Expression of PiT1 and PiT2 retroviral receptors and transduction efficiency of tumor cells.
Recombinant retroviral vectors are still the most common gene delivery vehicles for gene therapy purposes, especially for construction of genetically modified tumor vaccines (GMTV). However, these vehicles are characterized by relatively low titre and in the case of many tumor cell lines, low transduction efficiency. We constructed bicistronic retroviral vector pseudotypes of amphotropic murine leukemia virus (A-MuLV) and gibbon ape leukemia virus (GaLV), encoding enhanced green fluorescent protein (EGFP) as a rapid and easily detectable reporter gene. Transduction of five different human melanoma and four renal carcinoma cell lines by these two virus pseudotypes revealed differences in transduction efficiency, which wase markedly lower for the renal carcinoma cell lines. Stimulation of retroviral receptor expression (PiT1 and PiT2) by phosphate depletion induced a limited increase of receptor mRNA levels, but did not improve the gene transfer efficiency. In contrast, simultaneous transduction with both vector pseudotypes markedly increased the transduction efficiency, compared to GaLV or A-MuLV alone. The same effect could be achieved by several repeated exposures of target cells to fresh vector preparation. Overexpression of GaLV receptor (PiT1) in target cells significantly increased the transduction rate and enabled retrovirus mediated gene transfer into the cells which normally are not transducible by GaLV pseudotypes. We demonstrated that, using different transduction strategies, the relatively inefficient, widely used retroviral vector systems could be significantly improved.
D002292:Carcinoma, Renal Cell / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics / Q000502:physiology; D006801:Humans; D017989:Leukemia Virus, Gibbon Ape / Q000235:genetics / Q000502:physiology; D009052:Leukemia Virus, Murine / Q000235:genetics / Q000502:physiology; D008545:Melanoma / Q000235:genetics; D010710:Phosphates / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011991:Receptors, Virus / Q000235:genetics* / Q000378:metabolism*; D012190:Retroviridae / Q000235:genetics* / Q000502:physiology*; D014161:Transduction, Genetic / Q000379:methods*; D014407:Tumor Cells, Cultured
D010710:Phosphates; D011991:Receptors, Virus