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Construction of Multiple Gene Coexpression Lentiviral Vector System Using GFP as the Reporter Gene

Author: LiuJingWei
Tutor: XuYuMing；HanZhiQiang
School: Zhengzhou University
Course: Neurology
Keywords: Gene therapy Lentiviral vectors Titer determination siRNA
CLC: R346
Type: Master's thesis
Year: 2009
Downloads: 59
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Abstract

The nerve cells of transgenic technology is not only an important means to study the molecular mechanism of the disease of the nervous system, and brought new hope for gene therapy of diseases of the nervous system. The key is to choose one of the good gene transfer vector. Lentiviral vectors in recent years with its unique advantages and has been growing concern. Lentivirus vectors (LV, lentiviral vector) derived from the human immunodeficiency virus type 1 (HIV-1), the causative gene has been removed, it can infect non-dividing cells, including neurons, including, high infection efficiency, immunogen low transgene integration into the host cell genome, and can carry and long-term stable expression of the target gene carrying. Gene therapy because most of the nervous system diseases are caused by multiple genes together need to select (driven by a strong promoter) gene or cut (siRNA gene silencing) a plurality of target gene gene transfer vector can be simultaneously raised. Lentiviral vector research direction is to build a multi-gene co-expression vector. In addition, because of the slow viruses infect host cells without apparent infection markers, titer determination is extremely inconvenient, a shortfall of fast and stable titer determination method. In response to these issues, this study using molecular cloning methods to build a set of multi-gene co-expression of lentiviral vector construction system, this system can be used to build a multi-siRNA reporter gene GFP and the target gene co-expression of lentiviral vectors. GFP as a marker of infection, the use of standard the TCID ₅₀ calculate the virus titer, established a convenient the lentiviral titer determination method was established for the research and treatment of diseases of the nervous system utility. Objective: 1. Construct multiple siRNA expression cassette, GFP and the target gene co-expression lentiviral vector construction system pLKO-M and pSi-shutle. . Lentiviral envelope plasmid with helper plasmids were co-transfected into 293T cells to produce lentiviral particles expressing GFP and lentiviral titer TCID ₅₀ Rapid determination method. Method: 1. Existing lentiviral vector pLKO-1-puro polyclonal sites alterations: sequence designed and synthesized a new multicloning site (AgeI, EcoRI, XbaI, SalI and MluI) after annealing, is connected to the via AgeI and EcoRI double digestion pLKO-1-puro vector plasmid, the formation of the recombinant plasmid pLKO-1-Puro-MCS; transformed into E. coli (escherichia coli, E.coli) DH5α competent cells, and screening for positive colonies after amplification extract plasmid, MluI and BamHI double digestion. Start Sub-GFP-IRES-MCS element 2.CMV is cloned into pLKO-1-Puro-MCS: pGFP-IRES as template primers designed approximately 2.4kb fragment was amplified with a high fidelity DNA polymerase, XbaI and SalI digested cloned into pLKO-1-puro-MCS of the XbaI and SalI sites at, XbaI and lSalI of restriction endonuclease. 3. Supporting plasmid pSi-shutle build: the design INPTEN1 and INPTEN2 primers, PCR amplification pSilencer2.0, 421bp fragment cloned to pENTR-U6-con, too the pSi-shutle, SalI and XbaI restriction endonuclease positive colonies . Liposomes 2000 recombinant vector the plasmid PLKO-M helper plasmid the the pCMV-dR8.2 dvpr and envelope plasmid pCMV-VSV-G were co-transfected into 293T cells, resulting in the ability to infect lentiviral particles in 25000rpm at 4 ℃ centrifugation 90min concentrated in virus particles. 5.TCID ₅₀ Determination lentivirus titer: the virus particles were serially diluted 2 after infection of Vero cells in 96-well plates and after 48 hours in the inverted number of holes counted under a fluorescence microscope to produce green fluorescent and calculate the titer. Results: (1) with new multicloning site (MCS) of the oligonucleotide chain was successfully cloned into lentivirus vector plasmid pLKO-1-Puro with pLKO-1-Puro-MCS, after restriction endonuclease digestion proved Construction success. 2.CMV start the sub-GFP-IRES-MCS components were successfully cloned into pLKO-1-puro-MCS the restriction endonuclease correct. 3.pSilencer2.0 siRNA expression cassette plasmid was successfully cloned into pENTR-u6-con, digestion and sequencing analysis. 4 cotransfected 293T 48 hours after the visible expression of GFP lentiviral particles were harvested after 72 hours and concentrated. 5 after the serial dilution of the concentrated virus infection of Vero cells in 96-well plates 2, green fluorescence appear after 48 hours, with TCID ₅₀ France draw concentrated viral infection units (IU) mean 6.47X10 ^{6 TU / ml. Conclusion: 1. Successfully constructed the siRNA expression box and GFP-target gene co-expression lentiviral vector pLKO of-M. Successfully constructed supporting plasmid psi-Shuttle to pLKO-M, in its secondary can build multi-siRNA, a lentivirus carrier 3 co-expression of GFP and destination established as infection markers in green fluorescence with TCID ₅₀ Determination of lentiviral titer.}

Construction of Multiple Gene Coexpression Lentiviral Vector System Using GFP as the Reporter Gene

Abstract

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