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目的探讨遗传工程小鼠精子冷冻、复苏及体外受精率的效果,建立简便、经济的遗传工程小鼠保种体系。方法采用精子冷冻、体外受精和胚胎移植技术,比较了精子获能培养液、雄鼠周龄及精子冻存液等因素对于遗传工程小鼠精子冷冻保存的影响。结果用CPA精子冻存液冷冻保存精子,精子复苏后用PM精子培养液获能培养,体外受精率在82.49%~91.43%,而HTF精子培养液的体外受精率为14.46%~27.38%,同品系间差异极显著(P<0.01);10~35周龄的雄鼠精子均能成功冷冻、受精,移植后产仔;使用R18S3,CPM,CPA三种不同精子冻存液冷冻遗传工程小鼠精子,复苏后采用PM精子培养液体外受精,受精率分别是75.85%、88.89%和94.27%,移植后得到阳性小鼠;体外受精后的胚胎成功冷冻保存,移植后产仔。结论采用CPA精子冻存液冷冻保存精子,精子复苏后用PM精子培养液体外受精,能够更有效地保存遗传工程小鼠。
Objective To explore the effect of genetically engineered mouse sperm freezing, resuscitation and in vitro fertilization rate, and to establish a simple and economical genetically engineered mouse seed protection system. Methods The sperm cryopreservation, in vitro fertilization and embryo transfer techniques were used to compare the effects of sperm capacitation culture medium, male rat age and sperm cryopreservation on sperm cryopreservation in genetically engineered mice. Results The sperm were cryopreserved in CPA sperm cryopreservation solution. After sperm resuscitation, PM sperm culture fluid was able to be cultured, the in vitro fertilization rate was 82.49% ~ 91.43%, while the in vitro fertilization rate of HTF sperm culture fluid was 14.46% ~ 27.38% (P <0.01). The sperm of male mice aged 10-35 weeks were successfully frozen, fertilized and litter-borne, and the genetically engineered mice were frozen using three different sperm cryopreservation solutions of R18S3, CPM and CPA After sperm resuscitation, PM sperm culture liquid was fertilized by external fertilization. The fertilization rates were 75.85%, 88.89% and 94.27%, respectively. After transplanting, the positive mice were obtained. After in vitro fertilization, the embryos were cryopreserved and transplanted. Conclusion CPA sperm cryopreservation cryopreservation of sperm, sperm recovery with PM sperm liquid culture of external fertilization, can be more effective preservation of genetically engineered mice.