Construction Of Multiple Gene Coexpression Lentiviral Vector System Using Gfp As The Reporter Gene

Gene therapy brings us hopes to study and develop specific treatments for neurological disorders.One of the important issues of gene therapy is to choose an excellent transgene vector.Most recently,lentiviral vectors obtained researchers' attention because of its advantages.Lentiviral vector(LV) derived from human immunodeficiency virus type 1,and most of the toxic gene was deleted.It can infect nondividing cells,including neurons.Its infection efficiency is high and triggers no obvious immune reactions.It has the ability to transfer the gene of interest into host cells and integrate it with the genome of host cell.Because most kinds of neurological disorders are caused by abnormality of more than one single gene,so it is necessary to construct vectors that can upregulate(driven by strong promotors) or downregulate more than one genes simultaneously.So one of the focuses of lentiviral vector studies is to construct multiple gene coexpression vectors.Because lentiviral vectors have no obvious infection marker,we lack rapid and stable titration method for lentiviral vectors.This study established a multiple gene coexpressing lentiviral vector construction system which can be used to construct lentiviral vectors which can coexpress more than one siRNA,GFP and gene of interest.Green fluorescence can be used as an infection marker to titrate vectors with TCID₅₀ method through observing green fluorescence,a convenient titration method of lentivirus was established.This study provided an meaningful tool for neurological disorder studying and gene therapy.Objective:1.To establish a lentiviral vector construction system which can be used to construct lentiviral vectors that can coexpressing multiple siRNA,GFP and gene of interest.2.Cotransfect the helper plasmid and envelope plasmid with pLKO-M into 293T cells to produce GFP expressing lentiviral vectors and Titrate the lentiviral vectors with TCID₅₀ method.Methods:1.Modification of original lentiviral vector pLKO-1-puro:design oligonucleotides which contains the following restriction enzyme recognition sites: 5'AgeI,EcoRI,XbaI,SalI and MluI '3Annealing the two strands and clone it into AgeI and EcoRI digested plasmid pLKO-1-puro,produce pLKO-1-puro-MCS.Transform E.coli strain DH5α,screen positive clonies with EcoRI,XbaI,SalI,MluI and BamHI digestion.2.Clone CMV promotor-GFP-IRES-MCS element into pLKO-1-puro-MCS:Amplify the element with PrimeStar HS polymerase,the element is about 2.4kb, after digestion with XbaI and SalI,ligate it with XbaI and SalI digested pLKO-1-puro-MCS.Transform DH5α,screen positive colonies with XbaI and SalI digestion.3.Construction o support plasmid pSi-shutle:design primers to amplify siRNA expression cassette which is about 421bp,clone it into pENTR-U6-con and produce pSi-shutle.Transform E.coli and screen positive colonies with restriction enzyme digestion and sequencing.4.Cotransfect pLKO-M,helper plasmid pCMV-dR8.2 dvpr and envelope plasmid pCMV-VSVG into 293T cells with lipofectin 2000,produce infectious lentiviral vectors and concentrate it through centrifuging at 4℃,25000rpm for 90 minutes.5.Titration with TCID₅₀ method:infect Vero cells in 96-wells culture plates with serially diluted vector stock.Observe green fluorescence under inverted fluorescent microscope,count positive wells and calculate the titre.Result:1.Oligonucleotides contains new MCS was successfully cloned into original lentiviral vector pLKO-1-puro and produced pLKO-1-puro-MCS,which is confirmed with restriction digestion analysis. 2.CMV promotor-GFP-IRES-MCS element was successfully cloned into pLKO-1-puro-MCS and confirmed by restriction digestion analysis.3.The siRNA expression cassette of pSilencer2.0 was successfully amplified and cloned into plasmid pENTR-U6-CON,and confirmed by restriction digestion analysis and sequencing.4.Green fluorescence appeared 48 hours after contransfection of 3 plasmid into 293T cells,virus was harvested 72h after transfection and concentrated.5.Green fluorescence appeared 48 hours after infection Vero cells in two 96-well culture plates with serially diluted viral stock.The mean titre of concentrated viral stock was 6.47X10⁶ IU/ml.Conclusion:1.A lentiviral vector pLKO-M which can coexpress siRNA,GFP and gene of interest was successfully constructed.2.The support plasmid of pLKO-M,pSi-shutle was was successfully constructed, with the help of pSi-shutle,we can construct lentiviral vectors that can coexpress more than 1 siRNA,GFP and gene of interest.3.Green fluorescence could be used as a marker of infection to titrate lentiviral vector with TCID₅₀ method.