小鼠胚胎冷冻的研究
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摘要
随着生命科学的发展，作为研究生命科学基础条件之一的实验动物也在我国得到长足的发展。目前，全世界仅实验小鼠、遗传工程小鼠的品种、品系就有6000多种，并且每年以600—800种的速度在增加，我国为了与世界生命科学接轨，现正建设《国家遗传工程小鼠资源库》，引进和开发这些珍贵的实验动物，为了使这些珍贵的实验动物品种得以安全的保存，建立一种快速、经济、可靠的小鼠种质保存方法尤为重要。而胚胎冷冻保种技术是目前小鼠保种技术中最为成功的一项技术。
本文以EG40、ES40、EFS40为冷冻液，采用快速冷冻法、玻璃化冷冻法，用ICR小鼠对小鼠胚胎冷冻的条件进行了系统的探索，除用冷冻麦管进行冷冻外，首次用细胞冷冻管进行胚胎冷冻。EG40、ES40冷冻液进行胚胎快速冷冻，解冻后胚胎囊胚发育率可达56.4％、66.7％，胚胎移植着床率为41.4％、50％；用EFS40为冷冻液的进行玻璃化冷冻，解冻后囊胚发育率达87.2％，着床率为66.3％，与对照组差异不明显。用EFS40玻璃化冷冻液并用细胞冷冻管代替麦管进行胚胎冷冻，其结果与麦管法无差异。作者对一步法和二步法冷冻方法进行了比较，EG40、ES40冷冻液二步法冷冻效果比一步法好，差异显著；而EFS40玻璃化冷冻液二步法冷冻效果与一步法相比无差异。在此基础上，用冷冻麦管、细胞冷冻管分别采用一步法、二步法对Lats基因敲除小鼠胚胎进行玻璃化冷冻，胚胎解冻后，囊胚发育率分别为：90％、90.1％、89.2％、91.4％；移植后着床率分别：50.0％、47.1％、43.4％、46.5％，差异不显著；并且与对照组相比差异也不显著。而且通过基因检测，冷冻后代Lats基因未发生丢失。
因此以ES40、EFS40冷冻液的胚胎快速冷冻法和玻璃化冷冻法与EG40冷冻液相比，更具有应用价值，尤其是用细胞冷冻管进行玻璃化冷冻，方法
朱孝荣:小鼠胚胎冷冻的研究
更简单、实用，而且冷冻效果理想。
With the life science development ,as one of basis of life science,the laboratory animal has reached a great degree in our country as well. At present only the laboratory mouse and genectically engineered mouse are more than six thousand strains, and this number is increasing 600-800 per year. To be consistent with the development of the world life science, our country is constructing the "National Resource Library of Genectically Engineered Mice" to introduce and exploit these precious animals. It is very important to establish a rapid economic and reliable method for conservation of a valuble variety of strains in security.The technology of mice embryo cryopreservation is a reliable method on germplasm storage of the mice.
We use ICR mouse as an example to systematically optimize the condition of
mice embryo cryopreservation with solution EG40 ES40 and EFS40 by rapid
eryopreservative and vitrified method. Other than straw, the cell freezing tube
was firstly adopted. Using EG40 and ES40,The development ratio reached
66.4%x 66.7% and the implantation ratio reached 41.4% 50% respectively after
maw EFS40 was used as solution to be vitrified .The development ratio and
implantation ratio reached 87.2%, 66.3% respectively. The difference is not
emarkable compared to the control. We use EFS40 as vitrified solution and straw
was replaced by cell freezing tube, and the result is the same of the straw method.
Nuthors compared one-step and two-step method, found the effect of EG40, ES40
eryopreservative method is better than one-step method, and the difference is
remarkable, while the different effect of EFS40 two-step vitrified method is not
remarkable. Subsequently,with the straw and the cell freezing tube,we used
one-step and two-step method to vitrify the embryo of Lats gene knockout mice.
The development ratio reached 90% 90.1 % 89.2%, 91.4% and the implantation rauo reached 50.0%, 47.1% 43.4%, 46.5% respectively after thaw. The difference is not remarkable compared to the control.With genetic assay,we can receive the result that the Lats gene of cryopreservative offspring did not lose.
Taken together, the solution of ES40 and EFS40 are provided with more application than that of the solution of EG40.Especially, the vitrified method with cell freezing tube is even more simple and practical,but its result is outstanding
本文以EG40、ES40、EFS40为冷冻液，采用快速冷冻法、玻璃化冷冻法，用ICR小鼠对小鼠胚胎冷冻的条件进行了系统的探索，除用冷冻麦管进行冷冻外，首次用细胞冷冻管进行胚胎冷冻。EG40、ES40冷冻液进行胚胎快速冷冻，解冻后胚胎囊胚发育率可达56.4％、66.7％，胚胎移植着床率为41.4％、50％；用EFS40为冷冻液的进行玻璃化冷冻，解冻后囊胚发育率达87.2％，着床率为66.3％，与对照组差异不明显。用EFS40玻璃化冷冻液并用细胞冷冻管代替麦管进行胚胎冷冻，其结果与麦管法无差异。作者对一步法和二步法冷冻方法进行了比较，EG40、ES40冷冻液二步法冷冻效果比一步法好，差异显著；而EFS40玻璃化冷冻液二步法冷冻效果与一步法相比无差异。在此基础上，用冷冻麦管、细胞冷冻管分别采用一步法、二步法对Lats基因敲除小鼠胚胎进行玻璃化冷冻，胚胎解冻后，囊胚发育率分别为：90％、90.1％、89.2％、91.4％；移植后着床率分别：50.0％、47.1％、43.4％、46.5％，差异不显著；并且与对照组相比差异也不显著。而且通过基因检测，冷冻后代Lats基因未发生丢失。
因此以ES40、EFS40冷冻液的胚胎快速冷冻法和玻璃化冷冻法与EG40冷冻液相比，更具有应用价值，尤其是用细胞冷冻管进行玻璃化冷冻，方法
朱孝荣:小鼠胚胎冷冻的研究
更简单、实用，而且冷冻效果理想。
With the life science development ,as one of basis of life science,the laboratory animal has reached a great degree in our country as well. At present only the laboratory mouse and genectically engineered mouse are more than six thousand strains, and this number is increasing 600-800 per year. To be consistent with the development of the world life science, our country is constructing the "National Resource Library of Genectically Engineered Mice" to introduce and exploit these precious animals. It is very important to establish a rapid economic and reliable method for conservation of a valuble variety of strains in security.The technology of mice embryo cryopreservation is a reliable method on germplasm storage of the mice.
We use ICR mouse as an example to systematically optimize the condition of
mice embryo cryopreservation with solution EG40 ES40 and EFS40 by rapid
eryopreservative and vitrified method. Other than straw, the cell freezing tube
was firstly adopted. Using EG40 and ES40,The development ratio reached
66.4%x 66.7% and the implantation ratio reached 41.4% 50% respectively after
maw EFS40 was used as solution to be vitrified .The development ratio and
implantation ratio reached 87.2%, 66.3% respectively. The difference is not
emarkable compared to the control. We use EFS40 as vitrified solution and straw
was replaced by cell freezing tube, and the result is the same of the straw method.
Nuthors compared one-step and two-step method, found the effect of EG40, ES40
eryopreservative method is better than one-step method, and the difference is
remarkable, while the different effect of EFS40 two-step vitrified method is not
remarkable. Subsequently,with the straw and the cell freezing tube,we used
one-step and two-step method to vitrify the embryo of Lats gene knockout mice.
The development ratio reached 90% 90.1 % 89.2%, 91.4% and the implantation rauo reached 50.0%, 47.1% 43.4%, 46.5% respectively after thaw. The difference is not remarkable compared to the control.With genetic assay,we can receive the result that the Lats gene of cryopreservative offspring did not lose.
Taken together, the solution of ES40 and EFS40 are provided with more application than that of the solution of EG40.Especially, the vitrified method with cell freezing tube is even more simple and practical,but its result is outstanding
引文
1 Polge C, Simth AU. Revival of spermatozoa after vitrifcation and dehydration at low temperature. Nature, 1949,164:666
2 Whittingham DG, Leibo S P, Mazur P. Survival of mouse embryos frozen to-196 degree and -296 degree. Science, 1972,178:411-414
3 Rall WF. Fahy GM. Ice-free cryopreservation of mouse embryos at-196 degree by vitrification. Nature, 1985.313:573-575
4 Kasai M, Komi JH, Takakamo A. et al. A simple method for mouse embryo cryopreservation in low toxicity vitrification solution, without appreciable loss of viability. Joural of Reproducation and Fertilization ,1990, 89: 91-97
5 王光亚，段恩奎．山羊胚胎工程．陕西杨陵：天则出版社，1993：59-75
6 孙新明．小鼠胚胎进一部冷冻试验．陕西杨陵：西北农业大学，1993
7 Tada N, Saeki K. Inai M. et al. A simple and rapid method for cryopreservation of mouse 2-cell embryos by vitrification: benefical effect of sucrose and raffinose on their cryosurvival rate. Theriogenology, 1993, 40: 333-344
8 Valdez CA. Mazni OA. Takahashi Y, et al. Successful cryopreservation of zona-hatched mouse blastocysts. Journal of Reproduction and Fertility. 1996. 107: 37-42
9 朱士恩、王海彦、曾申明，等．小鼠桑椹胚玻璃化冷冻前后生物频谱处理对体外发育的影响．中国农业大学学报，1999，4：109-103
10 Takeda T. Elsden RP, Seidel GE. Cryopreservation of mouse embryos by direct plunging into liquid nitrogen. Theriogenology, 1984, 21: 266-268
11 王锋，蔡令波，王启风，等．小鼠胚胎超低温冷冻保存方法比较研究。草食家畜(增刊)；136-139
12 李劲松．孙强，巢琴美．等．HBV-C转基因小鼠快速冷冻方法的建立．中国实验动物学杂志．2001，11(1)：44-46
13 朱士恩，曾申明，张忠诚．小鼠胚胎玻璃化冷冻保存及保存时间对其体内外发的影响．中国畜牧杂志，1998，34(6)：12-14
14 努尔江，史洪才，黄俊成，等小鼠早期桑椹胚和致密桑椹胚玻璃化冷冻保存试验．草食家畜，2000(1)：27-29
15 Nakagata N. Cryopreservation of mouse strains by ultrarapid freezing. Experiment Animal (Tokyo), 1990. 39: 299-302
16 Landa V, Tepla O. Construction of aggregation chimeras from 8-cell mouse embryo cryopreservation for several years in liquid nitrogen. Folia Biologica. 1990. 36: 159-164
17 Pinnyes A. Wallave GA. Rall WF. Effect of genotype on the efficiency of mouse embryo cryopreservation by vitrufucation or slow freezing methods. Molecular reproduction and Development, 1995, 40: 429-435
18 左琴．岳秉飞．刘双环．等．近交系小鼠胚胎玻璃化冷冻技术研究．中国实验动物学杂志，2001，11(2)：82-84
19 孙青原，秦鹏春，徐立滨．哺乳动物卵母细胞的冷冻保存方法．生物技术，1993，3(1)：5-9
20 Shaw PW. Fuller BJ. Bernard A et al. Vitrification of mouse oocytes: improved rate of survival, fertilization and development to blastocysts. Molecular Reproducation and Development, 1991, 29: 373-378
21 Nakagata N Cryopreservation of embryos and gametes in mice. Experiment Animal. 1994, 43: 11-18
22 Bos Mikich A. Whittingham DG. Analysis of the chromosome complement of frozen-thawed oocytes after parthenogenetic activation. Molecular Reproduction and development, 1995, 42: 254-269
23 James J. Stachecki, Cohen Jacques, et al. Detrimental effect of sodium during mouse oocvte cryopreservation. Biology of Reproduction, 1998, 59: 395-400
24 Deanesly R. Egg survival in immature rat ovaries grafted after freezing and thawing. Proc R Soc Ling Ser B, 1957, 147: 412-421
25 Arp R, Leibach J, Black J, et al. Cryopreservation of murine ovarian tissue. Cryobiology, 1994, 31(4): 336-343
26 Cox S L. Shaw J. Jenkin G. Transplantation of cryopreservation fetal ovarian tissue to adult recipients in mice. Journal of Reproduction and fertility. 1996. 107: 315-322
27 Gunasena KT. Lakey JR. Villines PM. et al. Allogeneic and xenogeneic transplantation of cryopreserved ovarian tissue to athymic mice. Biol Reprod 1997. 57(3): 226-231
28 Sztein J. Sweet H. Farley J. Mobraaten L. Cryopreservation and orthotopic transplantation of mouse ovaries: new approach in gamete banking. Biol Reprod 1998. 58(4): 1071-1074
29 庞龙，王燕蓉．沈新升．等．小鼠卵巢组织的超速冻存法研究．中国实验动物学报，2002．10：142-147
30 Salle B. Lornage J. Demirci B, et al. Restoration of ovarian steroid and histologic assessment after freezing, thawing. and autogrfl of a hemi-ovary in sheep. Fertil Steril. 1999,72:366-370
31 Newton H. Aubard Y. Rutherford A, et al. Low temperature storage and grafting of human ovarian tissue. Hum Reprod, 1996, 11: 1481-1491
32 孙强．王晓辉，张静．等．利用高浓度乙二醇冷冻保存MMTV-WNT-1转基因小鼠胚胎．上海实验动物科学，2001．21(4)：205-208
33 刘丽均．徐平．小鼠桑椹期、囊胚期胚胎移植方法的探讨．上海实验动物科学．2000．20(2)：109-112
34 Tian Xu. Weiyi Wang, Sheng Zhang, et al. Identifying tumor suppressor genetic mosaics: the Drosophila lats gene encondes a putative protein kinase. Development, 1995, 121: 1053-1063.
35 St John MA, Tao W, Fukumoto R, et al. Mice deficient of Latsl develop softTissue sarcomas, ovarian tumours and pituitary dysfunction. Nat Genet, 1990. 21(2): 182-186.
36 朱孝荣，吴红，孙强，等．Latsl基因敲除小鼠保种的研究．中国实验动物学报，2002，10(4)：201-204
37 马新武，哺乳动物胚胎冷冻原理及研究进展．动物医学进展，2000(4)：112-115
38 Kono T, Kwon OY. Jchince K. et al. Effect of dilution on viability of vitrified-warmed mouse embryos. Japanese Journal of Animal Reproduction, 1989, 35: 211-216
39 Gal F, Baril G, Vallet J C, et al. In vivo and in vitro survival of goat embryos after freezing with ethylene glycol or glycekol. Theriogenoiogy, 1993, 40: 651-659
40 Dumoulin JM. Bergers Jansen JM. Pieters MHCE. et al. The protective effects of polymers in the cryopreservation of human and mouse zonae pellucidae and embryos. Fertility and sterility, 1994, 62: 793-798
41 Kasai M. Komi H,Takakamo H,et al. A simple method for mouse embryo cryopreservation in a low toxicity vitrification solution, without appreciable loss of viability. J Reprod Fert, 1990, 89: 91-97
42 Tada N, Sato M. Amann E, et al. A simple and rapid method for cryopreservation of mouse 2-cell embryos bv vitrification: beneficial effect of sucrose and raffinose on their cryosurvival rate. Theriodenotogy, 1993, 40: 333-344
43 Kasai M, Nishimori M, Zhu SE. et al. Survival of meuse morulae vitrified in
an ethylene glycol-based solution after exposure to the solution atvarious temperatures. Biology of Reproduction, 1992.47:1134-1130
44 Zhu SE. Kasai M. Otoge H. et al. Cryopreservation of expanded mouse blastocysts by vitrfication in ethylene glycol based solution. J Reprod Fertil. 1993. 98: 139-145
45 朱士恩，曾申明．张忠诚，等．小鼠胚胎玻璃化冷冻保存时间对其体内外发育率的影响．中国畜牧杂志．1998．34(6)：12-14
2 Whittingham DG, Leibo S P, Mazur P. Survival of mouse embryos frozen to-196 degree and -296 degree. Science, 1972,178:411-414
3 Rall WF. Fahy GM. Ice-free cryopreservation of mouse embryos at-196 degree by vitrification. Nature, 1985.313:573-575
4 Kasai M, Komi JH, Takakamo A. et al. A simple method for mouse embryo cryopreservation in low toxicity vitrification solution, without appreciable loss of viability. Joural of Reproducation and Fertilization ,1990, 89: 91-97
5 王光亚，段恩奎．山羊胚胎工程．陕西杨陵：天则出版社，1993：59-75
6 孙新明．小鼠胚胎进一部冷冻试验．陕西杨陵：西北农业大学，1993
7 Tada N, Saeki K. Inai M. et al. A simple and rapid method for cryopreservation of mouse 2-cell embryos by vitrification: benefical effect of sucrose and raffinose on their cryosurvival rate. Theriogenology, 1993, 40: 333-344
8 Valdez CA. Mazni OA. Takahashi Y, et al. Successful cryopreservation of zona-hatched mouse blastocysts. Journal of Reproduction and Fertility. 1996. 107: 37-42
9 朱士恩、王海彦、曾申明，等．小鼠桑椹胚玻璃化冷冻前后生物频谱处理对体外发育的影响．中国农业大学学报，1999，4：109-103
10 Takeda T. Elsden RP, Seidel GE. Cryopreservation of mouse embryos by direct plunging into liquid nitrogen. Theriogenology, 1984, 21: 266-268
11 王锋，蔡令波，王启风，等．小鼠胚胎超低温冷冻保存方法比较研究。草食家畜(增刊)；136-139
12 李劲松．孙强，巢琴美．等．HBV-C转基因小鼠快速冷冻方法的建立．中国实验动物学杂志．2001，11(1)：44-46
13 朱士恩，曾申明，张忠诚．小鼠胚胎玻璃化冷冻保存及保存时间对其体内外发的影响．中国畜牧杂志，1998，34(6)：12-14
14 努尔江，史洪才，黄俊成，等小鼠早期桑椹胚和致密桑椹胚玻璃化冷冻保存试验．草食家畜，2000(1)：27-29
15 Nakagata N. Cryopreservation of mouse strains by ultrarapid freezing. Experiment Animal (Tokyo), 1990. 39: 299-302
16 Landa V, Tepla O. Construction of aggregation chimeras from 8-cell mouse embryo cryopreservation for several years in liquid nitrogen. Folia Biologica. 1990. 36: 159-164
17 Pinnyes A. Wallave GA. Rall WF. Effect of genotype on the efficiency of mouse embryo cryopreservation by vitrufucation or slow freezing methods. Molecular reproduction and Development, 1995, 40: 429-435
18 左琴．岳秉飞．刘双环．等．近交系小鼠胚胎玻璃化冷冻技术研究．中国实验动物学杂志，2001，11(2)：82-84
19 孙青原，秦鹏春，徐立滨．哺乳动物卵母细胞的冷冻保存方法．生物技术，1993，3(1)：5-9
20 Shaw PW. Fuller BJ. Bernard A et al. Vitrification of mouse oocytes: improved rate of survival, fertilization and development to blastocysts. Molecular Reproducation and Development, 1991, 29: 373-378
21 Nakagata N Cryopreservation of embryos and gametes in mice. Experiment Animal. 1994, 43: 11-18
22 Bos Mikich A. Whittingham DG. Analysis of the chromosome complement of frozen-thawed oocytes after parthenogenetic activation. Molecular Reproduction and development, 1995, 42: 254-269
23 James J. Stachecki, Cohen Jacques, et al. Detrimental effect of sodium during mouse oocvte cryopreservation. Biology of Reproduction, 1998, 59: 395-400
24 Deanesly R. Egg survival in immature rat ovaries grafted after freezing and thawing. Proc R Soc Ling Ser B, 1957, 147: 412-421
25 Arp R, Leibach J, Black J, et al. Cryopreservation of murine ovarian tissue. Cryobiology, 1994, 31(4): 336-343
26 Cox S L. Shaw J. Jenkin G. Transplantation of cryopreservation fetal ovarian tissue to adult recipients in mice. Journal of Reproduction and fertility. 1996. 107: 315-322
27 Gunasena KT. Lakey JR. Villines PM. et al. Allogeneic and xenogeneic transplantation of cryopreserved ovarian tissue to athymic mice. Biol Reprod 1997. 57(3): 226-231
28 Sztein J. Sweet H. Farley J. Mobraaten L. Cryopreservation and orthotopic transplantation of mouse ovaries: new approach in gamete banking. Biol Reprod 1998. 58(4): 1071-1074
29 庞龙，王燕蓉．沈新升．等．小鼠卵巢组织的超速冻存法研究．中国实验动物学报，2002．10：142-147
30 Salle B. Lornage J. Demirci B, et al. Restoration of ovarian steroid and histologic assessment after freezing, thawing. and autogrfl of a hemi-ovary in sheep. Fertil Steril. 1999,72:366-370
31 Newton H. Aubard Y. Rutherford A, et al. Low temperature storage and grafting of human ovarian tissue. Hum Reprod, 1996, 11: 1481-1491
32 孙强．王晓辉，张静．等．利用高浓度乙二醇冷冻保存MMTV-WNT-1转基因小鼠胚胎．上海实验动物科学，2001．21(4)：205-208
33 刘丽均．徐平．小鼠桑椹期、囊胚期胚胎移植方法的探讨．上海实验动物科学．2000．20(2)：109-112
34 Tian Xu. Weiyi Wang, Sheng Zhang, et al. Identifying tumor suppressor genetic mosaics: the Drosophila lats gene encondes a putative protein kinase. Development, 1995, 121: 1053-1063.
35 St John MA, Tao W, Fukumoto R, et al. Mice deficient of Latsl develop softTissue sarcomas, ovarian tumours and pituitary dysfunction. Nat Genet, 1990. 21(2): 182-186.
36 朱孝荣，吴红，孙强，等．Latsl基因敲除小鼠保种的研究．中国实验动物学报，2002，10(4)：201-204
37 马新武，哺乳动物胚胎冷冻原理及研究进展．动物医学进展，2000(4)：112-115
38 Kono T, Kwon OY. Jchince K. et al. Effect of dilution on viability of vitrified-warmed mouse embryos. Japanese Journal of Animal Reproduction, 1989, 35: 211-216
39 Gal F, Baril G, Vallet J C, et al. In vivo and in vitro survival of goat embryos after freezing with ethylene glycol or glycekol. Theriogenoiogy, 1993, 40: 651-659
40 Dumoulin JM. Bergers Jansen JM. Pieters MHCE. et al. The protective effects of polymers in the cryopreservation of human and mouse zonae pellucidae and embryos. Fertility and sterility, 1994, 62: 793-798
41 Kasai M. Komi H,Takakamo H,et al. A simple method for mouse embryo cryopreservation in a low toxicity vitrification solution, without appreciable loss of viability. J Reprod Fert, 1990, 89: 91-97
42 Tada N, Sato M. Amann E, et al. A simple and rapid method for cryopreservation of mouse 2-cell embryos bv vitrification: beneficial effect of sucrose and raffinose on their cryosurvival rate. Theriodenotogy, 1993, 40: 333-344
43 Kasai M, Nishimori M, Zhu SE. et al. Survival of meuse morulae vitrified in
an ethylene glycol-based solution after exposure to the solution atvarious temperatures. Biology of Reproduction, 1992.47:1134-1130
44 Zhu SE. Kasai M. Otoge H. et al. Cryopreservation of expanded mouse blastocysts by vitrfication in ethylene glycol based solution. J Reprod Fertil. 1993. 98: 139-145
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