407. Comparison of Transgene Expression Driven from 4 Different Retroviral Long Terminal Repeats

Abstract

One of the major limitations of retroviral vectors is the low level of transgene expression in the target cells. To overcome this problem, several strategies have been employed, including the replacement of 3' long terminal repeat (LTR) with other viruses' LTR, the use of different leader sequences, and insertion of RNA export elements. In this study, we tried to evaluate the effect of LTR on transgene expression. For this purpose, we constructed a series of retroviral vectors, MT, MS, MF, and MP that had the same structure except 3' LTR: murine leukemia virus (MLV) LTR for MT, myeloproliferative sarcoma virus (MPSV) LTR for MS, spleen focus-forming virus (SFFV) LTR for MF, and murine stem cell virus (MSCV) LTR for MP. The level of gene expression from these vectors was evaluated using luciferase, stem cell factor (SCF), and enhanced green fluorescence protein (eGFP) as reporters in various cells. We found that luciferase activity driven by MSCV LTR was significantly lower than that driven by other LTRs in K562 and HT1080 cells. When SCF was used as a transgene, the SCF production from the vector containing MPSV LTR was significantly lower than that from other vectors. On the contrary, in experiments using eGFP, MPSV LTR drove the highest level of fluorescence intensity in K562 cells in spite of a low percentage of eGFP expressing cells. Our results strongly indicated that the transgene expression in retroviral vectors is influenced not only by the LTR but also by the nature of the transgenes.