Cloning And Expression Of Mouse GABRR2 Gene And CRISPR/Cas9 Mediated Gene Editing

BackgroundGamma-aminobutyric acid?GABA?is the main inhibitory neurotransmitter in the mammalian central nervous system?CNS?,and GABA receptors are also the key targets for many clinically useful neuroactive compounds.GABAc receptor is the key regulatory receptor in the retina,when activated they inhibit the release of glutamate from the bipolar neurons toward the ganglion cells,thus modulating the retina dynamic range.Optic nerve disease resulting from retinopathy is the emergency and in ophthalmology.However,the study of the new targets and new approach for the process of the retinopathy is still not very comprehensive.In this reasearch,we construct a structure-function screening platform based on GABAC recepter with GABAC recepter Rho2?GABRR2?gene overexpression and knock-out cell lines in mouse retinal ganglion cells?RGC-5?.The two cell lines will be useful tools to study the structure-function of GABAC recepter and the interaction between receptors and drugs,which will enrich the knowledge for the therapeutic applications and drug design.ObjectiveTo construct the GABRR2 gene overexpression and knock-out cell lines in mouse retinal ganglion cells?RGC-5?.To study the genotype and phenotype of the gene-edited diploid cells,and to study the effects of gene editing on the exon-splicing of the GABRR2 gene.Methods?1?pIRES-e GFP-mRho2 overexpression plasmid was constructed under the fastcloning method[1] using p CMV6-Entry-m Rho2 plasmid and p IRES-e GFP vector as the templates.Plasmid transfection was mediated into RGC-5 cells with Lipo6000^TM following the manufacturer's instructions to construct the GABRR2 gene overexpression cell line.GFP expression in transfected cells was examined using fluorescence microscope and Flow Cytometry,and the GABRR2 gene expressiong level was detected by Realtime-PCR method.?2?Seven sg RNAs targeting exon5 and exon6 of the GABRR2 gene were designed and synthesized,and then were ligated with Cas9/sg RNA expressing vector.Cas9/sg RNA1⁴ expression vectors were transfected into RGC-5cell,and the efficiency of the Cas9-sg RNA modification was investigated with T7E1 cleavage assay.The primary positive individual RGC-5 clones were obtained under the limiting dilution assay.Sequencing were applied for genomic DNA and m RNA sequences analysis.?3?Up and down homologous arms targeting exon5 and exon6 of the GABRR2 gene were designed and synthesized,and then were ligated with p Homo Recom vector to construct p Homo Recom-E5E6,co-transfected with Cas9/sg RNA4 expression plasmids.After the screen of G418 and GCV selection element,the primary positive individual RGC-5 clones were obtained by limiting diluted assay.Sequencing were applied for genomic DNA sequences analysis.Results?1?Successfully constructed p IRES-e GFP-m Rho2 overexpression plasmid and GABRR2 gene overexpression cell line.?2?Cas9/sg RNA1?Cas9/sg RNA2 and Cas9/sg RNA4 expressing vector can all edit the GABRR2 gene.Obtained 9 no-homologous recombination GABRR2 gene knock-out cell clones transfected with cas9/sgrna4 expression plasmids.Described the effects of CRISPR/Cas9-mediated gene editing on the splicing of GABRR2 exons?3?Successfully constructed p Homo Recom-E5E6 plasmid and obtained homologous recombination GABRR2 gene knock-out cell line.Conclusions Successfully constructed the GABRR2 gene overexpression and knock-out cell lines in mouse retinal ganglion cells?RGC-5?.They will be used to establish a cellular research platform for the gene function research and drug screening based on GABAC recepter,and provide important experimental basis for the reasearch during retinopathy.At the same time,we have further studied the application and rapid screening of CRISPR/Cas9 mediated gene editingin in diploid cell,and described the effects of gene editing technology on exon skippingof genes,providing a reference for the application of gene editing technologythe.