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Construction and identification of lentiviral vectors with cytosine deaminase and endostatin

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Author:
No author available
Journal Title:
Chinese Journal of Biomedical Engineering
Issue:
2
DOI:
10.3760/cma.j.issn.1674-1927.2012.02.010
Key Word:
胞嘧啶脱氨酶;血管内皮抑素;慢病毒;载体;聚合酶链反应;Cytosine deaminase;Endostatin;Lentiviral;Vectors;Polymerase chain reaction

Abstract: Objective To construct the lentiviral vectors with cytosine deaminase (CD) and endostatin (ES) genes.Methods The eukaryotic expression vectors pEGFP- CD and pDS- RED- ES encoding fluorescent proteins were constructed via designing and synthesizing the genes of CD and ES based on the sequence of E.coli provided by GenBank.After identification by digestion and sequencing,the vectors were immediately transfected into HEK293 cells followed by detection of CD and ES expression via real-time polymerase chain reaction.The lentiviral vectors were constructed using the gene segments EGFP-CD and DS-RED-ES via BP/LR recombination and were encapsulated by using 293FT cells.The viral stock solution was concentrated followed by detection of the viral titers.Results Construction of lentiviral vectors,pLenti6.3-CD-EGFP and pLenti6.3-ES-Monomer-DsRed,was verified by identification using enzyme digestion,gene sequencing and polymerase chain reaction.Expression of GFP and DsRed was noted via fluorescence microscope after transfection into 293FT cells.The concentrated titer of 2.2× 108 TU/ml and 6.5 × 107 TU/mlwas revealed in respective lentivirus.Conclusion The lentiviral vectors of CD and ES are successfully constructed in this study.

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