hnRNPU基因敲减小鼠preB A70细胞株的构建及鉴定

Construction and identification of hnRNPU-knockdown murine preB A70 cell line

目的构建shRNA-不均一核糖核蛋白U(heterogeneous nuclear ribonucleoprotein U,hnRNPU)慢病毒表达载体以及hnRNPU基因稳定敲减的小鼠preB A70细胞株。方法将靶向小鼠hnRNPU基因的shRNA干扰序列连接入pLKO.1-EBFP-PURO质粒中,测序检测。将构建成功的pLKO.1-hnRNPU shRNA-EBFP-PURO和慢病毒包装质粒共转染HEK293T细胞,收集病毒上清。将病毒上清感染A70细胞,利用倒置荧光显微镜观察病毒感染情况,利用Western blot检测hnRNPU蛋白的表达。通过有限稀释方法,构建单细胞克隆,并利用Western blot筛选hnRNPU基因稳定敲减的A70细胞株。结果成功构建了shRNA-hnRNPU慢病毒表达载体。表达shRNA-hnRNPU的慢病毒在HEK293T细胞中完成包装。包装好的病毒感染A70细胞72 h后,细胞内可以观察到蓝色荧光。感染后的细胞经过嘌呤霉素筛选后,细胞内的hnRNPU蛋白大约减少一半。通过有限稀释方法,构建了hnRNPU基因稳定敲减的细胞株。结论本研究成功构建了hnRNPU稳定敲减的A70细胞株,为后续研究hnRNPU在早期B细胞发育中的作用奠定了实验基础。

Objetive To construct shRNA-hnRNPU lentiviral expression vector and stable hnRNPU-knockdown(KD)murine preB A70 cell line.Methods shRNA against mouse hnRNPU gene was encapsulated in pLKO.1-EBFP-PURO vector.Identified by sequencing,pLKO.1-hnRNPU shRNA-EBFP-PURO vector was co-transfected with lentiviral packaging plasmids into HEK293T cells to form lentiviruses,and the lentiviral supernatant was collected.The packaged lentiviruses transduced A70 cells which were observed by an inverted fluorescence microscope.Western blot was used to test hnRNPU expression.Single cell clones were constructed by limiting dilution,stable hnRNPU-KD A70 cell line was formed and identified by Western blot.Results The shRNA-hnRNPU lentiviral expression vector was successfully constructed,and the lentiviruses expressing shRNA-hnRNPU were packaged in HEK293T cells.72 h after transduction,blue fluorescence was observed in the virus-infected A70 cells.HnRNPU expression in puromycin-treated cells decreased about a half.By limiting dilution,hnRNPU-KD cell lines were formed.Conclusion In this study,a stable hnRNPU knockdown A70 cell line was successfully constructed,which laid the experimental foundation for further study of the role of hnRNPU in early B cell development.