Construction Of Functional Vero-cre Cell Line Expressing Cre Recombinase

Cre-LoxP site specific recombination system is one kind of the allocation recombination system,which is widely used in the research and application at home and abroad.Cre-LoxP is a recombination system based on P1 bacteriophage which consists of Cre recombinase and LoxP sites.This system can not only cause site-direction integration between the exogenous genes and chromosome,but also delete specific DNA fragment.There are various application of this technology such as gene deletion,gene activation,gene transversion and group translocation.It also can be used in the construction of various vectors.Recently,various virus vectors are provided as important tools for the research of gene therapy.Herpes simplex virus(HSV) are recognized as such virus that can establish latent infection in neurons which are widely used in the gene therapy of neurological diseases.The application of BAC technology facilitates the construction of herpes simplex virus type 1(HSV-1) vector and makes such construction a new stage.BAC technology means cloning the HSV-1 genomes as bacterial artificial chromosomes (BACs) in Escherichia coli,which allows the stable maintenance of HSV genomes as BACs in Escherichia coli and manipulation of the viral genome in E.coli.There are LoxP sites between BAC which facilitates the recombination with the Cre recombinase in the process of vector construction and the deletion of BAC bones;In the construction of HSV-1 amplicon,the package signal can deleted by Cre-LoxP system.This study was aimed to construct the Cre recombinase lentivirus expression vector and establish the vero-Cre cell line expressing Cre recombinase.After infection of the HSV-1,the cell line can not only generate plenty of virus but also remove the BAC bone between the two LoxP sites.In the construction of HSV-1 amplicon,the package signal in the helper virus can deleted by Cre-LoxP system,which will generate lots of amplicon.By this way,it will be helpful to reduce the helper virus and increase the titer of amplicon.Object:This study was aimed to construct the Cre recombinase lentivirus expression vector and establish the vero-Cre cell line expressing Cre recombinase in order to facilitate the removal of the BAC backbone or the package signal in the procedure of HSV-1 vector construction by Cre-LoxP recombination system.Methods:1.Cre fragment about 2Kb was obtained by cleaving c66-pGEM-T-SV40-Cre with double restriction endonucleases NcoⅠand EcoRⅠ,which was cloned into lentivirus vector pLKO.1-puro that were cleaved by SalⅠand EcoRⅠby T4 ligase. The recombinant plasmid pLKO.1-puro-Cre was transformed into competent Escherichia coli DH5αcells.The positive recombinant clones were selected by ampicillin medium agar and identified by MluI and EcoRI.2.293T cells were cotransfected by recombinant vector plasmid of pLKO.1-puro-Cre,packaging plasmid ofΔ8.2 and envelop plasmid of VSV-G with FUGENE6 transfection reagent to produce lentivirus particles.3.Recombinant virus infected vero cells,then chose the cell line with the method of the puromycin screening at the least fatal dose to vero cells.Vero-Cre cell line was constructed by selection the singe clone of vero-Cre cells.4.The function of vero-Cre cell line could be identified by PCR and transfection BAC to the cell line.The sums of GFP positive cells were analysised by using non-parametric test of two dependent samples.Results:1.Cre recombinase was obtained by digestion of double restriction endonucleases.This segment was recombinated into lentivirus vector plasmid of pLKO-puro,then a positive recombinant colony was selected.2.Restriction enzyme digestion showed recombinant lentiviral vector plasmid PLKO.1-puro-Cre was correct.3.Lentiviral particles were made through three plasmid cotransfection of 293T cells.4.The least fatal dose of puromycin to veto cell was 8μg/ml,by which the cell line expressing Cre recombinase was selected.5.A segment about 560bp was obtained by PCR.After transfection BAC to vero cell line and vero-Cre cell line,significant differences were observed between vero cell and vero-Cre cell about sums of GFP positive cells.Conclusion:Vero-Cre cell line with stable expression of Cre recombinase was established which can be used for the construction and application of HSV-1 vector.