目的:构建KCTD10干扰RNA慢病毒载体.方法:合成KCTD10干扰序列,构建GV248-KCTD10-RNAi慢病毒干扰载体,利用PCR与测序验证阳性克隆.将KCTD10 siRNA干扰病毒载体与质粒pHelper1.0和质粒pHelper2.0三个载体共同转染293T细胞,使细胞合成并分泌释放病毒颗粒.收集病毒颗粒并检测病毒滴度.结果:GV248-KCTD10-RNAi慢病毒载体成功被连接转化,测序结果与设计序列一致;共转后收获KCTD10 siRNA慢病毒颗粒,检测其滴度为5×108TU/mL.结论:成功包装了KCTD10 siRNA慢病毒颗粒,为后续进一步研究KCTD10基因在糖尿病视网膜病变中的作用奠定了基础.%Objective To construct KCTD10 interference lentiviral vectors.Methods KCTD10 interference sequencewas synthesized to build GV248-KCTD10-RNAi lentivirus vectors, PCR and DNA sequencing were used to identify the positive clones. KCTD10 lentivirus vectors were cotransfected with pHelper 1.0 and pHelper 2.0 vectors into 293T cells, and viral par-ticles were produced and released from 293Tcells, then viral titer was detected. Results GV248-KCTD10-RNAi virus carrier are successfully connected and transformed. the sequencing results was consistent with the design of the sequence;Harvested the viral particles with a viral titer of 5×108 TU/ml after cotransfection. Conclusion The interference lentivirus particles of KCTD10 was packaged successfully, which laid a foundation for further study on the functions of KCTD10 gene in diabetic retinopathy.
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