摘要
对造血调控中发挥重要作用的IKAROS基因亚型IK4进行突变,并构建重组逆转录病毒表达载体及使其在白血病细胞中表达。采用重叠延伸PCR定点突变技术,成功构建了4个IK4单突变或多突变突变体。将这4个突变体克隆至Pmscvpuro逆转录病毒载体中,构建重组蛋白表达载体。基因测序验证突变序列正确后,将重组逆转录病毒载体与病毒包装质粒VSVG、Gag-Pol共同感染293T细胞,过滤收集病毒上清,将病毒上清转染白血病NB4细胞。用Puromycin对细胞进行筛选,并通过Western印迹法对突变体表达细胞系进行验证。通过重叠延伸PCR法成功构建了4个IK4的定点单突变和定点多突变体,利用逆转录病毒介导的转染方法使IK4的4个突变体在白血病细胞中成功稳定表达,为后续的功能和机制的研究奠定了基础。
IK4 is an isoform of IKAROS gene which plays a key role in the hematopoiesis, in this work, the construction of retro-virus vector of IK4 mutation and its expression in leukemic cells was investigated. PCR site-directed mutagenesis method based on overlap extension was used to construct four IK4 gene mutants of singleor multi-site mutations, which were then cloned into the retrovirus vector Pmscv-puro to form recombinant expression vector. The point mutations were verified by DNA sequencing. The recombinant expression vectors were co-transfected with packaging plasmids VSVG and Gag-Pol into 293T cells to produce retroviruses. Supernatants containing retroviruses were collected and were used to infect NB4 cells. Puromycin was used to select stable cell lines. The expression of mutants was verified by Western blotting analysis. Four IK4 gene mutants of singleor multi-site mutations were successfully constructed by overlap extension PCR, and the mutants were expressed in leukemic cells, which will provide basis for subsequent function and mechanism research.
出处
《中国科技论文》
CAS
北大核心
2016年第6期684-687,693,共5页
China Sciencepaper
基金
高等学校博士学科点专项科研基金资助项目(20133321120003)
国家自然科学基金资助项目(81400125)
浙江省自然科学基金资助项目(LQ13H080002)
