The Construction And Expression Of The Lentiviral Vectors LV-Rho-EGFP/CRX With Rat CRX Gene

Objective To investigate the transfection efficiency of the recombinant CRX gene-containing lentiviral vector(LV-Rho-EGFP/CRX) drived by the Rhodopsin promoter in the photoreceptor-deriving HXO-RB₄₄ cell line.Methods:1 The lentiviral vector(LV-Rho-EGFP/CRX)was reconstructed by replace of the EGFP gene fragment in the lentiviral vectors LV-Rho-EGFP with the EGFP/CRX gene fragment,which was isolated from pEGFP-C3/CRX vector.It was analysised with restriction digestion and bidirectional sequencing.2 The recombination lentiviral particles were produced by the packaging 293T cell by using the CaPO₄ method,the culture supernatant was harvested and the titration of them were calculated by the limiting dilution.3 The HXO-RB₄₄cells were transduced by the lentiviral particles, after 48h the GFP expression was observed under the fluorescence microscope.Results:1 The recombinant lentiviral vector LV-Rho-EGFP/CRX had been successfully constructed,the titration of lentivirus particles was 2×10⁴ IU/ml.2 The HXO-RB₄₄cells could be transduced by the recombinant lentiviral particles(2×10⁴ IU/ml).After 48h coculture,some HXO-RB₄₄ cells could express GFP.Conclusion:The recombination lentiviral vector LV-Rho-EGFP/CRX was successfully constructed.The HXO-RB₄₄cells could be transduced by lentiviral particles with the titration 2×10⁴ IU/ml in a certain degree.