KR100302316B1 - in vitro cultivation method of fertilized egg and use thereof - Google Patents
in vitro cultivation method of fertilized egg and use thereof Download PDFInfo
- Publication number
- KR100302316B1 KR100302316B1 KR1019990020458A KR19990020458A KR100302316B1 KR 100302316 B1 KR100302316 B1 KR 100302316B1 KR 1019990020458 A KR1019990020458 A KR 1019990020458A KR 19990020458 A KR19990020458 A KR 19990020458A KR 100302316 B1 KR100302316 B1 KR 100302316B1
- Authority
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- South Korea
- Prior art keywords
- fertilized
- fertilized eggs
- vitro
- culture
- eggs
- Prior art date
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/02—Breeding vertebrates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61D—VETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS
- A61D19/00—Instruments or methods for reproduction or fertilisation
- A61D19/04—Instruments or methods for reproduction or fertilisation for embryo transplantation
-
- 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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
본 발명은 수정란의 체외배양방법 및 이의 이용에 관한 것으로서, 보다 상세하게는, 생쥐 배아섬유아세포와 공동배양하는 공정을 포함하는 수정란의 체외배양방법과 이렇게 배양된 배반포기 수정란을 포유동물에 형질전환시켜 이로부터 생리활성물질을 생산하는 방법에 관한 것이다. 이러한 본 발명의 한 실시예에 따르면, 인체 락토페린 유전자 DNA를 미세주입한 수정란을 생쥐 배아섬유아세포와 공동배양하였을 때 수정란의 생존율을 종래 7.2%에서 16.7%까지 130% 이상 향상되었으며, 또한 정상적인 송아지 분만율을 종래 56.2%에서 82.6%까지 50% 이상 증가시킬 수 있었다. 그리고, 수정란을 동결 보존 후 융해하여 이식하여도 형질전환 젖소를 생산할 수 있으므로, 시기에 관계없이 수정란을 체외에서 장기보존할 수 있는 효과를 얻을 수 있었다.The present invention relates to an in vitro culture method of fertilized eggs and its use, and more particularly, to an in vitro culture method of fertilized eggs, including the step of co-culture with mouse embryonic fibroblasts and transformed into mammals blastocyst embryo fertilized culture so It relates to a method for producing a bioactive substance therefrom. According to one embodiment of the present invention, when the fertilized eggs microinjected with human lactoferrin gene DNA were co-cultured with mouse embryonic fibroblasts, the survival rate of the fertilized eggs was improved by more than 130% from 7.2% to 16.7%, and also normal calf delivery rate. It can be increased by more than 50% from 56.2% to 82.6%. In addition, since the embryos can be produced by cryopreservation and fusion of the fertilized eggs, transgenic cows can be produced. Therefore, the fertilized eggs can be stored in vitro for a long time regardless of the timing.
Description
본 발명은 수정란의 체외배양방법 및 이의 이용에 관한 것으로서, 보다 상세하게는, 생쥐 배아섬유아세포와 공동배양하는 공정을 포함하는 수정란의 체외배양방법과 이렇게 배양된 배반포기 수정란을 포유동물에 형질전환시켜 이로부터 생리활성물질을 생산하는 방법에 관한 것이다.The present invention relates to an in vitro culture method of fertilized eggs and its use, and more particularly, to an in vitro culture method of fertilized eggs, including the step of co-culture with mouse embryonic fibroblasts and transformed into mammals blastocyst embryo fertilized culture so It relates to a method for producing a bioactive substance therefrom.
일반적으로, 체외에서 수정된 정상적인 수정란이 형질전환용 포유동물의 자궁저부에 착상하기 위해서는 수정후 착상전에 충분한 난분할을 지속하여 배반포기에 이르러야 하며, 이는 배반포의 세포막인 영양막(trophoblast)의 노출된 세포내에만 자궁저부의 세포에 착상하는데 필요한 단백질 효소를 충분히 함유하고 있기 때문이다. 그러나, 체외수정된 수정란은 4 내지 8세포주기에 이르면 그 난분열을 멈춰 형질전환용 젖소의 자궁저부에 착상이 불가능하게 된다. 따라서, 이와 같은 문제를 해결하기 위하여 종래에는 섬유아세포[Voelkel et al., Theriogenology, 24, 271, 1985], 난관상피세포[Eystone et al., Biol. Reprod., 32, Suppl. 100, 1985; Fukui et al., J. Reprod. Fert., 86, 501, 1989; Gandolfi et al., ibid, 81, 23, 1987; Rexroad et al., J. Amin. Sci., 66, 947, 1988], 영양배엽세포[Heyman et al., Theriogenology, 27, 59, 1987; Pool et al., Ann. N.Y. Acad. Sci., 54, 407, 1987], 난구세포[Goto et al., J. Reprod. Fert., 83, 753, 1988], 과립막세포[Goto et al., J. Anim. Sci., 70, 1449, 1992; Han et al., Theriogenology, 42, 645, 1994], 기타 체세포[Carney et al., Mol. Reprod. Devel., 27, 209, 1990; Goto et al., J. Anim. Sci., 70, 1449, 1992] 등과 공동배양하여 수정란을 배반포기까지 난분할을 계속 진행하도록 수정란의 체외발달율을 증가시키는 방법을 이용하여 왔으나, 이들 방법은 여전히 수정란을 대리모에 이식할 수 있도록 배반포기 수정란으로의 발달율을 증가시키는데 한계가 있어 이러한 문제점에 대한 개선의 필요성이 있어왔다.In general, normal fertilized embryos in vitro fertilize the embryo at the base of the transgenic mammal to reach the blastocyst stage after sufficient fertilization after implantation, which is the exposure of the trophoblast, the cell membrane of the blastocyst. This is because only the cells contain enough protein enzymes to implant in the cells of the uterus. In vitro fertilized eggs, however, cease to divide when they reach the 4 to 8 cell cycles, making it impossible to implant them in the uterus bottom of the transgenic cow. Therefore, in order to solve this problem, fibroblasts (Voelkel et al., Theriogenology, 24, 271, 1985), tubal epithelial cells [Eystone et al., Biol. Reprod., 32, Suppl. 100, 1985; Fukui et al., J. Reprod. Fert., 86, 501, 1989; Gandolfi et al., Ibid, 81, 23, 1987; Rexroad et al., J. Amin. Sci., 66, 947, 1988], feeder germ cells [Heyman et al., Theriogenology, 27, 59, 1987; Pool et al., Ann. N.Y. Acad. Sci., 54, 407, 1987], oocytes [Goto et al., J. Reprod. Fert., 83, 753, 1988], granulosa cells [Goto et al., J. Anim. Sci., 70, 1449, 1992; Han et al., Theriogenology, 42, 645, 1994], and other somatic cells [Carney et al., Mol. Reprod. Devel., 27, 209, 1990; Goto et al., J. Anim. Sci., 70, 1449, 1992] have been using co-cultivation methods to increase the in vitro development rate of fertilized eggs so that the fertilization of the fertilized eggs continues to be divided into blastocysts. There is a limit to increasing the rate of development into fertilized eggs, there has been a need for improvement to this problem.
또한, 일반적으로 생리활성물질들은 인체내에서 매우 미량으로 생산, 분비되며, 여러 가지 대사작용 및 조절작용 등에 있어서 필수적인 역할을 한다. 지금까지 알려진 생리활성물질들로는 인슐린(insulin), 인터루킨(interlukins), 조혈생장인자(stem cell factor, granulocyte colony stimulating factor,erythropoietin 등), 각종 호르몬 등이 있다. 이러한 생리활성물질들은 그 중요성에 비해 지금까지 산업화되지 못하여 왔는데 이는 인체내에 극미량으로 존재하기 때문에 분리 및 정제가 힘들었기 때문이었다. 이러한 생리활성물질의 종래 생산방법으로는 대장균을 이용한 발현방법[대한민국 특허공고 제94-5585호]이 있으며, 이러한 대장균이나 다른 원핵생물을 이용한 발현시스템은 저렴한 비용으로 대량생산할 수 있는 장점이 있으나, 이 발현시스템은 원핵생물을 이용하기 때문에 진핵생물에서 단백질합성 이후에 이루어지는 당화(glycosylation)와 같은 번역후 변형(post-translational modification) 과정이 수행되지 않는다. 따라서, 이 시스템으로부터 생산된 EPO 등의 인체내 생리활성물질은 그 활성을 유지할 수 없는 경우가 많았고, 더욱이 인체에 투여하기 위하여 선결되어야 할 안정성 문제를 극복하지 못하는 문제점이 있었다.In addition, in general, bioactive substances are produced and secreted in very small amounts in the human body, and play an essential role in various metabolic and regulatory actions. Bioactive substances known to date include insulin (insulin), interlukins (stem cell factor, granulocyte colony stimulating factor, erythropoietin, etc.), various hormones. These bioactive substances have not been industrialized until now because of their importance, because they are present in the human body in trace amounts, making it difficult to isolate and purify them. Conventional production method of such a bioactive material is an expression method using E. coli [Korean Patent Publication No. 94-5585], the expression system using E. coli or other prokaryote has the advantage that can be mass-produced at low cost, Because the expression system uses prokaryotes, post-translational modifications such as glycosylation after protein synthesis in eukaryotes are not performed. Therefore, the physiologically active substances in the human body, such as EPO produced from this system was often unable to maintain its activity, and furthermore, there was a problem that cannot overcome the stability problem to be selected in order to administer to the human body.
또한, 또다른 생리활성물질의 종래 생산방법으로는 상기와 같은 문제점을 해결하기 위하여 진핵생물, 즉 동물세포를 이용한 발현시스템이 있다. 이러한 동물세포 발현시스템은 번역후 변형(post-translational modification) 과정이 수행되기 때문에 이로부터 생산된 생리활성물질의 활성은 유지할 수 있는 장점이 있으나, 여전히 동물세포의 배양을 위한 비용문제와 순수분리정제의 문제점이 있어 왔다.In addition, the conventional production method of another physiologically active substance is an expression system using eukaryotes, that is, animal cells in order to solve the above problems. This animal cell expression system has the advantage of maintaining the activity of physiologically active substances produced therefrom because post-translational modification process is carried out, but there is still a cost problem for the cultivation of animal cells and purified purification There has been a problem.
이에, 본 발명의 발명자들은 이러한 비용문제는 물론 순수분리정제의 문제점을 해결하기 위하여 한국산 재래종 산양 또는 젖소의 유선조직 발현시스템을 이용하여 생리활성물질을 생산하는 방법을 개발한 바 있다[대한민국 특허공개 제 96-31600호 및 대한민국 특허공개 제 98-73991호]. 이러한 형질전환 동물을 이용한 생리활성물질의 생산은 아직까지는 일부 연구소나 기업체에 국한된 기술이기는 하지만 가축번식학 분야에서의 기술발전을 바탕으로 가능하게 되었는데, 예를 들면, 형질전환 젖소를 생산하기 위해서는 난자 (난포란)의 체외성숙, 수정란의 체외수정, 수정란내에 목적하는 생리활성물질-코딩 유전자 미세주입, 체외배양, 형질전환 동물에의 수정란 이식 등[Bowen et al., Aust. J. Biol., 29, 125, 1994; Hill et al., Theriogenology, 37, 222, 1992; Hyttinen et al., Bio/Technol., 12, 606, 1994; Krimpenfort et al., ibid, 9, 844, 1991] 일련의 복합적 기술을 필요로 한다. 여기서, 형질전환 젖소 개발에 관한 과정을 간략히 설명하면, 도축장에서 폐기되는 난소로부터 회수한 난자를 체외에서 성숙시킨 다음(이를 '체외성숙과정(in vitro maturation)'이라 한다), 정자를 처리하여 체외에서 수정(이를 '체외수정과정(in vitro fertilization)'이라 함)하고, 생리활성물질-코딩 유전자를 클로닝시킨 발현벡터를 수정란내 미세주입(microinjection)한 다음, 상기 수정란을 체외에서 배양하는 단계(체외배양과정(in vitro culture))를 거쳐 제조한 수정란을 대리모 또는 수란우(recipient cow) 등에 이식(transplantation)하여 송아지를 생산하는 기술을 포함한다[도 1 참조]. 그러나, 이들 방법은 :Therefore, the inventors of the present invention have developed a method for producing a bioactive material using a mammary tissue expression system of native Korean goat or cow in order to solve such a problem of cost as well as purely purified tablets. 96-31600 and Korean Patent Publication No. 98-73991]. The production of physiologically active substances using these transgenic animals has been possible due to technological advances in the field of livestock breeding, although it is still limited to some research institutes and companies. For example, in order to produce transgenic cows, In vitro maturation of follicles, in vitro fertilization of fertilized eggs, microinjection of the desired bioactive substance-encoding gene in fertilized eggs, in vitro culture, transplantation of fertilized eggs into transgenic animals, etc. [Bowen et al., Aust. J. Biol., 29, 125, 1994; Hill et al., Theriogenology, 37, 222, 1992; Hyttinen et al., Bio / Technol., 12, 606, 1994; Krimpenfort et al., Ibid, 9, 844, 1991] require a series of complex techniques. Here, briefly describe the process for the development of transgenic cows, the oocytes recovered from the ovary discarded in the slaughterhouse are matured in vitro (this is called 'in vitro maturation'), and then treated with sperm in vitro. (In vitro fertilization) and the expression vector cloning the bioactive substance-encoding gene in microinjection (microinjection), and then incubating the fertilized egg in vitro ( It includes a technique for producing a calf by transplanting a fertilized egg prepared through in vitro culture) to a surrogate mother or a recipient cow (see FIG. 1). However, these methods are:
1). 도축우의 난소로부터 회수한 난자를 이용하여 유전자주입 수정란을 생산하는 과정 중에 이식에 공여되는 배반포기 수정란을 다량으로 얻을 수 없었기 때문에 새로운 배양체계의 개발이 필요하며,One). Development of a new culture system is necessary because a large amount of blastocyst embryos donated for transplantation could not be obtained during the production of transgenic fertilized eggs using eggs recovered from slaughtered ovaries.
2). 미세조작에 따른 물리적 손상 때문에 조작되지 않은 수정란의 임신율에비해 낮다는 문제점이 있었고[Hasler et al., Theriogenology, 43, 141, 1995; Kajihara et al., ibid, 37, 233, 1992],2). There was a problem of low fertility compared to the fertility rate of unmanipulated eggs due to physical damage following micromanipulation [Hasler et al., Theriogenology, 43, 141, 1995; Kajihara et al., Ibid, 37, 233, 1992],
3). 수란우에 수정란을 이식하는 과정인 수정란 이식의 최종단계에서 수란우의 발정일자와 일치할 때에는 채란한 수정란을 동결하지 않고 바로 이식할 수 있으나, 1주일에 불과한 수란우의 발정일자와 일치시켜 신선한 수정란을 준비하는 것은 작업 면에서 매우 어려운 일이며, 더욱이, 미세조작 등 실험을 행하는 실험실과 소를 사육하는 장소가 상당히 떨어져 있다는 문제점을 안고 있었다. 따라서, 이러한 문제점을 해결하기 위하여 배반포기의 수정란을 보다 다량으로 얻을 수 있는 방법과 수정란을 동결시켜 장기 보존하는 방법의 개발 필요성이 절실히 대두되고 있는 실정이었다.3). In the final stage of fertilized egg transplantation, which is the process of transplanting fertilized eggs into a fertilized cow, when it coincides with the estrous date of the fertilized cow, the fertilized egg can be transplanted without freezing. To do is very difficult in terms of work, and furthermore, there was a problem that the laboratory for experiments such as micromanipulation and the place for raising cattle were far apart. Therefore, in order to solve this problem, there is an urgent need for development of a method for obtaining a large amount of fertilized embryos and a method of freezing the embryos for long-term preservation.
이에, 본 발명의 발명자들은 상기와 같은 배반포기 수정란의 대량생산의 문제점과 수정란 장기보존의 문제점을 극복하기 위하여 예의 노력한 결과, 수정란을 생쥐 배아섬유아세포와 공동배양함으로써 다량의 수정란이 배반포기까지 발생하도록 할 수 있었으며, 또한 수정란을 완만하게 동결시켜 보존함으로써, 본 발명을 완성하였다.Therefore, the inventors of the present invention, as a result of intensive efforts to overcome the problems of mass production of blastocyst embryo fertilized eggs and problems of long-term preservation of fertilized eggs, co-cultivation of fertilized eggs with mouse embryonic fibroblasts, a large amount of fertilized eggs to blastocyst The present invention was completed by gently freezing the preserved eggs and preserving them.
결국, 본 발명의 주된 목적은 생쥐 배아섬유아세포와 공동배양하는 공정을 포함하는 수정란의 체외배양방법을 제공하는 것이다.After all, the main object of the present invention is to provide a method for in vitro culture of fertilized eggs, including the step of co-culture with mouse embryo fibroblasts.
또한, 본 발명의 또다른 목적은 수정란과 생쥐 배아섬유아세포를 공동배양하는 수정란의 체외배양공정을 포함하는 생리활성물질의 생산방법을 제공하는 것이다.In addition, another object of the present invention is to provide a method for producing a bioactive material comprising an in vitro culture process of fertilized eggs to co-culture fertilized eggs and mouse embryo fibroblasts.
도 1은 일반적인 형질전환 젖소의 개발과정의 모식도를 나타낸 것이다.Figure 1 shows a schematic diagram of the development of a general transgenic cow.
도 2은 동결수정란으로부터 태어난 형질전환 송아지의 게놈 DNA에 대한 서던블럿팅(Southern blotting)의 결과이다.FIG. 2 shows the results of Southern blotting of genomic DNA of transgenic calves born from cryo fertilized eggs.
본 발명은 생쥐 배아섬유아세포와 공동배양하는 공정을 포함하는 수정란의 체외배양방법에 관한 것이다.The present invention relates to a method for in vitro culture of fertilized eggs comprising a step of co-culture with mouse embryonic fibroblasts.
본 발명은 형질전환용 포유동물의 난소로부터 추출하여 체외성숙된 난포란을 정자와 체외수정을 거쳐 수정란을 얻은 다음, 전기 수정란이 배반포기까지 난분할을 계속하여 형질전환용 포유동물의 자궁저부에 안전하게 착상할 수 있도록 생쥐 배아섬유아세포와 동일배지내에서 공동배양한다. 이때, 수정란은 일반적인 포유동물의 수정란, 또는, 인슐린(insulin), 인터루킨(interlukins)은 물론, 스템셀펙터(stem cell factor), 그래뉼로사이트 콜로니자극인자(granulocyte colony stimulating factor), 에리스로포이에틴(erythropoietin) 등의 조혈생장인자, 또는 각종 호르몬 등이 코딩된 cDNA가 클로닝된 발현벡터가 미세주입된 수정란을 이용하며, 특히, 본 발명의 발명자들이 지난 95년의 관련 발명[대한민국 특허공개 제 98-73991호]의 출원 당시에 구축한 소의 베타-카세인 프로모터와 인체 락토페린-코딩 cDNA가 클로닝된 플라즈미드 pBL-1(KCTC 8630P)이 미세주입된 수정란을 이용할 수도 있다.The present invention extracts from the ovary of the mammal for transformation to obtain fertilized eggs through in vitro fertilization of the oocytes matured in vitro and then implanted safely in the uterus bottom of the mammal for transformation by continuing to divide the egg until blastocyst stage. Co-culture with mouse embryonic fibroblasts in the same medium. In this case, the fertilized egg is a general mammalian fertilized egg, or insulin (insulin), interlukins (interlukins), as well as stem cell factor (stem cell factor), granulocyte colony stimulating factor (granulocyte colony stimulating factor), erythropoietin (erythropoietin) Using a fertilized egg in which an expression vector cloned with a cDNA encoded by various hematopoietic growth factors, etc. or other hormones is microinjected, in particular, the inventors of the present invention relate to the invention of the past 95 years [Korean Patent Publication No. 98-73991]. Embryos microinjected with plasmid pBL-1 (KCTC 8630P) cloned from bovine beta-casein promoter and human lactoferrin-encoding cDNA constructed at the time of filing] may be used.
또한, 본 발명은 상기 생쥐 배아섬유아세포와 수정란을 공동배양하는 체외배양공정을 포함하는 생리활성물질의 생산방법에 관한 것이다. 즉, 도 1에 예시한 바와 같이, 본 발명은 통상의 방법에 따라 난소로부터 회수한 난자를 체외에서 성숙시키는 체외성숙(in vitro maturation)과정, 정자를 처리하여 체외에서 수정시켜 수정란을 수득하는 체외수정(in vitro fertilization)과정, 생리활성물질-코딩 유전자를 클로닝시킨 발현벡터를 수정란내에 도입하는 미세주입(microinjection)과정, 상기 수정란을 체외에서 배양하는 체외배양(in vitro culture)과정 및 체외배양된 수정란을 형질전환동물의 자궁내에 착상시키는 이식(Transplantation) 등의 일련의 과정로 이루어져 있다. 이러한 일련의 과정 중에 본 발명은 전기 미세주입된 수정란이 배반포기까지 난분할을 계속하여 형질전환용 포유동물의 자궁저부에 안전하게 착상할 수 있도록 생쥐 배아섬유아세포와 동일배지내에서 공동배양하고, 수득된 배반포기의 수정란을 다음 과정인 형질전환용 포유동물에의 이식에 이용한다. 여기서, 수정란은 일반적인 포유동물의 수정란, 또는 인슐린(insulin), 인터루킨(interlukins)은 물론, 스템셀펙터(stem cell factor), 그래뉼로사이트 콜로니자극인자(granulocyte colony stimulating factor), 에리스로포이에틴(erythropoietin) 등의 조혈생장인자, 또는 각종 호르몬 등을 코딩하고 있는 cDNA가 클로닝된 발현벡터를 미세주입한 수정란을 이용하며, 특히, 소의 베타-카세인 프로모터와 인체 락토페린-코딩 cDNA가 클로닝된 플라즈미드 pBL-1(KCTC 8630P)이 미세주입된 수정란을 이용할 수도 있다. 또한, 여기서 사용되는 형질전환용 포유동물로는 산양, 젖소는 물론, 통상적으로 유선조직이 발달된 동물이 포함되며, 이밖의 다른 공정은 당업자라면 누구나 용이하게 알 수 있는 통상의 방법을 이용한다.The present invention also relates to a method for producing a physiologically active substance comprising an in vitro culture process for co-culturing the mouse embryo fibroblasts and fertilized eggs. That is, as illustrated in FIG. 1, the present invention is an in vitro maturation process in which an egg recovered from an ovary is matured in vitro according to a conventional method, and an in vitro treatment of sperm in vitro to obtain fertilized eggs in vitro. In vitro fertilization process, microinjection process for introducing the expression vector cloned with the bioactive substance-coding gene into the fertilized egg, in vitro culture process for incubating the fertilized egg in vitro and in vitro culture It consists of a series of processes such as implantation (transplantation) to implant the fertilized egg in the uterus of the transgenic animal. During this series of processes, the present invention co-cultivates in the same medium as mouse embryonic fibroblasts so that the electric microinjected fertilized eggs can be safely implanted in the uterine bottom of the transformed mammal by continuing to divide into the blastocyst stage. Embryos of the blastocyst stage are used for the next procedure for transplantation into a transgenic mammal. Here, the fertilized egg is a common mammalian fertilized egg, or insulin (insulin), interlukins (interlukins), as well as stem cell factor (stem cell factor), granulocyte colony stimulating factor (granulocyte colony stimulating factor), erythropoietin (erythropoietin), etc. A fertilized egg microinjected with a cDNA cloned expression vector encoding hematopoietic growth factor or various hormones, and in particular, plasmid pBL-1 (KCTC) cloned from bovine beta-casein promoter and human lactoferrin-encoding cDNA 8630P) may also be used to instill fertilized eggs. In addition, the mammals for transformation used herein include goats, dairy cows, as well as animals that are usually developed in the mammary gland tissue, and other processes using conventional methods that can be easily known to those skilled in the art.
또한, 본 발명은 추가적으로 상기 수정란을 동결보존시키는 공정을 포함할 수도 있다. 즉, 본 발명은 상기 과정에서 수득한 수정란 또는 배반포기의 수정란을 완만하게 동결시켜 보존한 다음, 이들 수정란을 수란우의 발정시기에 따라 해동시켜 사용할 수 있도록 개선하고 있다. 이때의 수정란은 체외수정된 후이거나 또는 체외배양 후의 것을 이용한다.In addition, the present invention may further include a process of cryopreserving the fertilized egg. That is, the present invention improves so that the fertilized eggs obtained in the above process or ovulation blast embryos gently frozen and preserved, and then thawed using the fertilized eggs according to the estrous time of the eggs. The fertilized egg at this time is used after the in vitro fertilization or in vitro culture.
이상에서와 같은 본 발명의 결과, 한 실시예에 따르면, 인체 락토페린 유전자 DNA를 미세주입한 수정란을 생쥐 배아섬유아세포와 공동배양하였을 때 수정란의 생존율을 종래 7.2%에서 16.7%까지 130% 이상 향상되었으며, 또한 정상적인 송아지 분만율을 종래 56.2%에서 82.6%까지 50% 이상 증가시킬 수 있는 등의 효과가 있었다. 따라서, 이밖에도 인슐린(insulin), 인터루킨(interlukins)은 물론, 스템셀펙터(stem cell factor), 그래뉼로사이트 콜로니자극인자(granulocyte colony stimulating factor), 에리스로포이에틴(erythropoietin) 등의 조혈생장인자, 또는 각종 호르몬과 같은 생리활성물질을 보다 효율적으로, 그리고 보다 경제적으로 수득할 수 있으며, 또한 장기 보존할 수 있는 효과도 얻을 수 있게 된다.As a result of the present invention as described above, according to one embodiment, when the fertilized egg microinjected with human lactoferrin gene DNA was co-cultured with mouse embryonic fibroblasts, the survival rate of the fertilized egg was improved by more than 130% from 7.2% to 16.7%. In addition, there was an effect such that the normal calf delivery rate can be increased by 50% or more from 56.2% to 82.6%. Therefore, other hematopoietic growth factors, such as insulin, interlukins, stem cell factor, granulocyte colony stimulating factor, erythropoietin, or various hormones Bioactive substances such as can be obtained more efficiently and more economically, and also the effect of long-term preservation can be obtained.
이하, 본 발명을 실시예에 의하여 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 이들 실시예에 의하여 본 발명의 범위가 한정되지 않는다는 것은 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.
실시예 1: 수정란의 체외배양Example 1 In Vitro Culture of Fertilized Eggs
1. 체외 성숙공정(In-vitro maturation)1. In-vitro maturation
도살장에서 홀스타인 암소의 난소을 적출하고, 0.9% 생리식염수에 담가 22 내지 25℃로 유지시켜 실험실로 옮긴 다음, 주사기를 사용하여 난포로부터 미수정란을 회수하고, 세척용 배양액(10mM hepes-buffered TCM-199(Gibco BRL사 제품) with Eagle's salt and L-glutamine supplemented with 0.3% BSA and 2mM NaHCO3) 3ml로 3회, 체외배양용 배양액(TCM-199 with Eagle's salts and L-glutamine supplemented with 10% heat-inactivated FBS and 25mM NaHCO3)으로 1회 세척하여 미수정란들을 준비하였다.The Holstein cow's ovary was extracted from the slaughterhouse, soaked in 0.9% saline, maintained at 22 to 25 ° C, and transferred to the laboratory. The embryo was recovered from the follicle using a syringe, and the washed culture solution (10 mM hepes-buffered TCM-199). (From Gibco BRL) with Eagle's salt and L-glutamine supplemented with 0.3% BSA and 2 mM NaHCO3 3 times in 3 ml, in vitro culture medium (TCM-199 with Eagle's salts and L-glutamine supplemented with 10% heat-inactivated FBS and 25 mM NaHCO 3) once washed to prepare unfertilized eggs.
10 내지 20개의 미수정란을 1 ug/ml 에스트라디올(estradiol; Sigma사 제품)과 1 ug/ml FSH-P(Schering-Plough Animal Health사 제품)을 첨가한 체외배양용 배양액 0.5ml에 옮긴 다음, 38℃, 5% CO2에서 22-24시간 배양하였다.10 to 20 unfertilized eggs were transferred to 0.5 ml of in vitro culture medium with 1 ug / ml estradiol (produced by Sigma) and 1 ug / ml FSH-P (produced by Schering-Plough Animal Health). Incubated for 22-24 hours at 38 ℃, 5% CO2.
2. 체외수정(In-vitro fertilization)2. In-vitro fertilization
2-1. 정액의 준비[Parrish et al., Biol. Reprod., 38, 1171, 1988]2-1. Preparation of Semen [Parrish et al., Biol. Reprod., 38, 1171, 1988]
37℃에서 1분간 녹인 홀스타인 소의 동결 정액을 45 내지 90% 퍼콜구배(Percoll gradient)를 이용하여 700 × g에서 30분간 원심분리하여 침전물을 회수한 다음, 정자수의 농도가 ml 당 2 × 106개가 되도록 수정용 배양액(Parrish et al., Biol. Reprod., 38, 1171, 1988)으로 희석시켰다.The precipitate was recovered by centrifuging frozen frozen semen of Holstein bovine, dissolved at 37 ° C. for 1 minute at 700 × g for 30 minutes using 45-90% Percoll gradient, and the concentration of sperm water was 2 × 10 6 per ml. Dogs were diluted with fertilization broth (Parrish et al., Biol. Reprod., 38, 1171, 1988).
2-2. 수정 및 배양2-2. Fertilization and culture
배양된 미수정란 10개를 수정용 배양액 50ul drop에 분산시키고, 정액을 혼합하고, 헤파린(heparin) 2 ug/ml, 20uM 페니실아민(penicillamine), 10uM 하이포타우린(hypotaurine), 1uM 에피네프린(epinephrine)을 첨가하여 18시간 배양한 다음, 수정란에 붙어 있는 난구세포(cumulus cells)을 제거하였다.Ten cultured unfertilized eggs were dispersed in 50ul drop of fertilization broth, mixed with semen, 2 ug / ml heparin, 20uM penicillamine, 10uM hypotaurine, 1uM epinephrine After incubation for 18 hours, the cumulus cells attached to the fertilized egg were removed.
3. 미세주입(Microinjection)3. Microinjection
3-1. 발현벡터의 준비3-1. Preparation of Expression Vectors
소의 베타-카세인 프로모터와 인체의 락토페린을 코딩하는 cDNA 유전자가 클로닝된 플라스미드 pBL-1[KCTC 8630P; Kim et al., Mol. Cells, 4, 355, 1994; 한국특허출원 95-2423호]를 제한효소 SacI 과 SalI으로 처리하여 5.5kb의 개환 플라스미드 벡터를 제조하였다. 이때 사용한 용액은 0.1mM EDTA를 첨가한 10mM Tris-Cl(pH 8.0)으로, 락토페린 발현 유전자의 최종농도를 4 ug/ml로 조정하였다.Plasmid pBL-1 [KCTC 8630P cloned with bovine beta-casein promoter and cDNA gene encoding human lactoferrin; Kim et al., Mol. Cells, 4, 355, 1994; Korean Patent Application No. 95-2423] was treated with restriction enzymes SacI and SalI to prepare a ring-opening plasmid vector of 5.5 kb. The solution used was 10 mM Tris-Cl (pH 8.0) to which 0.1 mM EDTA was added, and the final concentration of the lactoferrin expression gene was adjusted to 4 ug / ml.
3-2. 주입3-2. Injection
상기 공정에서 수득한 개환 플라스미드 벡터를 미세주입법[Simmons et al., Bio/Technol., 6, 179, 1986]으로 체외수정란내에 주입하였다. 즉, 상기에서 준비한 체외수정란을 12,000 × g 로 7분간 원심분리한 다음, 체외수정 후 21 내지 25시간째에 각각 수정란에 유전자를 미세주입하였는데, 이때 200배 배율의 현미경하에서 전핵이 부푼 것으로 주입되었음을 확인하였다.The ring-opening plasmid vector obtained in the above process was injected into the in vitro fertilized egg by microinjection method (Simmons et al., Bio / Technol., 6, 179, 1986). In other words, the in vitro fertilized eggs prepared above were centrifuged at 12,000 × g for 7 minutes, and then the genes were microinjected into the fertilized eggs 21 to 25 hours after in vitro fertilization. Confirmed.
BSA(Sigma, A6003) 3 mg/ml을 첨가한 CR1aa 배양액 50 ul에 생존한 수정란10개씩 분주하고, 38.5℃에서 2일간 5% CO2배양기에서 배양하여 4 또는 8세포기 수정란으로 발달시켰다. 이때 사용한 배양액은 CR1aa 배양액[Rosenkrans & First, Theriogenology, 35, 266, 1991]에 1 × MEM 필수 아미노산(essential amino acid; Gibco BRL사), 1 × MEM 비필수아미노산(non-essential amino acid; Gibco BRL사), BSA 3 mg/ml을 첨가한 것이었다.Ten surviving eggs were dispensed into 50 ul of CR1aa culture solution containing 3 mg / ml of BSA (Sigma, A6003), and cultured in a 5% CO 2 incubator at 38.5 ° C. for 2 days to develop into 4 or 8 cell embryos. At this time, the culture medium used was 1 × MEM essential amino acid (Gibco BRL), 1 × MEM non-essential amino acid (Gibco BRL) in CR1aa culture [Rosenkrans & First, Theriogenology, 35, 266, 1991]. G) BSA 3 mg / ml was added.
4. 체외배양(In-vitro culture)4. In-vitro culture
4-1. 생쥐 배아섬유아세포의 준비4-1. Preparation of Mouse Embryonic Fibroblasts
조이너(Joyner)의 방법[Gene Targeting: a practical approach, Oxford Press, NY, 1993]에 따라, 임신 14 내지 16일된 생쥐로부터 적출하여 얻은 태아를 파쇄하고, 인산완충액으로 3회 세척하고, 0.1% 트립신과 0.05% EDTA 용액으로 37℃에서 10분간 3회 처리하여 단일세포로 분리하였다. 세포부유액을 여과기 (Ikemoto사 제품)로 걸러내고, 다시 700 × g 에서 5분간 원심분리하여 얻은 침전물을 인산완충액으로 3회 세척하고, 10% FBS를 첨가한 DMEM에서 37℃, 5% CO2배양기에서 48시간 배양하였다. 0.1% 트립신과 0.05% EDTA 용액으로 37℃에서 10분간 3회 처리하여 회수하고, 다시 원심분리한 다음, 50% FBS와 10% DMSO(Sigma사)를 첨가한 DMEM과 혼합하여 제조한 동결배양액에 부유시키고, 영하 70℃의 액체질소 용기에서 보관하였다.According to Joyner's method [Gene Targeting: a practical approach, Oxford Press, NY, 1993], fetuses obtained from 14 to 16 days old mice were crushed, washed three times with phosphate buffer, 0.1% trypsin And 0.05% EDTA solution was treated three times for 10 minutes at 37 ℃ was isolated into single cells. The cell suspension was filtered using a filter (manufactured by Ikemoto Co., Ltd.), and the precipitate obtained by centrifugation at 700 x g for 5 minutes was washed three times with phosphate buffer, and then incubated at 37 ° C and 5% CO 2 in DMEM with 10% FBS. Incubated for 48 hours. After treatment with 0.1% trypsin and 0.05% EDTA solution three times at 37 ° C. for 10 minutes, the mixture was centrifuged again and then mixed with DMEM added with 50% FBS and 10% DMSO (Sigma) to a freeze culture prepared. It was suspended and stored in a liquid nitrogen container at minus 70 ° C.
동결보관된 생쥐 배아섬유아세포를 37℃ 온수에서 융해하여 DMEM 배양액에서2일간 배양하고, 마이토마이신(mitomycin) C(Sigma사) 10 ug/ml를 2.5시간 처리하여 생쥐 배아섬유아세포를 불활성화시키고, 인산완충액으로 3회 세척하였다. 6 × 105cells/ml 되도록 분산시켜 1시간 정치하여 단층을 형성시키고, DMEM을 제거한 다음, 10% FBS를 첨가한 CR1aa 배양액으로 대체하였다.The cryopreserved mouse embryonic fibroblasts were fused in warm water at 37 ° C. and incubated in DMEM culture for 2 days, incubated with 10 ug / ml of mitomycin C (Sigma) for 2.5 hours to inactivate the mouse embryonic fibroblasts. , Washed three times with phosphate buffer. Dispersed to 6 × 10 5 cells / ml and allowed to stand for 1 hour to form a monolayer, DMEM was removed, and then replaced with a CR1aa culture added with 10% FBS.
4-2. 생쥐 배아섬유아세포와 체외 공동배양(In-vitro culture) 14-2. Mouse embryonic fibroblasts and in vitro coculture (In-vitro culture) 1
상기 과정을 통해 제조한 생쥐 배아섬유아세포 단세포층을 함유하는 CR1aa배양액에 발현백테-클로닝후 생존한 2516개의 생존 수정란 중 1805개의 배양 수정란을 넣고서 38.5℃, 5% CO2에서 5일간 배양하고, 다시 5.56mM 글루코스(glucose), 1 × GMS-X 첨가물(supplement; insulin 1.0 mg/ml, Na selenate 0.67 ug/ml, transferrin 0.55 mg/ml, ethanolamine 0.2 mg/ml; Sigma사)를 첨가한 배양액에서 배양하여 배반포까지의 발달율을 현미경으로 관찰하였다. 그리고, 그 결과는 다음 표 1에 요약하여 나타내었다.Into the CR1aa culture medium containing the mouse embryonic fibroblast single cell layer prepared through the above procedure, 1805 cultured fertilized eggs out of 2516 surviving embryos survived after expression back-cloning and cultured at 38.5 ° C. and 5% CO 2 for 5 days, and again. Incubate in culture with 5.56 mM glucose, 1 × GMS-X supplement (supplement; insulin 1.0 mg / ml, Na selenate 0.67 ug / ml, transferrin 0.55 mg / ml, ethanolamine 0.2 mg / ml; Sigma) The development rate up to blastocyst was observed under a microscope. The results are summarized in Table 1 below.
비교예 1: 단순배양 1Comparative Example 1: Simple Culture 1
1804개의 배양 수정란을 사용한 점과 생쥐 배아섬유아세포와 공동배양하지 않은 점을 제외하고는 상기 실시예 1과 동일한 방법으로 수정란을 배양하여 배반포까지의 발달율을 현미경으로 관찰하고, 그 결과를 다음 표 1에 요약하여 나타내었다.Except for using 1804 cultured fertilized eggs and not co-cultured with mouse embryonic fibroblasts, the embryos were cultured in the same manner as in Example 1 to observe the development rate up to blastocyst under a microscope. It is shown in summary.
이상에서와 같이, 실시예 1의 생쥐 배아섬유아세포와 공동배양한 경우의 유전자주입 수정란의 배반포까지 발달율은 16.7%(302/1805)인데 반하여, 비교예 1의 단순배양한 경우의 유전자주입 수정란의 배반포까지 발달율은 7.2%(131/1804)를 나타내었다(P<0.001). 이러한 발달율은 유전자주입된 수정란의 체외발달율이 통상 5% 내지 10%라는 다른 연구자의 보고[Perua et al., Theriogenology, 42, 433, 1994]와 일치하므로, 본 발명의 생쥐 배아섬유아세포와 공동배양을 통하여 유전자주입 수정란을 체외배양하는 경우 배반포까지 발달율을 두 배 정도 높일 수 있는 효과가 있음을 알 수 있었다.As described above, the development rate up to blastocyst of the embryo injected embryos when co-cultured with mouse embryonic fibroblasts of Example 1 was 16.7% (302/1805), whereas the embryo injected embryos of Comparative Example 1 were cultured. The development rate until blastocyst was 7.2% (131/1804) (P <0.001). This development rate is co-cultured with the mouse embryonic fibroblasts of the present invention, consistent with the reports of other researchers (Perua et al., Theriogenology, 42, 433, 1994) that the in vitro development rate of transgenic fertilized eggs is typically 5% to 10%. Through in vitro culture of fertilized embryos, it was found that there was an effect of doubling the development rate up to blastocysts.
실시예 2: 생쥐 배아섬유아세포와 체외 공동배양(In-vitro culture) 2Example 2 In Vitro Culture with Mouse Embryonic Fibroblasts (In-vitro Culture) 2
실시예 1에서 체외성숙, 체외수정 및 미세주입된 663개 난자 중 48시간째 분할된 난자는 498개로 75.1%이었다. 이 중 배양된 수정란 각각 4-세포주기의 수정란 96개, 8-세포주기의 수정란 105개를 이용하여 생쥐 배아섬유아세포와 공동배양하고, 배반포까지의 발달율을 현미경으로 관찰하고, 그 결과를 다음 표 2에 요약하여 나타내었다.In Example 1, 498 eggs divided by 48 hours among 663 eggs in vitro matured, in vitro fertilized and microinjected were 75.1%. Among them, the cultured embryos were co-cultured with mouse embryonic fibroblasts using 96 four-cell cycle fertilized eggs and 105 eight-cell cycle fertilized eggs, respectively, and the development rates up to blastocysts were observed under a microscope. 2 is summarized.
비교예 2: 단순배양 2Comparative Example 2: Simple Culture 2
배양된 수정란 각각 4-세포주기의 수정란 91개, 8-세포주기의 수정란 103개를 이용하여 단순배양하고, 배반포까지의 발달율을 현미경으로 관찰하였다. 그리고, 그 결과를 다음 표 2에 요약하여 나타내었다.Cultured fertilized eggs were cultured using 91 embryos of 4-cell cycles and 103 embryos of 8-cell cycles, respectively, and the development rate up to blastocysts was observed under a microscope. The results are summarized in Table 2 below.
이상에서와 같이, 실시예 2의 경우, 체외수정란을 생쥐 배아섬유아세포와 공동배양한 결과 배반포까지의 발달율은 24.4% (49/201)인데 반하여, 비교예 2의 단순배양한 경우는 14.4%(28/194)으로, 보다 높은 발달율을 나타냈다(P<0.05).As described above, in Example 2, the in vitro fertilized embryos were co-cultured with mouse embryonic fibroblasts, and the development rate up to blastocyst was 24.4% (49/201), whereas in the simple culture of Comparative Example 2, 14.4% ( 28/194), showing a higher development rate (P <0.05).
실시예 3: 생쥐 배아섬유아세포와 체외 공동배양한 배반포기의 수정란 이식(Transplantation)Example 3: Transplantation of Mouse Embryonic Fibroblasts and In Vitro Co-cultured Blastocysts
충남 안면도에 소재한 두산목장에서 관리하고 있는 14 내지 18개월된 자연발정된 미경산 젖소 131마리를 대상으로 0.25ml 들이 스트로를 장착한 수정란 이식기구(IMV, France)를 멸균한 나일론필름(Chemise Sanitaire, France)으로 둘러싼 다음, 자궁경관을 통과하여 생쥐 배아섬유아세포와 체외 공동배양한 배반포기의 수정란을 자궁각 선단에 이식하였다. 이후, 임신이 지속되도록 대리모를 잘 관리하여 송아지를 분만하게 되었다. 그리고, 그 결과는 다음 표 3에 요약하여 나타내었다.Nylon film sterilized with a fertilized egg implantation device (IMV, France) equipped with 0.25 ml straw for 131 14- to 18-month-old unestablished dairy cows managed by Doosan Ranch, Anmyeon-do, Chungcheongnam-do. France, and then fertilized embryos of blastocysts, which were co-cultured in vitro with mouse embryonic fibroblasts through the cervix, were implanted at the tip of the uterus. Since then, the surrogate mother was well managed to give birth to the calf. The results are summarized in Table 3 below.
비교예 3: 단순배양한 배반포기의 수정란 이식(Transplantation)Comparative Example 3: Transplantation of Simple Cultured Blastocysts
단순배양한 배반포기의 수정란을 이용한 점과 미경산 젖소 93마리를 대상으로 한 점을 제외하고는 상기 실시예 3과 동일한 과정을 통하여 수정란 이식과정을 수행하고, 이후, 임신이 지속되도록 대리모를 잘 관리하여 송아지를 분만하게 되었다. 그리고, 그 결과는 다음 표 3에 요약하여 나타내었다.Except for the use of fertilized embryos of culturing blastocysts and 93 uncultivated cows, fertilized egg transplantation was carried out through the same process as in Example 3, after which the surrogate mother was well maintained so that pregnancy continued. Care took place and calf delivery. The results are summarized in Table 3 below.
이상에서와 같이, 유전자주입된 수정란의 수태율은 실시예 3의 생쥐 배아섬유아세포와 체외 공동배양한 배반포기의 수정란의 경우 27.4%(23/84)인데 반하여, 비교예 3의 단순배양한 배반포기의 수정란을 이용한 경우는 24.2%(16/66)로서 상호 유사하게 나타났다. 그러나, 비교예 3의 단순배양한 배반포기의 수정란을 이용한 경우는 3마리가 유산되고 4마리가 사산되어 결국, 최종 9마리가 태어나 정상 분만율은 56.2%인데 반하여, 실시예 3의 경우는 유산과 사산이 각각 2마리였으며, 그 결과, 최종 19마리가 태어나 정상 분만율은 82.6%로 매우 높음을 알 수 있었다.As described above, the fertility rate of the fertilized embryos injected with mouse embryo fibroblasts of Example 3 was 27.4% (23/84) in fertilized blastocysts in vitro, whereas the embryonic blastocysts of Comparative Example 3 were cultured. Using fertilized eggs of 24.2% (16/66) showed similar results. However, in the case of using the fertilized egg of the culturing blastocyst in Comparative Example 3, three were aborted and four were still born, resulting in the final nine being born and the normal delivery rate was 56.2%. There were two stillbirths, and as a result, the final 19 were born and the normal rate of delivery was 82.6%.
실시예 4: 수정란 동결에 따른 효과Example 4 Effect of Embryo Freezing
완만동결법[Massip et al., J. Reprod. Fert., 97, 65, 1967]으로 유전자주입 수정란을 동결하였다. 즉, 유전자주입 수정란 2 내지 5개를 50% FBS와 10% 글리세롤을 첨가한 인산완충액으로 조성되는 동결액에 10분간 분산시키고, 0.25 ml 들이 스트로에 넣은 다음, 스트로 분말(FHK, Fujihara Ind.)로 봉합하고, 자동세포동결기(SACO ACF-101, San Cheon Tech-Ind.)를 사용하여 영하 5℃에서 빙정형성을 유도한 다음, 분당 0.5℃씩 하강시켜 영하 33℃까지 하강시키고, 액체질소용기에서동결보관하였다.Slow freezing method [Massip et al., J. Reprod. Fert., 97, 65, 1967] was used to freeze transgenic fertilized eggs. That is, two to five transgenic eggs were dispersed in a freeze solution composed of phosphate buffer solution containing 50% FBS and 10% glycerol for 10 minutes, 0.25 ml of straw, and then straw powder (FHK, Fujihara Ind.) Suture, ice crystal formation at 5 ° C below zero using an automatic cell freezer (SACO ACF-101, San Cheon Tech-Ind.), Then drop to 0.5 ° C per minute to 33 ° C below liquid nitrogen. Frozen in containers.
37℃ 물에 약 20초간 흔들어 녹인 다음, 스트로 내용물을 페트리접시에 방출시키고, 융해된 수정란을 6%, 3%, 0% 글리세롤을 첨가한 희석액(0.25M sucrose, 0.5% BSA를 첨가한 인산완충액)에서 각각 5분간 침지시키고, 30%와 15% FBS를 첨가한 인산완충액으로 각각 세척한 다음, 생쥐 배아섬유아세포 단세포층이 포함된 CR1aa배양액에서 12시간 이상 공동배양하였다. 그리고, 그 결과는 다음 표 4에 요약하여 나타내었다.Shake and dissolve in water at 37 ° C for about 20 seconds, release the contents of the straw into a petri dish, and dilute the dissolved fertilized egg with 6%, 3%, and 0% glycerol (0.25M sucrose, phosphate buffer with 0.5% BSA). Immersed for 5 minutes, washed with phosphate buffer added with 30% and 15% FBS, respectively, and co-cultured for more than 12 hours in CR1aa culture solution containing a mouse embryonic fibroblast single cell layer. The results are summarized in Table 4 below.
이상에서와 같이, 동결·융해 후 유전자주입 수정란의 생존율은 58.5%(72/123)로서, 유전자 주입하지 않은 수정란의 생존율 61%와 유사하였다[Han et al., Theriogenology, 46, 769, 1994]. 따라서, 수정란의 동결여부는 수정란의 생존율에 거의 영향이 없음을 알 수 있었다.As described above, the survival rate of the transgenic embryos after freezing and thawing was 58.5% (72/123), which was similar to the survival rate of 61% of the fertilized eggs without gene injection [Han et al., Theriogenology, 46, 769, 1994]. . Therefore, it could be seen that the freezing of the fertilized egg had little effect on the survival rate of the fertilized egg.
실시예 5: 형질전환 여부의 확인Example 5: Confirmation of transformation
호겐 등의 방법[Hogan et al., Manipulating the mouse embryo: a laboratory manual, CSH, N.Y., 1986]에 따라 태어난 송아지로부터 채혈한 혈액을1,500 × g로 원심분리하여 상층부에서 얻은 백혈구세포층을 세포분해액 (proteinase K 100 ug/ml, 0.1mM EDTA, 0.5% SDS in 10mM Tris-Cl, pH 8.0)에 넣어 55℃에서 용해시키고, 페놀-에탄올법으로 게놈(genomic) DNA를 분리하였다. DNA 10ug을 제한효소 DraI, EcoRI, HindIII로 각각 처리하고, 0.8% 아가로스겔에서 전기영동하고, 나일론막에 흡착시켰다.The cell lysate was obtained by centrifugation of 1,500 × g of blood collected from calves born according to Hogen et al., Manipulating the mouse embryo: a laboratory manual, CSH, NY, 1986. (proteinase K 100 ug / ml, 0.1 mM EDTA, 0.5% SDS in 10 mM Tris-Cl, pH 8.0) was dissolved at 55 ℃, genomic DNA was isolated by phenol-ethanol method. 10 ug of DNA was treated with restriction enzymes DraI, EcoRI and HindIII, respectively, electrophoresed on 0.8% agarose gel and adsorbed onto nylon membrane.
인체 락토페린-코딩 cDNA 중 2.6kb 크기의 EcoRI 절편을 [α-32P]dCTP로 표지하여 제조한 프로브를 사용하여, 65℃에서 하이브리드 반응시켰다. 하이브리드 막은 2 × SSC, 0.1% SDS 용액으로 실온에서 세척하고, 같은 용액으로 65℃에서 10분간 세척하였다. 다시 1 × SSC, 0.1% SDS 용액으로 실온에서 세척하고, 같은 용액으로 65℃에서 10분간 세척한 다음, 영하 70℃에서 3일간 X-선 필름에 감광시키고, 포스포이메이저 분석(Phosphoimazer analysis)을 통해 정량하였다. 그리고, 그 결과를 다음 표 5 및 도 2에 나타내었다.2.6 kb EcoRI fragments in human lactoferrin-encoding cDNA were hybridized at 65 ° C. using a probe prepared by labeling with [α- 32 P] dCTP. Hybrid membranes were washed at room temperature with 2 × SSC, 0.1% SDS solution, and the same solution at 65 ° C. for 10 minutes. Again washed at room temperature with 1 × SSC, 0.1% SDS solution, washed for 10 minutes at 65 ° C. with the same solution, and then photosensitive to X-ray film for 3 days at minus 70 ° C., followed by Phosphoimazer analysis. Quantification through And the result is shown in following Table 5 and FIG.
이상에서와 같이, 동결수정란으로부터 태어난 12마리 송아지의 게놈 DNA에서 인체 락토페린 유전자의 삽입여부를 서던블럿(Southern blot)으로 분석하였을 때,2 마리가 인체 락토페린 유전자를 가지고 있는 것으로 확인되었다. 또한, 동결수정란으로부터 태어난 형질전환 젖소(보람이)는 인체 락토페린 유전자를 4카피 가지고 있는 것으로 확인되었다[도 2 참조]. 따라서, 상기와 같은 완만법으로 수정란을 동결시켜 보존하더라도 충분한 형질전환능을 지니고 있음을 확인할 수 있었다.As described above, when the human lactoferrin gene was inserted into the genomic DNA of 12 calves born from cryo fertilized eggs by Southern blot, it was confirmed that the two had human lactoferrin gene. In addition, the transgenic cow (Boramyi) born from cryofertilized eggs was confirmed to have four copies of the human lactoferrin gene (see FIG. 2). Therefore, it could be confirmed that even when the fertilized eggs were frozen and preserved by the gentle method as described above, they had sufficient transformation capacity.
이상에서 상세히 설명한 바와 같이, 본 발명은 수정란의 체외배양방법 및 이의 이용에 관한 것으로, 이러한 본 발명의 한 실시예에 따르면, 인체 락토페린 유전자 DNA를 미세주입한 수정란을 생쥐 배아섬유아세포와 공동배양하였을 때 수정란의 생존율을 종래 7.2%에서 16.7%까지 130% 이상 향상되었으며, 또한 정상적인 송아지 분만율을 종래 56.2%에서 82.6%까지 50% 이상 증가시킬 수 있었다. 그리고, 수정란을 동결 보존 후 융해하여 이식하여도 형질전환 젖소를 생산할 수 있으므로, 시기에 관계없이 수정란을 체외에서 장기보존할 수 있는 효과를 얻을 수 있었다.As described in detail above, the present invention relates to an in vitro culture method of fertilized eggs and its use, according to one embodiment of the present invention, the fertilized egg microinjected with human lactoferrin gene DNA co-cultured with mouse embryonic fibroblasts The survival rate of fertilized eggs was improved by 130% or more from 7.2% to 16.7%, and the normal calf delivery rate could be increased by 50% or more from 56.2% to 82.6%. In addition, since the embryos can be produced by cryopreservation and fusion of the fertilized eggs, transgenic cows can be produced. Therefore, the fertilized eggs can be stored in vitro for a long time regardless of the timing.
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| KR100386880B1 (en) * | 2000-04-28 | 2003-06-11 | 주식회사 비엠아이 코리아 | Cell culture media |
| KR20230094546A (en) | 2021-12-21 | 2023-06-28 | 이숭재 | Spray-type treatment device for pet excrement and pests |
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| KR100386880B1 (en) * | 2000-04-28 | 2003-06-11 | 주식회사 비엠아이 코리아 | Cell culture media |
| KR20230094546A (en) | 2021-12-21 | 2023-06-28 | 이숭재 | Spray-type treatment device for pet excrement and pests |
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