A modified lentiviral vector construction system

Objective: To develop a lentiviral vector system, which can be used to co-express multiple genes of interest, such as siRNAs cassettes, reporter gene and resistant gene simultaneously and to facilitate to titrate lentiviral vector stock with TCID₅₀ method. Method: Synthesis new multiple cloning sites(MCS)to replace the original MCS of pLKO-1-puro and produced an intermediate plasmid pLKO-1-puro-MCS; Destination plasmid pLKO-M was constructed through transfering the CMV promoter-GFP-IRES element into pLKO-1-puro-MCS; The support plasmid pENTR-U6-20 was produced through transfering the siRNA-Expression cassette of pSilencer 2.0 into pENTR-U6-con. pLKO-M was cotransfected with pCMV-dR8.2 dvpr and pCMV-VSVG into 293T cells to produce lentiviral vectors. Harvest the virus 72 hours after contransfection and titrate it on Vero cells with TCID₅₀ method.Result: Restriction enzyme digestion analysis of pLKO-M showed that it was constructed successfully; restriction enzyme digestion analysis and sequencing result of pENTR-U6-20 indicated that it is constructed successfully. The GFP gene in pLKO-M can express normally and the titre caculated with TCID₅₀ method is 6.47X10⁶ IU/ml. Conclusion: The multiple gene coexpressing system which contains two plasmid, pLKO-M and pENTR-U6-20, was established and the titre of pLKO-M virus can be measured with TCID₅₀ method.