Abstract
BackgroundThe primary goal of any recombinant protein produc-tion is to achieve successful gene transfer and expres-sion in a target cell. There are two general categories ofdelivery vehicles/vectors employed in protein expressionprotocols. The first category includes the non-viral vec-tors, ranging from direct injection of DNA to complex-ing DNA with cationc lipds, polylysine, etc. The secondcategory comprises DNA and RNA viral vectors.Viruses have evolved specific mechanism to delivertheir genetic material to target cell nuclei. Virus mem-bers of family Retroviridae, e.g. retroviruses and lenti-viruses, are among the most widely used viral vectors.The use of lentiviral vectors has been increasing becausethe vector system has attractive features. Lentiviruseshave an advantage over retroviruses in that they caninfect both dividing and non-dividing cells and thereforehave attracted much attention regarding the potential asvectors for gene delivery/therapy. Once integrated intothe genome, recombinant cell lines are selected usingdifferent selection mechanisms.ResultsLentivirus particles were produced by simultaneous co-transfection of HEK 293T cells with four plasmids. Thepackaging construct (pMDLg/pRRE) [1], the VSV-G-expressing construct (pMD.G) [2], the Rev-expressingconstruct (pRSV-Rev) [1], and the self-inactivating (SIN)lentiviral vector construct containing the green fluores-cent protein (GFP) reporter gene (pLV-PLK-eGFP). Themedium containing lentiviral particles was collected 48h after transfection, clarified by centrifugation 10 min at2000 rpm and then stored at -80°C. To determine viraltiters, HEK 293T cells were seeded at 3 x 10
Highlights
The primary goal of any recombinant protein production is to achieve successful gene transfer and expression in a target cell
HEK 293T cells were seeded at a density of 6 x 104 cell/ml in 6-well plates and after 24 h the supernatant was replaced by 1 ml medium containing lentiviral vectors. 96 h post-transduction the cells were analyzed by flow cytometry (t=0); the cells were incubated with the puromycin selection agent to obtain stable recombinant cell lines
Once the cells were resistant to different concentrations of puromycin, recombinant cell lines were
Summary
The primary goal of any recombinant protein production is to achieve successful gene transfer and expression in a target cell. There are two general categories of delivery vehicles/vectors employed in protein expression protocols. The first category includes the non-viral vectors, ranging from direct injection of DNA to complexing DNA with cationc lipds, polylysine, etc. The second category comprises DNA and RNA viral vectors. Viruses have evolved specific mechanism to deliver their genetic material to target cell nuclei. Virus members of family Retroviridae, e.g. retroviruses and lentiviruses, are among the most widely used viral vectors. Lentiviruses have an advantage over retroviruses in that they can infect both dividing and non-dividing cells and have attracted much attention regarding the potential as vectors for gene delivery/therapy. Once integrated into the genome, recombinant cell lines are selected using different selection mechanisms
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