Transcription Activator-like Effectors Nucleases Mediate CCR5Gene Expression In Hela Cells

Objictive: TALENs（Transcription activator-like effectors nucleases）consisting ofthe TALEN F16.5and the TALEN R18.5which is included in recombinant shuttleplasmid pDC316, editing CCR5target sequence,resulting CCR5structural frameshiftchange, which may confirm it has gene directional editing function.Methods: construt recombinant plasmid expressring TALENs. Jurkat cells amdHela cells were divided into four groups: the first group is added with recombinantshuttle plasmid including TALEN F16.5/recombinant shuttle plasmid includingTALEN R18.5/CVU55762; the second group is tested with recombinant shuttleplasmid including TALEN F16.5/CVU55762; the third is blank group; the fourth isgreen fluorescent protein control (GFP). Hela cells transfected with editing functionTALENs produced by recombinant shuttle plasmid including TALEN F16.5/recombinant shuttle plasmid including TALEN R18.5/CVU55762, targeting on CCR5gene. The fluorescence intenxity was used to determine the efficiency of transfection,which was photographed by fluorescence microscopy. The tested hela cells wereextracted and their CCR-5gene were sequenced.Results: While transfected Hela and Jurkat cells showed very week fluorescencesignal, fluorescence intensity in hela cells were relatively brighter in which genesequencing results also showed CCR5gene mutations when the recombinant shuttleplasmid including TALEN F16.5/recombinant shuttle plasmid including TALENR18.5collaborated within the hela cells. The first group of transfected hela cellsshowed higher fluorescence efficiency as compared with other groups, in which theinsertion or deletion mutations in CCR5gene were relatively higher as well.Conclusions: At first, the recombinant shuttle plasmid pDC316including TALENF16.5and TALEN R18.5encoded the TALENs that can functionally knockout CCR5 by changing CCR5structure. Secondly, hela cells are good cell type in testing theediting effect of TALENs.Thirdly, TALENs mediated gene editing with highspecificity and tolerance, TALENs determines the editing site.