Preliminary Research On The Establishment Of Cell Lines For VEGF-A Drug Screening Using Genome-targeted Modification Technology

VEGF(Vascular Endothelial Growth Factor,VEGF)family is mitogen specific to endothelial cells,and they could regulate angiogenesis and vasculogenesis.This family,which contains six subgroups of proteins,VEGF-A,-B,-C,-D,-E and placenta,plays an important role in the development of blood vessels.Among all of them,VEGF-A is the pivotal molecular of angiogenesis and vasculogenesis.Based on the alternative splicing of VEGF-A mRNA,it could be translated into five isoforms.The molecular weight of all these isoforms varies from 35-44kDa.These isoforms function differently between each other,but all are related with the generation and differentiation of vessel,lymph and other human vascular.The level of VEGF-A expression is closely related not only with the development of tumor,but also with the development of vascular diseases.The study on VEGF-A could help us clarify its molecular mechanism,and screen out drugs that affect the expression of VEGF-A.Hence,these studies mean a lot for both basic research and therapy upon disease relates with this molecule.So far,the experiments to explore the function of VEGF-A are mainly conducted in a cell line carrying a reporter gene controlled by a VEGF-A promoter,followed by establishing a cell line by transfecting.However,there are some defects with the aforementioned method,that is,the cell lines built this way could hardly reflect the regulation of gene expression by endogenous VEGF-A promoter.The evolution upon artificial nucleases technology lays important foundation for precious genome edition.Genome editing based on the double strand breaks on targeted sites introduced by artificial nucleases could induce endogenous homologous recombination repair mechanism of the cell.Through homologous recombination,we could introduce wanted genes into target genome.That is,via this homologous recombination,it is possible for us to integrate a reporter gene on the downstream of VEGF-A promoter in the genome.Cell lines built use this method could better reflect endogenous regulation in cells,so that we could reach a deeper and more accurate research of VEGF-A expression.By now,there are three types of most widely used artificial endonucleases,zinc finger nucleases(ZFN),transcription activator-like effector nuclease(TALEN)and CRISPR/Cas system.They are different in three aspects of recognition,specificity and construction.Compared with two other endonucleases,TALEN has more difficulty in construction,but it has a much higher specificity.And the specificity is a very important characteristic in area of therapeutic research.In the project,TALEN technology will be used to knock-in a sensitive reporter gene downstream in site of VEGF-A promoter.In this way we could build cell lines to screen drugs affecting VEGF-A expression,which will lay a foundation for understanding VEGF-A associated signal pathway and tumor clinic treatment.The contents of this research are as follows.(1)Designing,assembling and activity testing of TALENs targeting VEGF-A genome.In order to use endogenous VEGF-A promoter to control the reporter gene,we applied two genome editing strategies.The first one,we inserted reporter gene right after the downstream of VEGF-A promoter.In this situation,the inserted reporter gene will destroy the endogenous expression of VEGF-A on this chromosome.The other strategy is to insert the reporter gene at the end of VEGF-A gene,which could produce both VEGF-A and reporter by T2A self-cleaving peptides.By the information of VEGF-A promoter and structure,TALENs are designed for targeting two areas for the strategies above using ZiFiT software.After designing,TALEs are assembled according to the sequences of targeting sites.Then TALEs were cloned into adenovirus E1 shuttle vector generated by our laboratory that carrying N terminal,C terminal of TALEs and FokI endonuclease of TALENs,which could generate wanted TALENs.Genome DNA was extracted from HEK293 cell line transfected with the TALENs 72hours post-transfection,and then used for testing TALENs' activity using T7E1 endonuclease.(2)Constructing targeting vectors containing reporter gene.In order to insert a reporter gene in the genome controlled by endogenous VEGF-A promoter,we designed two different targeting vectors for this purpose.The first one is to insert luciferase reporter gene right behind endogenous VEGF-A promoter,second one is to insert luciferase gene at the end of VEGF-A gene using T2A self-cleaving peptide between VEGF-A and reporter gene.For the first strategy,targeting vector consists of up homologous arm ends right after VEGF-A promoter,then follows IRES element associating with luciferase reporter gene,a CMV-eGFP-T2A-Neo-pA positive selection cassette,down homologous arm and TK negative selection cassette for preventing non-specific intergration of this vector.In the vector targeting the end of VEGFA gene,targeting vector consists of up homologous arm ends right before the terminal codon TGA of VEGF-A,T2A self-cleaving small peptide fused with luciferase reporter gene right after the downstream of the up homologous arm to separate VEGF-A and luciferase reporter,neomycin positive selection cassette and down homolodous arm.Finally,the targeting vectors above were used for building cell lines.(3)Constructing VEGF-A promoter luciferase knock-in HEK293 and VEGF-A luciferase knock-in HEK293 cell lines carrying reporter gene regulated by endogenous VEGF-A promoter.Co-transfection of targeting vectors that carrying luciferase reporter gene and screening gene into HEK293 cell lines with corresponding expression vectors carrying TALENs,respectively.After transfection,GCV and G418 were used for screening cell lines.Once cell lines stable expressing resistance gene were obtained,genome DNA were extracted for identifying the cell lines use PCR and sequencing methods.(4)In vitro study on HEK293 cell line carrying reporter gene regulated by endogenous VEGF-A promoter.After PCR and sequencing identification,we got two cell lines with the reporter gene inserted after endogenous VEGF-A promoter.The cell lines were named promoter VEGF-A luciferase knock-in HEK293 and VEGF-A luciferase knock-in HEK293 respectively.The lysate of them were gatehered and the relative luciferase activities were measured.After the measurement,we confirmed that the inserted luciferase gene were successfully endogenous expressed.Then the promoter VEGF-A luciferase knock-in HEK293 cell line with relative lower luciferase activity were monclonaled use limited dilute method.Based on the statement above,the results were obtained as follows.(1)Successfully obtained two pairs of TALENs targeting area located in downstream of human VEGF-A promoter and end of human VEGF-A gene separately.Constructed expression vectors carrying TALENs,and confirmed all the TALENs have a favorable activity on a genome wide scale.(2)Successfully constructed two kinds of targeting vectors for the construction of cell lines with reporter genes regulated by endogenous VEGF-A promoter.The targeting vector targeted just downstream of endogenous VEGF-A promoter,which has IRES-luciferase-SV40pA reporter cassette and CMV-eGFP-T2A-Neomycin-SV40pA positive screening cassette.Outside of the homologous arms,it also has a PGK-TK negative screening cassette.The targeting vector targeted end of VEGF-A gene,which has T2A-luciferase T2A-Neomycin-SV40pA cassette,and this vector does not have a negative screening cassette.(3)Successfully obtained cell lines by co-transfecting expression vectors carrying TALENs targeting human VEGF-A gene with targeting vectors carrying reporter gene and screening genes into HEK293 cell line,followed by the screening of the transfected cell lines using GCV and G418.(4)Successfully detected the endogenous expression of inserted luciferase.After limited dilute method,the relative luciferase activity of promoter VEGF-A luciferase knock-in HEK293 was significantly improved.