国家科技支撑计划（2014BAI03B01）。
目的 在细胞水平筛选狨猴B2m基因的有效沉默靶点，并进行验证。方法 查询人源B2m验证过的有效siRNA靶位点序列，与狨猴B2m基因序列进行同源性比较，选择匹配靶点合成shRNA序列。将体外合成的2条干扰序列分别与慢病毒载体FUGW-TDT连接，构建FUGW-TDT-shb2m干扰表达质粒，在聚乙烯亚胺（polyethylenimine，PEI）介导下转染293T细胞，转染后48h，用实时荧光定量法检测转染细胞中B2m基因mRNA的水平。结果 筛选出2个与狨猴完全同源的B2m沉默靶位点，分别位于B2m mRNA的290~310 bp，665~685 bp；B2m两个靶点在转录水平的沉默效率分别为（46.54±7.91）%（P < 0.05）和（83.22±4.37）%（P< 0.0001），差异有显著性。结论 成功构建成FUGW-TDT-shb2m重组质粒；在细胞水平筛选得到2个有效的B2m基因沉默靶点；为后续有关介导狨猴B2m基因沉默的研究奠定了基础。
Objective To screen and determine the effective silencing targets of β2-microglobulin(B2m)gene at the cellular level in marmoset.Methods By homology comparison of the b2m gene in human and the B2m gene in marmoset, choose homology small hairpin RNA(shRNA)sequences targeting marmoset B2m gene were designed, We choose homology small hairpin RNA(shRNA)sequences targeting designed B2m gene to make homology analysis, and insert into lentivirus-based gene silencing constructs FUGW-TDT. The vectors were transfected into HEK293T cells induced by polyethylenimine(PEI). The suppression of B2m mRNA was detected by real-time PCR. Results Two gene-silencing sequences were screened that lied in 290~310 bp and 665~685 bp of the marmoset B2m mRNA, and have statistical significance in the silencing rate:(46.54±7.91)% (P< 0.05) and(83.22±4.37)%(P< 0.0001). Conclusions Two effective silencing target sequences are screened at cellular level, which can be further used in studies on gene silencing in marmoset.
邓怡晨,张晨,向志光,滕永康,刘云波.狨猴B2m基因沉默位点在细胞水平的验证[J].中国比较医学杂志,2017,27(5):37~41.
复制