GDF11过表达稳转U251细胞株的构建

Construction of GDF11-overexpressing stable glioma U251 cell line

目的 :构建过表达生长分化因子11(GDF11)的神经胶质瘤U251稳转细胞株。方法 :合成GDF11全长序列,构建HBLV-h-GDF11-3×flag-LUC-PURO慢病毒载体。将其与质粒pAX2和pMD2G共同转染293T细胞,收集上清并浓缩,获得慢病毒浓缩液。利用该慢病毒感染U251细胞,经嘌呤霉素筛选获得稳定过表达GDF11的细胞株,通过RT-PCR鉴定稳转细胞株中GDF11 mRNA的表达水平。结果 :测序结果与设计序列一致,慢病毒浓缩液滴度为3.0×10^(8) TU/mL,RT-PCR结果显示在该稳转细胞株中GDF11表达水平上调154倍。结论 :成功构建了稳定过表达GDF11的U251细胞株,为进一步研究GDF11在神经胶质瘤中的作用奠定了基础。

Objective To construct a glioma cell line with stable growth differentiation factor 11(GDF11) overexpression.Methods The full-length GDF11 sequence was synthesized to construct HBLV-h-GDF11-3×flag-LUC-PURO lentiviral vector. It was co-transfected with plasmids pAX2 and pMD2G into 293T cells, and then the supernatant was collected and concentrated to obtain a lentiviral concentrate. U251 cells were infected with the lentivirus and screened by puromycin to obtain a GDF11-overexpressing stable cell line. The expression level of GDF11 mRNA in this stable cell line was identified by RTPCR. Results The sequencing results were consistent with the designed sequence. The titer of the lentivirus concentrate was 3.0×10~8 TU/m L. RT-PCR results showed that the expression level of GDF11 was up-regulated 154-fold in the lentivirus-infected U251 cells. Conclusion The glioma cell line stably overexpressing GDF11 has been successfully constructed, which may lay the foundation for further research on the role of GDF11 in glioma.