摘要
本实验旨在探讨鸡精原干细胞(SSCs)经体外遗传修饰后移植获得转基因精子的方法。将人抗病毒基因MxA重组慢病毒体外感染鸡SSCs,获得可表达MxA的SSCs;同时将80日龄公鸡经白消安处理30 d后作为无生精能力的受体鸡。MxA基因转染后的SSCs移植到受体鸡,10 d后开始采精,经PCR检测精液DNA中的MxA基因;取移植后10 d的睾丸组织分离培养并检测外源基因表达。结果表明:MxA重组慢病毒感染SSCs后,可见其特征性荧光表达;白消安处理后的受体鸡睾丸组织曲细精管中内源性生精细胞已基本去除;实验组有1只受体鸡的精液DNA连续4次扩增出MxA特异性片段;睾丸组织细胞的RT-PCR及免疫组化均检测到MxA基因的表达。结果显示,重组慢病毒感染后的SSCs能成功移植至受体组织,并继续分化为携带MxA外源基因的精子。本研究为下一步通过人工授精获得抗病毒转基因鸡奠定了良好基础。
To establish the foundation of new strategy for preparation of transgenic chicken, the spermatogonial stem cells （SSCs） modified with exogenous gene in vitro were transplanted into recipient to generate transgenic sperms. In this study, chickens SSCs were infected by the recombinant lentiviral containing anti-virus gene MxA, and SSCs carrying MxA gene were obtained. Meanwhile, rooster chicken was treated with busulfan as recipient without spermatogenesis. Transgenic SSCs were transplanted into the recipient chicken testicular tissue. Screens were collected every two days from 10 d after transplanted, and used for detection of MxA gene by PCR. The cells of recipient testicular tissue after transplanted on 10 d were isolated and cultured for analysis of MxA gene expression by RT-PCR and immunohistochemical stain. The results showed that SSCs with MxA-EGFP fusion protein in vitro were observed by fluorescence; the histology of testis biopsy implied the endogenous germ cells in seminiferous tubules of recipient treated by busulfan has been basically removed; MxA specific fragments were amplified from 4 consecutive semen samples from one fourth of recipient; MxA expression in testicular cells of transplanted recipient has been detected by RT-PCR and immunohistochemistry. The data indicated that SSCs infected by MxA recombinant lentiviral were successfully transplanted into the recipient and sequentially differentiated into sperm carrying exogenous gene, which laid a good foundation for preparation antiviral transgenic chickens through the artificial insemination.
出处
《中国畜牧杂志》
CAS
北大核心
2013年第19期81-85,共5页
Chinese Journal of Animal Science
基金
国家自然科学基金项目(30500270)
浙江省钱江人才计划项目(2012R10076)
