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SV40PolyA片段串联拷贝数目影响GFP报告基因表达

, PP. 187-194

Keywords: 拷贝数,SV40PolyA,重复序列,GFP,Alu

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Abstract:

Alu元件(约280bp)是人类基因组中的重要重复序列,串联Alu呈长度依赖性下调GFP报告基因表达,在Alu串联序列上游正向或反向插入SV40PolyA(简称PolyA,240bp),解除Alu串联序列对GFP基因的抑制作用.PCR法扩增PolyA反序(PolyAas)不同位置的60bp片段,插入pAlu14质粒(14个Alu正向串联插入pEGFP-C1)的GFP基因和Alu14之间,瞬时转染HeLa细胞,通过荧光显微镜观察和Northern检测,1F1R(PolyAas5′端第1个60bp片段)和4F4R(自PolyAas5′端计算第4个60bp片段)不能活化基因;2F2R和3F3R(PolyAas中间的2段)可以解除Alu14对GFP基因的抑制作用.将2F2R和3F3R各自反复首尾串联,分别插入pAlu14质粒GFP基因和Alu串联序列之间,瞬时转染HeLa细胞,用插入4个2F2R的pAlu28,插入4个3F3R的pAlu18,插入4个3F3R的pAlu28作长度对照,以排除由于插入片段长度增加可能对结果造成的干扰,发现2~4个拷贝的2F2R和3F3R活化基因作用高于一个拷贝的同样片段,但是多于8拷贝以后,活化GFP基因作用减弱.本实验证明,SV40PolyAas至少含有两段活化基因序列,活化基因序列(2F2R,3F3R)的最适活化基因条件需要合适的拷贝数.

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