睾丸注射慢病毒生产转基因鸡的研究

Transgenic chicken generated by testis injection of recombinant lentivirus

【目的】采用睾丸注射慢病毒的方法生产转基因鸡以提高转基因效率,使转基因鸡的批量生产变得简单可行。【方法】用磷酸钙沉淀法包装慢病毒,对慢病毒包装体系中的质粒总量和HN Buffer的pH值进行优化,以包装出滴度较高的慢病毒;然后将慢病毒注射到受精鸡蛋的胚胎中验证其活性;最后,将慢病毒注射到8日龄公鸡的睾丸内,待其性成熟以后,将精液DNA呈阳性的公鸡与非转基因母鸡进行交配生产出转基因鸡。【结果】当90mm培养皿中质粒总量为50μg、HN Buffer的pH值为7.05时慢病毒的包装效率最高,在该条件下包装出的慢病毒滴度高达7.5×107 TU/mL;采用慢病毒胚胎注射途径成功地生产出增强绿色荧光蛋白(enhanced Green Fluorescent Protein,eGFP)转基因鸡;采用慢病毒睾丸注射途径生产的转基因鸡后代中能检测到eGFP目的基因,但未检测到其相关的表达。【结论】慢病毒睾丸注射途径能够将外源基因整合到精原干细胞中,并遗传给后代。

【Objective】We applied testis injection with recombinant lentivirus to improve transgene efficiency to make generation of transgenic chicken simple.【Method】Plasmid quantity and pH of HN buffer in the packaging system were optimized to produce recombinant lentiviruses by calcium phosphate precipitation with high titers.Then,the activity of recombinant lentiviruses was tested by being injected into embryos of new fresh fertilized eggs.Testis of 8-day-old male chicken was injected with lentivirus.After being sexual mature,chicken with transgenic sperm were verified by examining sperm DNA and were mated with normal female chicken to generate transgenic offspring.【Result】The titers of lentivirus was up to 7.5×10^7 TU/mL when the plasmid quantity was 50μg in 90 mm petri dish and the pH of HN Buffer was 7.05.Transgenic chicken were successfully generated.Target eGFP gene was verified in the transgenic offspring generated by testis injection method based on lentivirus,but no protein was detected.【Conclusion】The exogenous gene could be integrated into spermatogonial stem cells and transferred to offspring through testis injection approach based on lentivirus.