【摘要】 Cre介导的片段交换技术利用重组酶Cre的位点特异性重组特性 ,在基因组的特定位点进行靶片段与目的片段的交换。运用互为反向的Lox位点 ,在鼠红白血病MEL细胞中进行靶载体的整合和交换载体的交换 ,探讨在特定的染色质环境下红系特异性顺式作用元件的功能。电穿孔转染MEL细胞后从含有潮霉素 (hygromycin)的选择性半固体培养基中挑取MEL细胞单克隆 ,通过PCR和Southern杂交鉴定整合完整性和拷贝数 ,获得三种整合有靶载体p1L HyTk L1 β EGFP neo的细胞株A ,B和D。交换载体pL1 HS2 1L(含有 732 bp的人β 珠蛋白基因簇 5′DNaseI高敏位点 2核心片段 )和Cre表达载体pBS185共转染细胞株A ,9 (1,3 二羟 2丙氧甲基 )鸟嘌呤 (gancyclovir)负筛选后挑取单细胞克隆A HS。PCR检测显示HS2片段以反方向进行了交换。流式细胞仪分析显示平均的荧光细胞百分比 (2 .4 2 % )低于未交换的细胞株A (35 .94 % )。A HS中EGFP的低表达可能是处于非容许方向的HS2片段出现方向依赖性基因沉默所致。更多还原

【Abstract】 Cre-mediated cassette exchange has been developed to perform site-specific chromosomal integration using Cre recombinase. Here, site-specific integration with inverted Lox sites was used to investigate the erythroid cis -acting DNA element in specific chromatin contexts in mouse erythroleukemia cells. Single hygromycin-resistant clones were obtained from the selective semi-solid medium containing hygromycin post-electroporation. PCR and Southern blotting analysis showed single-copy integration of target vector in clones A, B and D. Site-specific cassette exchange was performed in clone A with exchange vector and Cre expression plasmid, followed by gancyclovir selection. Flow cytometry was used for analysis of EGFP gene expression. A 732-bp fragment of human β-globin gene cluster 5′ DNase I hypersensitive site 2(HS2) was exchanged and integrated into clone A in an anti-genomic orientation. The low EGFP expression in clone A-HS may be due to the orientation-dependent gene silencing caused by integration of HS2 in a non-permissive orientation.更多还原