Quantitative assessment on the cloning efficiencies of lentiviral transfer vectors with a unique clone site.
Lentiviral vectors (LVs) are powerful tools for transgene expression in vivo and in vitro. However, the construction of LVs is of low efficiency, due to the large sizes and lack of proper clone sites. Therefore, it is critical to develop efficient strategies for cloning LVs. Here, we reported a combinatorial strategy to efficiently construct LVs using EGFP, hPlk2 wild type (WT) and mutant genes as inserts. Firstly, site-directed mutagenesis (SDM) was performed to create BamH I site for the inserts; secondly, pWPI LV was dephosphorylated after BamH I digestion; finally, the amounts and ratios of the insert and vector DNA were optimized to increase monomeric ligation. Our results showed that the total percentage of positive clones was approximately 48%±7.6%. Using this method, almost all the vectors could be constructed through two or three minipreps. Therefore, our study provided an efficient method for constructing large-size vectors.
Department of Medicine, Centre for Research in Neurodegenerative Diseases, University of Toronto, 6 Queen's Park Crescent West, Toronto, ON, M5S 3H2, Canada. gang.zhang@utoronto.ca
Gang
G
Zhang
Anurag
A
Tandon
D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D015245:Deoxyribonuclease BamHI / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D014162:Transfection / Q000379:methods*; D019076:Transgenes / Q000235:genetics*
D049452:Green Fluorescent Proteins; C496126:PLK2 protein, human; D017346:Protein Serine-Threonine Kinases; D015245:Deoxyribonuclease BamHI; D015252:Deoxyribonucleases, Type II Site-Specific; C038565:GCGGCCGC-specific type II deoxyribonucleases