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目的掏建转IΚBα突变型基因水生化神经前体细胞抹(INPCs)。方法限制性内切酶双酶切含IΚBα突变型基因的质粒pBluescript/CNP/IΚBαM,得到目的基因片断IΚBα突变型基因,克隆入真核表达载体pcDNA3．1中,酶切反应及DNA测序鉴定莆组载体pcDNA3．1/IΚBαM。利用脂质体介导的基因转染技术分别将载体pcDNA3．1及pcDNA3．1/IΚBαM转染INPCs．经G418(150μg/ml)筛选,挑选阳性克隆并扩大培养．2周后用RT-PCR和Western blot检测阳性细胞株中IΚBα的表达,用受NF-ΚB特异性启动表达的荧光素酶报告基因检测阳性细胞株中NF-κB的活性。结果经限制性酶切分析及DNA测序证实重组载体pcDNA3．1/IκBαM中含有IκBα突变型基因．转pcDNA3．1/IκBαM永生化神经前体细胞中IκBα的总mRNA表达增高,Western blot可检测到突变型IκBα蛋白的表达,且细胞中NF-κB活性下降。结论成功构建了,转IκBα突变型基因INPCs,该细胞株能通过IκBα突变型基因的表达来下调NF-κB的活性
Objective To dig into IKBα mutant gene hydro-neural precursor cells (INPCs). Methods The plasmid pBluescript / CNP / IKBαM containing the IKBα mutant gene was digested by restriction enzymes. The mutant gene of IKBα gene was cloned into the eukaryotic expression vector pcDNA3.1 and identified by DNA sequencing. Group vector pcDNA3.1 / IKBαM. The vectors pcDNA3.1 and pcDNA3.1 / IκBαM were transfected into INPCs respectively by liposome-mediated gene transfection. The positive clones were selected by G418 (150 μg / ml) and expanded. After 2 weeks, the expression of IκBα in positive cell lines was detected by RT-PCR and Western blot. The activity of NF-κB in positive cell lines was detected by luciferase reporter gene specifically activated by NF-κB. Results Restriction analysis and DNA sequencing confirmed that the recombinant plasmid pcDNA3.1 / IκBαM contained IκBα mutant gene. The total mRNA expression of IκBα in pcDNA3.1 / IκBαM immortalized neural progenitor cells increased. The expression of mutant IκBα protein was detected by Western blot, and the activity of NF-κB in cells decreased. Conclusion INPCs, a mutant of IκBα gene, was successfully constructed and down-regulated the activity of NF-κB through the expression of IκBα mutant gene