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《遗传多样性:前沿与挑战——中国的遗传学研究(2013-2015)——2015中国遗传学会大会论文摘要汇编》2015年
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Construction of TALEN Plasmids and The General Vector of Multiple Locus Gene Targeting of Human Cells

刘婉霞  严爱芬  LIU Fang  FENG Cui-lan  LIU Jing  张雅洁  唐冬生  
【摘要】:Construction of TALEN plasmids to knock out rDNA and the general vector of multiple locus gene targeting.Using online software to design suitable TALEN cutting the corresponding loci in human genome rDNA gene,Five TALEN plasmids were constructed using a TALEN construction kit and they were transfected into HEK293T cells.After positive screening by puromycin,the genomic DNA of cells was extracted as the template,and the PCR products containing target sequence were sequenced.The results showed that the cleavage efficiency of each TALEN pair and pick up the significant one TALEN-L1R2.Meanwhile,amplify two homogenous recombination arms DS1、DS2 by PCR using human genome as a template.The fragment was inserted into pUC-19。Then,using In-fusion enzyme,the pCMV-MCS-EGFP DNA fragment amplified from the plasmid of pEGFP-N1 by PCR was connected with pUC-DS1-DS2.After enzyme identification,sequencing and transfecting it into HEK293T cells,all results showed that the recombinant expression vector pUC-DS1-EGFP-DS2 was successfully constructed.The multiple locus gene targeting combined with TALENs provides the possibility for site-specific transgenic animals and human gene therapy.It is also a new attempt to improve the efficiency of gene targeting.

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