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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。
The Cre-mediated Fragment Swap Exchange utilizes the site-specific recombination of recombinase Cre to exchange target and target segments at specific sites in the genome. Using the Lox sites in opposite directions, the target vector integration and exchange vectors were exchanged in murine erythroleukemic MEL cells to investigate the function of erythroid-specific cis-acting elements in specific chromatin environments. After electroporation transfection of MEL cells, MEL cell clones were picked from hygromycin-containing selective semi-solid medium, and the integration integrity and copy number were identified by PCR and Southern blotting to obtain three kinds of target vectors Cell lines A, B and D of p1L HyTk L1 β EGFP neo. The vector pL1 HS2 1L (containing a 732 bp human β-globin gene cluster) was co-transfected into the Cre expression vector pBS185 cell line A, 9 (1,3 dihydroxy 2-propoxymethyl ) Single-cell clone A HS was picked after negative selection by gancyclovir. PCR assays revealed that HS2 fragments were swapped in the opposite direction. Flow cytometry analysis showed that the average percentage of fluorescent cells (2.42%) was lower than that of untransformed cell line A (35.94%). The low expression of EGFP in A HS may be due to the direction-dependent gene silencing of the HS2 fragment in an impermissible direction.