摘要
为了得到超量表达胶质细胞源神经营养因子(glial cell line-derived neuotrophic factor,GDNF)的NIH-3T3细胞株,用于制作精原干细胞(spermatogonial stem cells,SSCs)培养的饲养层,通过RT-PCR方法成功地从幼年小鼠睾丸中克隆了gdnf基因,构建了真核表达载体pcDNA3.1-gdnf,并用其转染NIH-3T3细胞.对筛选出的阳性细胞克隆进行的免疫荧光染色、RT-PCR和Western blotting的结果表明,获得了超量表达gdnf基因的NIH-3T3细胞株,这为精原干细胞的培养奠定了基础.
In order to obtain the feeder cells expressing the glial cell line-derived neurotrophic factor （GDNF） to culture spermatogonial stem cells （SSCs）, the gdnf gene from the testes of normal immature mice was cloned by RT-PCR method, the eukaryotic expression vector of pcDNA3. 1-gdnf was constructed and then transfected into NIH-3T3 cells. Sequence analysis identified that 723 bp eDNA of gdnf was obtained precisely, and the transgenetie over-expression were also demonstrated with immunofluorescence detection, RT-PCR and Western blotting. Thus, the NIH-3T3 feeders over-expressing gdnf was obtained successfully and the basis of SSCs culture was established.
出处
《生命科学研究》
CAS
CSCD
2009年第1期55-59,共5页
Life Science Research
基金
山东省自然科学基金资助项目(Y2007079)
潍坊市科技发展计划项目(2007028)
