睾丸内注射和体内电穿孔法建立转基因小鼠的实验研究

目的 探讨在体睾丸生精小管注射合并体内电穿孔建立转基因小鼠的可行性.方法 将两种不同处理的转染液分别注射到4组SPF级雄性昆明小鼠睾丸生精小管内,然后对同一种转染液注射的两组动物中的一组进行体内电穿孔,另一组则不进行体内电穿孔;2周后各组SPF级雄性昆明小鼠分别与超排处理的SPF级雌性昆明小鼠合笼交配,最后对各组新生仔鼠进行PCR-Southern检测.结果 PCR检测,四组之间存在显著差异(P＜0.01),并且以A组最高(45%);Southern blotting检测,4组之间差异不显著(P＞0.05),但A组的整合效率(25%)仍高于其他3组.结论 运用在体睾丸生精小管内注射合并体内电穿孔技术可以显著地提高外源基因的转染效率,该方法是否可广泛用于生产转基因动物还有待于进一步深入研究.

Generation of transgenic mice by intratesticular injection and electroporation in vivo

Objective To evaluate the feasibility of establishing transgenic mice by means of seminiferous tubule microinjection and electroporation (EP) in vivo. Methods Specific pathogen-free (SPF) male Kunming mice divided into 4 groups were subjected to microinjection of two different transfection solutions labeled with enhance green fluorescent protein (EGFP) into the seminiferous tubule of the testis, and in one of the two groups receiving the identical transfection solutions, EP in vivo was performed. After two weeks, the male mice of each group were mated with SPF female Kunming mice with superovulation treatment, and PCR coupled with Southern blotting was performed for the offspring mice. Results The results of PCR suggested significant difference in the efficiency of exogenous gene integration between the 4 groups (P<0.01), among which group A achieved the greatest efficiency (45%). Southern blotting did not identify significant difference between the 4 groups (P>0.05), but still suggested the highest efficiency in group A (25%). Conclusion Seminiferous tubule microinjection in conjunction with subsequent EP in vivo can remarkably enhance the integration efficiency of exogenous genes into the host genome, but this new method needs to be further tested for its potential utility in transgenic animal generation.