Abstract：Gene targeting is a new molecular biology technique, which introduces heritable genetic modification on the basis of DNA homologous recombination. Compared with classic approaches, homologous recombination using a promoter trap vector could yield higher targeting frequencies. It is very important for the correct expression of exogenous gene. In this study, neor gene was amplified by PCR technology from plasmid pIRES2-EGFP and BamHⅠ and BglⅡ restriction enzyme sites were added to the upstream and downstream, respectively; then the PCR fragment was cloned into pMD19-T simple Vector, named pMD-neo. Plasmids pMD-neo and pEGFP-C1 were digested by BamHⅠ and BglⅡ, and ligated together to obtain intermediate plasmid pEGFP-neo. Then pEGFP-neo and pIRES2-EGFP were digested by Bsp1407Ⅰ and SfiⅠto constitute another intermediate plasmid pIRES-EGFPneo. At last, multiple cloning sites (MCS) were added to pIRES-EGFPneo after digestion of pIRES-EGFPneo and pMD-MCS with BamHⅠand MluⅠ, and the final gene trap vector pMCS-IEN was constituted. To test the expression of green fluorescence protein (GFP) and G418 resistance gene, the PGK promoter was added to the upstream of MCS of the pMCS-IEN and the reconstitute plasmid was transfected to Hela cells. GFP and G418 resistance colonies were obtained after screening by microscopy and G418 selection. When the homologous arms were inserted into the basic vector pMCS-IEN and formed the promoter trap vector，it could be used to target the objective gene. Therefore, this vector will become a useful tool to generate gene modification animals.
CAPECCHI MR. 1989. Altering the genome by homologous recombination. Science 244: 1288-1292.DOETSCHMAN T, GREGG RG, MAEDA N, HOOPER ML, MELTON DW, THOMPSON S AND SMITHIES O. 1987. Targetted correction of a mutant HPRT gene in mouse embryonic stem cells. Nature 330: 576-578.ERRO E ET AL. 2013. Bioengineering the liver: scale-up and cool chain delivery of the liver cell biomass for clinical targeting in a bioartificial liver support system. BioResearch open access 2: 1-11.FRIEDEL RH ET AL. 2005. Gene targeting using a promoterless gene trap vector ("targeted trapping") is an efficient method to mutate a large fraction of genes. Proceedings of the National Academy of Sciences of the United States of America 102: 13188-13193.HENTZE MW, CAUGHMAN SW, CASEY JL, KOELLER DM, ROUAULT TA, HARFORD JB AND KLAUSNER RD. 1988. A model for the structure and functions of iron-responsive elements. Gene 72: 201-208.KHAN KH. 2013. Gene Expression in Mammalian Cells and its Applications. Advanced pharmaceutical bulletin 3: 257-263.KOMAR AA, MAZUMDER B AND MERRICK WC. 2012. A new framework for understanding IRES-mediated translation. Gene 502: 75-86.LEAVITT AD AND HAMLETT I. 2011. Homologous recombination in human embryonic stem cells: a tool for advancing cell therapy and understanding and treating human disease. Clinical and translational science 4: 298-305.LI J, HAN C, DENG J AND WANG H. 2010. [Construction and functional analysis of a common gene targeting vector with double-selection markers]. Sheng wu gong cheng xue bao = Chinese journal of biotechnology 26: 1696-1703.MIAO X. 2013. Recent advances in the development of new transgenic animal technology. Cellular and molecular life sciences : CMLS 70: 815-828.MORTENSEN R. 2007. Overview of gene targeting by homologous recombination. Current protocols in neuroscience / editorial board, Jacqueline N Crawley [et al] Chapter 4: Unit 4 29.PEREZ-CAMPO FM, SPENCER HL, ELDER RH, STERN PL AND WARD CM. 2007. Novel vectors for homologous recombination strategies in mouse embryonic stem cells: an ES cell line expressing EGFP under control of the 5T4 promoter. Experimental cell research 313: 3604-3615.PORTEUS MH AND BALTIMORE D. 2003. Chimeric nucleases stimulate gene targeting in human cells. Science 300: 763.PRINJHA R AND TARAKHOVSKY A. 2013. Chromatin targeting drugs in cancer and immunity. Genes & development 27: 1731-1738.SCHWARTZBERG PL, GOFF SP AND ROBERTSON EJ. 1989. Germ-line transmission of a c-abl mutation produced by targeted gene disruption in ES cells. Science 246: 799-803.SHEN W, YANG ZT AND DENG JX. 2003. [Production of mammary gland bioreactor by gene targeting of somatic cells]. Sheng wu gong cheng xue bao = Chinese journal of biotechnology 19: 767-770.STEWART H, WALTER C, JONES D, LYONS S, SIMMONDS P AND HARRIS M. 2013. The non-primate hepacivirus 5' untranslated region possesses internal ribosomal entry site activity. The Journal of general virology 94 Pt 12: 2657-2663.TICHY ED, PILLAI R, DENG L, LIANG L, TISCHFIELD J, SCHWEMBERGER SJ, BABCOCK GF AND STAMBROOK PJ. 2010. Mouse embryonic stem cells, but not somatic cells, predominantly use homologous recombination to repair double-strand DNA breaks. Stem cells and development 19: 1699-1711.TONG C, HUANG G, ASHTON C, LI P AND YING QL. 2011. Generating gene knockout rats by homologous recombination in embryonic stem cells. Nature protocols 6: 827-844.TSAKIRIDIS A, TZOUANACOU E, RAHMAN A, COLBY D, AXTON R, CHAMBERS I, WILSON V, FORRESTER L AND BRICKMAN JM. 2009. Expression-independent gene trap vectors for random and targeted mutagenesis in embryonic stem cells. Nucleic acids research 37: e129.UHM SJ, KIM NH, KIM T, CHUNG HM, CHUNG KH, LEE HT AND CHUNG KS. 2000. Expression of enhanced green fluorescent protein (EGFP) and neomycin resistant (Neo(R)) genes in porcine embryos following nuclear transfer with porcine fetal fibroblasts transfected by retrovirus vector. Molecular reproduction and development 57: 331-337.张映辉. 2001. 在体细胞中实现基因打靶. 生物技术通讯: 63-66.
