小鼠胚胎冷冻的研究

【作者】 朱孝荣；

【导师】 李厚达；

【作者基本信息】 扬州大学 ， 动物学， 2003， 硕士

【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。更多还原

【Abstract】 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 ofmice embryo cryopreservation with solution EG40 ES40 and EFS40 by rapideryopreservative and vitrified method. Other than straw, the cell freezing tubewas firstly adopted. Using EG40 and ES40,The development ratio reached66.4%x 66.7% and the implantation ratio reached 41.4% 50% respectively aftermaw EFS40 was used as solution to be vitrified .The development ratio andimplantation ratio reached 87.2%, 66.3% respectively. The difference is notemarkable compared to the control. We use EFS40 as vitrified solution and strawwas 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, ES40eryopreservative method is better than one-step method, and the difference isremarkable, while the different effect of EFS40 two-step vitrified method is notremarkable. Subsequently,with the straw and the cell freezing tube,we usedone-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更多还原