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Quantitative assessment on the cloning efficiencies of lentiviral transfer vectors with a unique clone site

Scientific Reports · 2012 · 1K citations · 29 references

Open access

TL;DR

Lentiviral vectors are powerful for transgene expression but are difficult to construct efficiently because of their large size and lack of suitable clone sites. The study aims to develop a more efficient cloning strategy for lentiviral vectors. The authors introduced a BamHI site via site‑directed mutagenesis, dephosphorylated the pWPI vector after BamHI digestion, and optimized insert‑to‑vector DNA ratios to promote monomeric ligation. The optimized method yielded about 48 % positive clones (±7.6 %) and enabled construction of nearly all vectors within two or three minipreps, demonstrating an efficient approach for large‑size lentiviral vectors.

Abstract

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.