摘要
利用慢病毒干扰系统,对东北梅花鹿角柄骨膜干细胞(PP细胞)P21基因进行干扰。结果表明:筛选出的2条针对梅花鹿P21基因的siRNA与载体质粒PLVTHM连接成功,并与pMD2.G、pCMV-dr8.9质粒共转染到293t细胞,获得重组慢病毒;通过感染PP细胞并利用流式细胞仪进行分选,获得了纯度90%以上的感染细胞;荧光定量RT-PCR检测表明P21基因的mRNA水平大幅度下调,干扰效率达到70%。表明成功干扰了P21基因在PP细胞中的表达,获得了低表达P21的PP细胞系。
P21 gene of the pedicle periosteal cells of sika deer was interfered using RNAi in lentiviral vector system .The results showed that：（1 ）Two sequences of small interfering RNAs,targeting P21 gene of sika deer,were successfully reassembled into the lentiviral plasmids（Plvthm）.Positive clones were identified based on the results of both PCR and sequencing .Recombinant lentivirus was acquired by each positive plasmid co-transfecting into 293 T cells with the plasmids pMD2 .G and pCMV-dr8.9;（2）Recombinant lentivirus was successfully interfered into the PP cells,and the GFP positive cell pro-portion obtained by flow cytometry （FCM）sorting was about 90%;（3）The result of RT-PCR showed that the expression level of P21 mRNA in cells infected with recombinant lentiviral was obviously de-creased,and the interferential efficiency was about 70%.Therefore,in this study,we successfully inter-fered the expression of P21 gene in the pp cells,and obtained pp cell line with decreased expression of P21 gene,which lays foundation for revealing the regulatory mechanism of P21 underlying antler regener-ation .
出处
《吉林农业大学学报》
CAS
CSCD
北大核心
2014年第1期116-121,共6页
Journal of Jilin Agricultural University
基金
"973"前期研究专项(2011CB111515)
吉林省自然科学基金项目(20101575)
关键词
鹿茸再生
P21
基因
角柄骨膜
慢病毒表达载体
RNAi
antler regeneration
P21 gene
RNAi
pedicle periosteum
lentiviral expression vector
