg ggggcactac attgcattat attttgtgtc 2160 cattgactat cactcaattt atttataaaa aattcatcca tgttgtttct gtgacagtaa 2220 ctcattcaca ttaattgtaa tatctcattg cattgtatac tacaatttat ttatacaaaa 2280 tactattatt cacacttctg ttgattttaa tttggaacat caacaataac gtggctgaga 2340 agcttctttc tttagtatat tgttaaggat ttccttgatc aagattttac ctacttttct 2400 ggtccaattg gtgagagaca gtcataagga aatgctgtgt ttattgcaca atatgtaaag 2460 catcttcctg agaaaataaa agggaaatgt tgaatgggaa ggatatgctt tcttttgtat 2520 tccttttctg agaaatcaga ctttttcacc ttggccttgg ccaccaaaag ctaacaaata 2580 aaggcatatg aagtagccaa ggccttttct agttatatct atgacactga gttcatttca 2640 tcatttattt tcctgacttc ctcctgggtc catatgagca gtcttagaat gaatattagc 2700 tgaataatcc aaatacatag tagatgttga tttgggtttt ctaagcaatc caagacttgt 2760 atgacagtaa gatgtattac catccaacac acatctcagc atgatataaa tgcaaggtat 2820 attgtgaaga aaaattttta attatgtcaa agtgcttact ttagaaggtc atctatctgt 2880 cccaaagctg tgaatatata tattgaaggt aatgaataga tgaagctaac cttgtaaaaa 2940 tgagtagtgt gaaatacaac tacaattatg aacatctgtc actaaagagg caaagaaact 3000 tgaagattgc ttttgcaaat gggctcctat taataaaaag tacttttgag gtctggctca 3060 gactctattg tagtacttag ggtaagaccc tcctcctgta tgggctttca ttttctttct 3120 tgcttccctc atttgccctt ccatgaatac tagctgataa acattgacta taaaagatat 3180 gaggccaaac ttgagctgtc ccattttaat aaatctgtat aaataatatt tgttctacaa 3240 aagtattatc taaataaatg ttactttctg tcttaaaatc cctcaacaaa tccccactat 3300 ctagagaata agattgacat tccctggaat cacagcatgc tttgtctgcc attatctgac 3360 ccctttctct ttctctcttc tcacctccat ctactccttt ttccttgcaa ttcatgaccc 3420 agattcactg tttgatttgg cttgcatgtg tgtgtgctga gttgcgtctg actgttatca 3480 accccatgaa tgatagtcca ccaggctcta ctgtccatga aattttccag tcaagaatac 3540 tggagtggat tgcatttcct actccatttg attaatttag tgacttttaa atttcttttt 3600 ccatattcgg gagcctattc ttccttttta gtctatactc tcttcactct tcaggtctaa 3660 ggtatcatcg tgtgcttgtt agcttgttac tttctccatt atagcttaag cactaacaac 3720 tgttcaggtt ggcatgaaat tgtgttcttt gtgtggcctg tatatttctg ttgtgtatta 3780 gaatttaccc caagatctca aagacccact gaatactaaa gagacctcat tgtggttaca 3840 ataatttggg gactgggcca aaacttccgt gcatcccagc caagatctgt agctactgga 3900 caatttcatt tcctttatca gattgtgagt tattcctgtt aaaatgctcc ccagaatttc 3960 tggggacaga aaaataggaa gaattcattt cctaatcatg cagatttcta ggaattcaaa 4020 tccactgttg gttttatttc aaaccacaaa attagcatgc cattaaatac tatatataaa 4080 cagccactaa atcagatcat t 4101 <210> 3 <211> 632 <212> DNA <213> Woodchuck hepatitis B virus <220> <221> misc_signal <222> (1)..(632) <223> Woodchuck heptitis virus posttranscriptional regulatory element <400> 3 accaggttct gttcctgtta atcaacctct ggattacaaa atttgtgaaa gattgactgg 60 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta 120 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct 180 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt 240 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac 300 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg 360 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaagctgac 420 gtcctttcca tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg 480 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct 540 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc 600 ctccccgcct gtttcgcctc gggctcctcg ag 632 <210> 4 <211> 1916 <212> DNA <213> Artificial Sequence <220> <223> neomycin resistance gene <400> 4 gcggccgcgc gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt 60 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct 120 tcaataatat tgaaaaagga agagtcctga ggcggaaaga accagctgtg gaatgtgtgt 180 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat 240 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg 300 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg 360 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt 420 tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt 480 tttggaggcc taggcttttg caaagatcga tcaagagaca ggatgaggat cgtttcgcat 540 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg 600 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc 660 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca 720 agacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct 780 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga 840 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg 900 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat 960 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga 1020 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg 1080 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg 1140 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat 1200 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct 1260 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga 1320 cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg 1380 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt 1440 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc 1500 caccctaggg ggaggctaac tgaaacacgg aaggagacaa taccggaagg aacccgcgct 1560 atgacggcaa taaaaagaca gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc 1620 ggggttcggt cccagggctg gcactctgtc gataccccac cgagacccca ttggggccaa 1680 tacgcccgcg tttcttcctt ttccccaccc caccccccaa gttcgggtga aggcccaggg 1740 ctcgcagcca acgtcggggc ggcaggccct gccatagcct caggttactc atatatactt 1800 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat 1860 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc cgatcg 1916 <210> 5 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> WPRE forward primer <400> 5 accaggttct gttcctgtta atcaacctc 29 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> WPRE reverse primer <400> 6 ctcgaggagc ccgaggcgaa acaggcg 27 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Neomycin resistant gene forward primer <400> 7 gcggccgcgc gcgtcaggtg gcac 24 <210> 8 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Neomycin resistant gene reverse primer <400> 8 cgatcggacg ctcagtggaa cgaaaactc 29 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mouse GnT-V Forward primer <400> 9 cactgttaat tcgcccacct 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mouse GnT-V Reverse primer <400> 10 gcttggtcct cctgactctg 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CHO GnT-V Forward primer <400> 11 gtacagagtg acctgccaaa 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CHO GnT-V Reverse primer <400> 12 gtctttgcat agggccactt 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mouse GnT-III Forward primer <400> 13 gctcaggcct ctagtaatct 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mouse GnT-III Reverse primer <400> 14 tcctgacccc taacctactc 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CHO GnT-III Forward primer <400> 15 gcatctactt caarctcgtg 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CHO GnT-III Reverse primer <400> 16 gtgctcrtgg gctcccggta 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mouse & CHO Fuc-TIV Forward primer <400> 17 cacacdgtgg cccgctacaa 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mouse & CHO Fuc-TIV Reverse primer <400> 18 tcccagaarg argtgatgtg 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for transgene check <400> 19 ctccttggca gaaggaagcc 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for transgene check <400> 20 cagccatgga aaggacgtca 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hEPO specific forward primer <400> 21 gtagaagtct ggcagggcct 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hEPO specific reverse primer <400> 22 tcatctgtcc cctgtcctgc 20 <110> CHO-A PHARM Co., LTD. <120> Mammary gland-specific human erythropoietin expression vector,          transgenic animal and method for producing human erythropoietin          using same <130> 05p-295 <160> 22 <170> KopatentIn 1.71 <210> 1 <211> 2148 <212> DNA <213> Homo sapiens <220> <221> gene (222) (1) .. (2148) <223> human erythropoietin <400> 1 atgggggtgc acggtgagta ctcgcgggct gggcgctccc gccgcccggg tccctgtttg 60 agcggggatt tagcgccccg gctattggcc aggaggtggc tgggttcaag gaccggcgac 120 ttgtcaagga ccccggaagg gggagggggg tggggcagcc tccacgtgcc agcggggact 180 tgggggagtc cttggggatg gcaaaaacct gacctgtgaa ggggacacag tttgggggtt 240 gaggggaaga aggtttgggg gttctgctgt gccagtggag aggaagctga taagctgata 300 acctgggcgc tggagccacc acttatctgc cagaggggaa gcctctgtca caccaggatt 360 gaagtttggc cggagaagtg gatgctggta gctgggggtg gggtgtgcac acggcagcag 420 gattgaatga aggccaggga ggcagcacct gagtgcttgc atggttgggg acaggaagga 480 cgagctgggg cagagacgtg gggatgaagg aagctgtcct tccacagcca cccttctccc 540 tccccgcctg actctcagcc tggctatctg ttctagaatg tcctgcctgg ctgtggcttc 600 tcctgtccct gctgtcgctc cctctgggcc tcccagtcct gggcgcccca ccacgcctca 660 tctgtgacag ccgagtcctg gagaggtacc tcttggaggc caaggaggcc gagaatatca 720 cggtgagacc ccttccccag cacattccac agaactcacg ctcagggctt cagggaactc 780 ctcccagatc caggaacctg gcacttggtt tggggtggag ttgggaagct agacactgcc 840 cccctacata agaataagtc tggtggcccc aaaccatacc tggaaactag gcaaggagca 900 aagccagcag atcctacggc ctgtgggcca gggccagagc cttcagggac ccttgactcc 960 ccgggctgtg tgcatttcag acgggctgtg ctgaacactg cagcttgaat gagaatatca 1020 ctgtcccaga caccaaagtt aatttctatg cctggaagag gatggaggtg agttcctttt 1080 tttttttttt tcctttcttt tggagaatct catttgcgag cctgattttg gatgaaaggg 1140 agaatgatcg ggggaaaggt aaaatggagc agcagagatg aggctgcctg ggcgcagagg 1200 ctcacgtcta taatcccagg ctgagatggc cgagatggga gaattgcttg agccctggag 1260 tttcagacca acctaggcag catagtgaga tcccccatct ctacaaacat ttaaaaaaat 1320 tagtcaggtg aagtggtgca tggtggtagt cccagatatt tggaaggctg aggcgggagg 1380 atcgcttgag cccaggaatt tgaggctgca gtgagctgtg atcacaccac tgcactccag 1440 cctcagtgac agagtgaggc cctgtctcaa aaaagaaaag aaaaaagaaa aataatgagg 1500 gctgtatgga atacattcat tattcattca ctcactcact cactcattca ttcattcatt 1560 cattcaacaa gtcttattgc ataccttctg tttgctcagc ttggtgcttg gggctgctga 1620 ggggcaggag ggagagggtg acatgggtca gctgactccc agagtccact ccctgtaggt 1680 cgggcagcag gccgtagaag tctggcaggg cctggccctg ctgtcggaag ctgtcctgcg 1740 gggccaggcc ctgttggtca actcttccca gccgtgggag cccctgcagc tgcatgtgga 1800 taaagccgtc agtggccttc gcagcctcac cactctgctt cgggctctgg gagcccaggt 1860 gagtaggagc ggacacttct gcttgccctt tctgtaagaa ggggagaagg gtcttgctaa 1920 ggagtacagg aactgtccgt attccttccc tttctgtggc actgcagcga cctcctgttt 1980 tctccttggc agaaggaagc catctcccct ccagatgcgg cctcagctgc tccactccga 2040 acaatcactg ctgacacttt ccgcaaactc ttccgagtct actccaattt cctccgggga 2100 aagctgaagc tgtacacagg ggaggcctgc aggacagggg acagatga 2148 <210> 2 <211> 4101 <212> DNA <213> goat <220> <221> promoter (222) (1) .. (4101) <223> beta-casein promoter <400> 2 ggatccctcg acctgcaggt caacggatca caacaaactg gaaaattctt caagagaaga 60 ataccagacc accctacctg cttcctgaga aatctgtttg ctgctcagaa gcaacagtta 120 gaaccagaca tggaacaaca gactggttcc aaatcaggaa aggagtatgt caaggctgta 180 tatcgtcacc ctgattattt aacttatatg catagtacat aatacaaaat gccaggctgg 240 atgaatcgca agctggaatc aagatttctg ggagaaatat caataaacga gatacaaaga 300 tacaccacac ttatggcaga aaactaagaa gaactaaaga gcctcttgat gaaagtgaaa 360 gaggagagtg aaaaagccag cttaaaaccc aacattcaaa atcaagatca tcatttcatg 420 gcaaataaat ggggaaacaa tggaaacagt gagagacttt attttcttgg gctccaaaat 480 cactgcagat tgtgactaca gccatgatta aaagatgctt gctccttgga agagaagcta 540 ttaccaaact agaaagcata ttaaaaagca gagacgttac tttgctgact aagttctgtc 600 tagtcaaacc tatggttttt ccagtagtca tatatggatg tgagttgaac tataaagaaa 660 gctgagcacc aaagaattga tgcttttgaa atttggtgtt ggagaagtct cttgagagtc 720 ccttgaacct gcaaggagat ccaaccagtc catcctaaag gaaatcagtc ctgaatattc 780 attggaagga ctgatgctga aattgaagat taacgttttg gactcaccta atgcagaaga 840 gccaactcac tagaaaagac cccatgttgg caaaaattga agccaggaag agaagtgaat 900 gacagaggat gagatggttg gatggcatcg ttgactgaat ggacatgagt ctgatcaagt 960 tccgggagac agcaaaggac agggctgcct ggtctgctgc agtccatggg gttgcaaaga 1020 gtcggtctca aatgagtaac taaacaacaa ccaagcagta gaaaaataaa taaaatttgt 1080 ctctgagatc tcagtacctc tttctgtgca tatccgtctc ctgttattgt actttgtctt 1140 ctgcttgtaa taaagctgtc ctgttagtaa aatctgtttg ggtcctctga attcttttag 1200 ctatcaaaaa tggaaggtga ttattgtgca atgtccacct ctgagtaata tacagagaat 1260 aaaagaaggg agaaattatg tgcaagttct ctctcatctc ctgcttctca tttaaaagat 1320 tctacctcag tgggggctaa aactccacat ttaacagtag caaaaaccaa tattccatag 1380 cttcttagga aaccattttt tatactcttg tatgtaatta cattcaagct caaaagcaaa 1440 gaagtgattc tgcgttggtg aaggcccaac catagaaaag aggaagaaaa taggccacat 1500 actgtgcttc ccccatagct cagttggtaa agaatctacc tacaatgcag gaggcctggg 1560 cttgatccct gggtaaggga gatcccctgg agaaggaaat ggtaacccac tccagtactc 1620 ttgcctgtaa atcccatgga cggaggagcc tggcagctac agccttgggg tggcaagagt 1680 tggacatgat taacaactaa accactgcca ccactccaca tactgagtgc tccccagtgg 1740 cactagtggt aaagaaccac ctgccggtgc agaagacatt aaagacactg gctctatccc 1800 tgcttgggaa gtagggaaga tcccctagag agggaaatag caacccactc cagaattctt 1860 gcctggaaaa tcccatgaat gaagactggc gggctgtagt aactggggtc acaaagagtt 1920 aaacatgatt tagcaactaa acatcaccac attaaaaaaa ttaccaccaa aatagtcata 1980 ttccaggcta aggggaataa tagcactagt acctgagaga actttctcag attctctgtc 2040 aagttcttcc ttctctcata taaccagtag tctagtttac ctcatcagat attaactact 2100 catcgattct aaattatcta attatggggg ggggcactac attgcattat attttgtgtc 2160 cattgactat cactcaattt atttataaaa aattcatcca tgttgtttct gtgacagtaa 2220 ctcattcaca ttaattgtaa tatctcattg cattgtatac tacaatttat ttatacaaaa 2280 tactattatt cacacttctg ttgattttaa tttggaacat caacaataac gtggctgaga 2340 agcttctttc tttagtatat tgttaaggat ttccttgatc aagattttac ctacttttct 2400 ggtccaattg gtgagagaca gtcataagga aatgctgtgt ttattgcaca atatgtaaag 2460 catcttcctg agaaaataaa agggaaatgt tgaatgggaa ggatatgctt tcttttgtat 2520 tccttttctg agaaatcaga ctttttcacc ttggccttgg ccaccaaaag ctaacaaata 2580 aaggcatatg aagtagccaa ggccttttct agttatatct atgacactga gttcatttca 2640 tcatttattt tcctgacttc ctcctgggtc catatgagca gtcttagaat gaatattagc 2700 tgaataatcc aaatacatag tagatgttga tttgggtttt ctaagcaatc caagacttgt 2760 atgacagtaa gatgtattac catccaacac acatctcagc atgatataaa tgcaaggtat 2820 attgtgaaga aaaattttta attatgtcaa agtgcttact ttagaaggtc atctatctgt 2880 cccaaagctg tgaatatata tattgaaggt aatgaataga tgaagctaac cttgtaaaaa 2940 tgagtagtgt gaaatacaac tacaattatg aacatctgtc actaaagagg caaagaaact 3000 tgaagattgc ttttgcaaat gggctcctat taataaaaag tacttttgag gtctggctca 3060 gactctattg tagtacttag ggtaagaccc tcctcctgta tgggctttca ttttctttct 3120 tgcttccctc atttgccctt ccatgaatac tagctgataa acattgacta taaaagatat 3180 gaggccaaac ttgagctgtc ccattttaat aaatctgtat aaataatatt tgttctacaa 3240 aagtattatc taaataaatg ttactttctg tcttaaaatc cctcaacaaa tccccactat 3300 ctagagaata agattgacat tccctggaat cacagcatgc tttgtctgcc attatctgac 3360 ccctttctct ttctctcttc tcacctccat ctactccttt ttccttgcaa ttcatgaccc 3420 agattcactg tttgatttgg cttgcatgtg tgtgtgctga gttgcgtctg actgttatca 3480 accccatgaa tgatagtcca ccaggctcta ctgtccatga aattttccag tcaagaatac 3540 tggagtggat tgcatttcct actccatttg attaatttag tgacttttaa atttcttttt 3600 ccatattcgg gagcctattc ttccttttta gtctatactc tcttcactct tcaggtctaa 3660 ggtatcatcg tgtgcttgtt agcttgttac tttctccatt atagcttaag cactaacaac 3720 tgttcaggtt ggcatgaaat tgtgttcttt gtgtggcctg tatatttctg ttgtgtatta 3780 gaatttaccc caagatctca aagacccact gaatactaaa gagacctcat tgtggttaca 3840 ataatttggg gactgggcca aaacttccgt gcatcccagc caagatctgt agctactgga 3900 caatttcatt tcctttatca gattgtgagt tattcctgtt aaaatgctcc ccagaatttc 3960 tggggacaga aaaataggaa gaattcattt cctaatcatg cagatttcta ggaattcaaa 4020 tccactgttg gttttatttc aaaccacaaa attagcatgc cattaaatac tatatataaa 4080 cagccactaa atcagatcat t 4101 <210> 3 <211> 632 <212> DNA <213> Woodchuck hepatitis B virus <220> <221> misc_signal (222) (1) .. (632) <223> Woodchuck heptitis virus posttranscriptional regulatory element <400> 3 accaggttct gttcctgtta atcaacctct ggattacaaa atttgtgaaa gattgactgg 60 tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta 120 tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct 180 gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt 240 tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac 300 tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg 360 ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaagctgac 420 gtcctttcca tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg 480 ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct 540 gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc 600 ctccccgcct gtttcgcctc gggctcctcg ag 632 <210> 4 <211> 1916 <212> DNA <213> Artificial Sequence <220> <223> neomycin resistance gene <400> 4 gcggccgcgc gcgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt 60 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct 120 tcaataatat tgaaaaagga agagtcctga ggcggaaaga accagctgtg gaatgtgtgt 180 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat 240 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg 300 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg 360 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt 420 tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt 480 tttggaggcc taggcttttg caaagatcga tcaagagaca ggatgaggat cgtttcgcat 540 gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg 600 ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc 660 gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca 720 agacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct 780 cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga 840 tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg 900 gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat 960 cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga 1020 gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg 1080 cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg 1140 ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat 1200 agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct 1260 cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga 1320 cgagttcttc tgagcgggac tctggggttc gaaatgaccg accaagcgac gcccaacctg 1380 ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt 1440 ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc 1500 caccctaggg ggaggctaac tgaaacacgg aaggagacaa taccggaagg aacccgcgct 1560 atgacggcaa taaaaagaca gaataaaacg cacggtgttg ggtcgtttgt tcataaacgc 1620 ggggttcggt cccagggctg gcactctgtc gataccccac cgagacccca ttggggccaa 1680 tacgcccgcg tttcttcctt ttccccaccc caccccccaa gttcgggtga aggcccaggg 1740 ctcgcagcca acgtcggggc ggcaggccct gccatagcct caggttactc atatatactt 1800 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat 1860 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc cgatcg 1916 <210> 5 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> WPRE forward primer <400> 5 accaggttct gttcctgtta atcaacctc 29 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> WPRE reverse primer <400> 6 ctcgaggagc ccgaggcgaa acaggcg 27 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Neomycin resistant gene forward primer <400> 7 gcggccgcgc gcgtcaggtg gcac 24 <210> 8 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Neomycin resistant gene reverse primer <400> 8 cgatcggacg ctcagtggaa cgaaaactc 29 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mouse GnT-V Forward primer <400> 9 cactgttaat tcgcccacct 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mouse GnT-V Reverse primer <400> 10 gcttggtcct cctgactctg 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CHO GnT-V Forward primer <400> 11 gtacagagtg acctgccaaa 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CHO GnT-V Reverse primer <400> 12 gtctttgcat agggccactt 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mouse GnT-III Forward primer <400> 13 gctcaggcct ctagtaatct 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mouse GnT-III Reverse primer <400> 14 tcctgacccc taacctactc 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CHO GnT-III Forward primer <400> 15 gcatctactt caarctcgtg 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CHO GnT-III Reverse primer <400> 16 gtgctcrtgg gctcccggta 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mouse & CHO Fuc-TIV Forward primer <400> 17 cacacdgtgg cccgctacaa 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mouse & CHO Fuc-TIV Reverse primer <400> 18 tcccagaarg argtgatgtg 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for transgene check <400> 19 ctccttggca gaaggaagcc 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for transgene check <400> 20 cagccatgga aaggacgtca 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hEPO specific forward primer <400> 21 gtagaagtct ggcagggcct 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hEPO specific reverse primer <400> 22 tcatctgtcc cctgtcctgc 20

Claims (13)

유선 특이적 프로모터인 베타-카제인 프로모터 염기서열, 그 3'쪽으로 인간 에리트로포이에틴(hEPO)을 코딩하는 DNA 서열 및 hEPO 유전자의 3'쪽으로 WPRE DNA 서열을 포함하는 pBC1-hEPO-WPRE 벡터.A pBC1-hEPO-WPRE vector comprising a sequential specific beta-casein promoter sequence, a DNA sequence encoding human erythropoietin (hEPO) towards 3 'and a WPRE DNA sequence towards 3' of hEPO gene. 제 1항에 있어서, 상기 hEPO 을 코딩하는 DNA 서열은 서열번호 1에 기재된 것인 pBC1-hEPO-WPRE 벡터.The pBC1-hEPO-WPRE vector of claim 1, wherein the DNA sequence encoding hEPO is set forth in SEQ ID NO: 1. 제2항에 있어서, 상기 베타-카제인 프로모터 DNA 서열은 서열번호 2에 기재된 것인 pBC1-hEPO-WPRE 벡터.3. The pBC1-hEPO-WPRE vector of claim 2, wherein the beta-casein promoter DNA sequence is set forth in SEQ ID NO: 2. 제3항에 있어서, 상기 WPRE DNA 서열은 서열번호 3에 기재된 것인 pBC1-hEPO-WPRE 벡터.4. The pBC1-hEPO-WPRE vector of claim 3, wherein the WPRE DNA sequence is set forth in SEQ ID NO: 3. 제 1항의 벡터에 선택적 표지 유전자로 네오마이신 저항성 유전자를 추가로 포함하는 pBC1/hEPO/NEO 벡터.A pBC1 / hEPO / NEO vector further comprising a neomycin resistance gene as a selective marker gene in the vector of claim 1. 제5항에 있어서, 상기 네오마이신 저항성 유전자는 서열번호 4에 기재된 것인 pBC1/hEPO/NEO 벡터.The pBC1 / hEPO / NEO vector of claim 5, wherein the neomycin resistance gene is set forth in SEQ ID NO: 4. 7. 제6항에 있어서, 기탁번호 KACC 95043P로 기탁된 것인 pBC1/hEPO/NEO 벡터.The pBC1 / hEPO / NEO vector according to claim 6, deposited with accession number KACC 95043P. 제 1항 내지 제7항 중 어느 한 항의 발현 벡터를 도입시켜 형질전환시킨 체세포.       A somatic cell transformed by introducing the expression vector of any one of claims 1 to 7. 제 1항 내지 제7항 중 어느 한 항의 발현 벡터를 도입시켜 형질전환시킨 체세포를 탈핵된 난자에 핵이식하여 만든 수정란.       A fertilized egg made by transfecting somatic cells transformed by introducing the expression vector of any one of claims 1 to 7 into denuclearized eggs. 제 1항 내지 제7항 중 어느 한 항의 발현 벡터를 도입시켜 형질전환시킨 동물.       An animal transformed by introducing the expression vector of any one of claims 1 to 7. 제10항에 있어서, 상기 형질전환 동물은 돼지, 생쥐, 소, 염소, 양 및 닭 중 선택된 하나인 형질전환 동물.The transgenic animal of claim 10, wherein the transgenic animal is one selected from pigs, mice, cows, goats, sheep, and chickens. 제10항의 형질전환동물로부터 생산된 유즙 중 발현된 에리트로포이에틴을 분리, 정제하는 단계로 이루어진 인간 에리트로포이에틴의 생산방법.A method for producing human erythropoietin, comprising separating and purifying erythropoietin expressed in milk produced from the transgenic animal of claim 10. 제12항에 있어서, 상기 형질전환 동물은 돼지, 생쥐, 소, 염소, 양 및 닭 중 선택된 하나인 인간 에리트로포이에틴의 생산방법.The method of claim 12, wherein the transgenic animal is one selected from pigs, mice, cattle, goats, sheep, and chickens.
KR1020060013485A 2006-02-13 2006-02-13 Mammary gland-specific human erythropoietin expression vector transgenic animal and method for producing human erythropoietin using same KR100769291B1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
KR1020060013485A KR100769291B1 (en) 2006-02-13 2006-02-13 Mammary gland-specific human erythropoietin expression vector transgenic animal and method for producing human erythropoietin using same

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
KR1020060013485A KR100769291B1 (en) 2006-02-13 2006-02-13 Mammary gland-specific human erythropoietin expression vector transgenic animal and method for producing human erythropoietin using same

Publications (2)

Publication Number Publication Date
KR20070081531A true KR20070081531A (en) 2007-08-17
KR100769291B1 KR100769291B1 (en) 2007-10-24

Family

ID=38611414

Family Applications (1)

Application Number Title Priority Date Filing Date
KR1020060013485A KR100769291B1 (en) 2006-02-13 2006-02-13 Mammary gland-specific human erythropoietin expression vector transgenic animal and method for producing human erythropoietin using same

Country Status (1)

Country Link
KR (1) KR100769291B1 (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009020252A1 (en) * 2007-08-08 2009-02-12 Cho-A Pharm Co., Ltd. Mammary gland-specific human erythropoietin expression vector, transgenic animal and method for producing human erythropoietin using same
KR101033819B1 (en) * 2008-06-30 2011-05-13 조아제약주식회사 A Gene of porcine beta casein, a Promoter of the same and Use thereof
WO2011081343A3 (en) * 2009-12-30 2011-11-17 한화엘앤씨 주식회사 Transgenic pig in which ho-1 genes and tnfr1-fc genes are simultaneously expressed, and method for producing same
KR101219794B1 (en) * 2010-08-20 2013-01-10 국립암센터 Composition for diagnosing non-small cell lung cancer comprising an agent for determining level of methylation of HOXA11 gene and a method for diagnosing non-small cell lung cancer using the same

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010002160A2 (en) 2008-06-30 2010-01-07 조아제약주식회사 Gene of porcine alpha-s1 casein, a promoter of the same and use thereof

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5959171A (en) 1994-08-17 1999-09-28 Pharming B.V. Method for the production of biologically active polypeptides in a mammal's
KR100358754B1 (en) * 2000-02-14 2002-11-07 대한민국 The production method of transgenic porcine producing human erythropoietin and the transgenic porcine
KR100552634B1 (en) * 2002-11-04 2006-02-20 조아제약주식회사 Porcine uroplakin ? promoter and the production method of useful proteins using said promoter
KR20040101793A (en) * 2003-05-27 2004-12-03 재단법인서울대학교산학협력재단 Transgenic cloned cow producing human erythropoietin and method for producing the same

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009020252A1 (en) * 2007-08-08 2009-02-12 Cho-A Pharm Co., Ltd. Mammary gland-specific human erythropoietin expression vector, transgenic animal and method for producing human erythropoietin using same
KR101033819B1 (en) * 2008-06-30 2011-05-13 조아제약주식회사 A Gene of porcine beta casein, a Promoter of the same and Use thereof
WO2011081343A3 (en) * 2009-12-30 2011-11-17 한화엘앤씨 주식회사 Transgenic pig in which ho-1 genes and tnfr1-fc genes are simultaneously expressed, and method for producing same
KR101312829B1 (en) * 2009-12-30 2013-09-27 한화엘앤씨 주식회사 Transgenic pig expressing HO-1 and TNFR1-Fc and the method of producing thereof
US9018439B2 (en) 2009-12-30 2015-04-28 Hanwha Advanced Materials Corporation Transgenic pig in which HO-1 and TNFR1-Fc are simultaneously expressed, and method for producing same
KR101219794B1 (en) * 2010-08-20 2013-01-10 국립암센터 Composition for diagnosing non-small cell lung cancer comprising an agent for determining level of methylation of HOXA11 gene and a method for diagnosing non-small cell lung cancer using the same

Also Published As

Publication number Publication date
KR100769291B1 (en) 2007-10-24

Similar Documents

Publication Publication Date Title
Cui et al. Inactivation of Stat5 in mouse mammary epithelium during pregnancy reveals distinct functions in cell proliferation, survival, and differentiation
Park et al. Recombinant human erythropoietin produced in milk of transgenic pigs
CN104561095A (en) Preparation method for transgenic mice capable of producing human nerve growth factor
EP1893756A2 (en) Elimination of n-glycolylneuraminic acid from mammalian products for human use
AU652355B2 (en) Improved expression of polypeptides
EP1870472A1 (en) Model animal of Alzheimer&#39;s disease and use thereof
KR20070081531A (en) Mammary gland-specific human erythropoietin expression vector, transgenic animal and method for producing human erythropoietin using same
US20110077208A1 (en) Expression of secreted human alpha-fetoprotein in transgenic animals
CN101855352A (en) Mammary gland-specific human erythropoietin expression vector, transgenic animal and method for producing human erythropoietin using same
CN106978416B (en) Gene positioning integration expression system and application thereof
KR100552634B1 (en) Porcine uroplakin ? promoter and the production method of useful proteins using said promoter
US10717991B2 (en) Transgenic pig which simultaneously expresses HO-1 gene and TNFR1-Fc gene, and comprises knocked-out GGTA1 gene, and use thereof
JP2004500879A (en) Renal regulatory elements and methods of their use
US7115796B2 (en) Transgenic pig containing heat shock protein 70 transgene
JP5771240B2 (en) Immunodeficient pig
KR100222916B1 (en) A lacteal gland specific expression system which uses betacasein promoter of korean goat
JP2002142610A (en) Als model rat
KR20040101793A (en) Transgenic cloned cow producing human erythropoietin and method for producing the same
EP3020273B1 (en) A method of producing biotechnological drugs using transgenic bovine animals
US10036041B2 (en) Method for positioning and integrating transgene and use thereof
Li et al. Inactivation of Stat5 in Mouse Mammary
Proetzel Functional analysis of transforming growth factor-beta-3 using gene targeting
WO2002102998A1 (en) Animal with gene hypoexpression
MXPA03001281A (en) Use of a transgene encoding a vertebrate phytase to increase capacity to utilize phytic acid in livestock feed.
Fine Expression of Escherichia coli beta-galactosidase in transgenic mice: An approach to cell lineage analysis

Legal Events

Date Code Title Description
A201 Request for examination
E902 Notification of reason for refusal
N231 Notification of change of applicant
E902 Notification of reason for refusal
E701 Decision to grant or registration of patent right
GRNT Written decision to grant
FPAY Annual fee payment

Payment date: 20130108

Year of fee payment: 7

FPAY Annual fee payment

Payment date: 20140107

Year of fee payment: 8

FPAY Annual fee payment

Payment date: 20160108

Year of fee payment: 10

FPAY Annual fee payment

Payment date: 20170109

Year of fee payment: 11

FPAY Annual fee payment

Payment date: 20180108

Year of fee payment: 12

FPAY Annual fee payment

Payment date: 20190108

Year of fee payment: 13

FPAY Annual fee payment

Payment date: 20200108

Year of fee payment: 14