[1]章波,王燕,许雪青,等.一种真核生物染色质隔离子筛选方案的初步建立[J].第三军医大学学报,2007,29(17):1688-1691.
 ZHANG Bo,WANG Yan,XU Xue-qing,et al.Construction of screen system for chromosome insulator in eukaryotes[J].J Third Mil Med Univ,2007,29(17):1688-1691.
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一种真核生物染色质隔离子筛选方案的初步建立(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
29卷
期数:
2007年第17期
页码:
1688-1691
栏目:
论著
出版日期:
2007-09-15

文章信息/Info

Title:
Construction of screen system for chromosome insulator in eukaryotes
作者:
章波王燕许雪青白云杨晓亚
第三军医大学基础医学部医学遗传学教研室
Author(s):
ZHANG BoWANG YanXU Xue-qingBAI YunYANG Xiao-ya
Department of Medical Genetics, College of Medicine, Third Military Medical University,Chongqing 400038,China
关键词:
染色质隔离子增强子基因表达
Keywords:
chromatin insulator enhancer gene expression
分类号:
Q756; Q78
文献标志码:
A
摘要:
目的  探讨建立染色质隔离子的正负筛选工具的可行性。 方法  合成引物从鸡基因组cHS4区域扩增了1段特异的DNA,将PCR产物克隆到T-载体后测序,验证其对增强子的阻断功能;对pEGFP-N1载体进行一系列改造,评价其正负筛选效果。 结果  成功地获得已知的隔离子片段,初步结果表明该片断能阻断增强子的转录激活作用。将TK基因作为负向筛选标记引入pEGFP-N1载体后获得了改造后的载体,转染COS-7细胞经过G418筛选10d后获得了稳定转染的细胞系,在不同浓度GCV药物作用下细胞杀伤效果有差异。 结论  正负筛选方法被成功地引入到筛选系统,为进一步进行染色质隔离子的筛选与利用奠定了基础。
Abstract:
Objective    To further investigate the function of chromatin insulators and to construct a screen system for chromatin insulators.     Methods    A primer set targeting chicken cHS4 locus was designed and synthesized. The specific PCR product using this primer set was obtained and sequenced. In order to test its blocking activity, we further inserted this fragment between enhancer and promotor. Then TK gene was inserted into modified pEGFP-N1 plasmid, which was tested for its negative and positive selection.     Results    The result of  luciferase activity showed that insulators could function when it was located on plasmid. Then vector pEGFP-N1 was modified as a screening system for chromatin insulator, in which phosphotransferase gene and thymidine kinase gene could function as selection marker. In order to evaluate its efficiency and viability, the recombinant vector was transfected into COS-7 cells. After selected with G418 for 10 d, stable transfected cells with G418 resistance were obtained. The sensitivity to pro-drug of GCV of these cells was different from that of its control cells by MTT.     Conclusion    This study provides an early research to further screen and utilize chromatin insulator in eukaryotes.

参考文献/References:

章波,王燕,许雪青,等.  一种真核生物染色质隔离子筛选方案的初步建立[J].第三军医大学学报,2007,29(17):1988-1691.

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更新日期/Last Update: 2008-07-21