TCF25干扰慢病毒载体的构建与鉴定

Construction and Characterization of a Lentiviral Vector for RNA Interference of TCF25

为构建一个针对TCF25(transcription factor 25)基因的慢病毒干扰载体,设计并合成3组针对TCF25的短发夹RNA序列(shRNA),通过基因重组技术连入p LL3.7载体,酶切鉴定及DNA测序后,重组正确的p LL3.7质粒与病毒包装质粒共转染293FT细胞,培养48 h后,分别收集细胞培养上清液,感染H9C2细胞,Western-blot检测TCF25在H9C2细胞中的表达.结果显示TCF25蛋白在H9C2细胞中的表达被抑制.这说明TCF25的慢病毒干扰载体构建成功.

Objective To construct a lentiviral vector for RNA interference of TCF25（ transcription factor25） gene and identify it. Methods Three pairs of complementary short hairpin RNA（ shRNA） oligonucleotides targeting the TCF25 gene were designed,synthesized,and then inserted into p LL3. 7 vectors by gene recombination technology. The recombinant plasmid was identified by enzyme digestion and DNA sequencing. Correct recombinant plasmids were cotransfected with packaging plasmids into 293 FT cells. The cell culture supernatant was obtained after 48 hours,and then applied to infect H9C2 cells. The expression of TCF25 in H9C2 cells was detected by western-blot. Results The recombinant plasmid was successfully constructed. The western-blot showed that the expression of TCF25 in H9C2 cells was inhibited after the cells were successfully infected with the lentiviral vector supernatant. Conclusion The lentiviral vector interfering TCF25 was constructed successfully.