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African Journal of Biotechnology

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Construction of lentiviral shRNA expression vector targeting phospholipase D2 (PLD2) gene△

XF Lian, CX Yu, XL He, JJ Lin, YZ Chen, L Zhu

Abstract


The aim of this research was to construct a lentiviral shRNA vectors targeting phospholipase D2 (PLD2) gene, thus providing information for further study of the biological functions of PLD2 and clinical treatments of leukemia. Specific siRNA targets with short hairpin frame were designed using the siDESIGN software and synthesized according to cDNA sequence of PLD2 (GenBank accession number: NM_002663). DNA oligo was cloned into lentiviral expression vector, and then polymerase chain reaction (PCR) and sequencing analyses were conducted to verify the constructs. The verified vectors were co-transfected into 293FT cells that could produce lentiviral. shRNA lentiviruses from the selected constructs were propagated and harvested with a virus packaging system, and the virus titers were determined by flow cytometry. After the lentiviral packing with PLD2-shRNA2 transfecting 293FT cells, using fluorescence microscope, we found the lentiviral interference vectors, which have GFP report genes expression accompanied with the host genes in 293FT cells. The optimal interfering target was then selected, while the titer of lentiviral packing PLD2-shRNA was 3.47 × 104 TU/ml and the virus was successfully packaged. PCR and sequencing analyses revealed that lentiviral shRNA vectors of three targeting PLD2 gene were successfully constructed.

Key words: RNA interference (RNAi), phospholipase D2 (PLD2), lentiviral vectors.




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