定量诱导表达PRDM1永久细胞系的建立

Establishment of quantitatively inducible PRDMI-expressing stable cell line

  • 摘要: 目的 建立可定量诱导表达PRDM1的永久细胞系,为进一步研究PRDM1的功能创建平台。方法 将野生型PRDM1基因的cDNA全长克隆到pMEP4表达质粒中,电穿孔法将pMEP4-PRDM1瞬时转染到细胞系SALT3,经潮霉素B筛选后挑选可增殖的单克隆细胞并大量扩增,在不同浓度的硫酸镉(CdSO4)诱导下检测PRDM1蛋白的表达状况。结果 成功构建了pMEP4-PRDM1表达载体,在最佳电穿孔条件下将其转染到SALT3细胞,经潮霉素B筛选后扩增出6个细胞克隆。CdSO4诱导后运用免疫印迹法验证了PRDM1蛋白的表达与诱导剂CdSO4存在量效关系。结论 运用pMEP4载体可建立定量诱导表达目的基因PRDM1的永久细胞系,为进一步研究PRDM1的功能奠定了基础。

     

    Abstract: Objective To establish the quantitatively inducible PRDM1-expressing stable cell line in order to create a platform for the study of PRDM1 function. Methods Full-length PRDM1 cDNA was inserted into the multiple cloning site of pMEP4 vector.A pMEP4-PRDM1 expression vector was instantaneously transfected into SALT3 cell line by electroporation,followed by hygromycin B selection.Hygromycin-resistant stable clones were expanded and PRDM1 protein expression was detected at different doses of CdSO4 by Western blot. Results The pMEP4-PRDM1 expression vector was successfully constructed and subsequently transfected into the SALT3 cell line under optimal electroporation conditions.Six hygromycin-resistant stable clones were established and expanded.The PRDM1 protein expression in these stable cell lines was proved to be quantitatively correlated with the dose of CdSO4. Conclusion Quantitatively inducible PRDM1-expressing stable cell line can be established using the pMEP4 vector,thus laying a good foundation for further analysis of the function of this gene.

     

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