Functional analysis of various promoters in lentiviral vectors at different stages of in vitro differentiation of mouse embryonic stem cells.
Given the therapeutic potential offered by embryonic stem (ES) cells, it is critical to optimize stable gene delivery and expression at different developmental stages of ES cell differentiation. Here, we systematically analyzed lentiviral vectors containing the following promoters: the human elongation factor 1alpha (EF1alpha) promoter, the human cytomegalovirus (CMV) immediate early region enhancer-promoter, the composite CAG promoter (consisting of the CMV immediate early enhancer and the chicken beta-actin promoter), the human phosphoglycerate kinase 1 (PGK) promoter, the murine stem cell virus (MSCV) long terminal repeat (LTR), or the gibbon ape leukemia virus (GALV) LTR. Our results show that the EF1alpha promoter directed robust transgene expression at every stage of mouse ES cell differentiation, whereas the CMV promoter drove transgene expression only during late stages. Similarly, the CAG and PGK promoters drove transgene expression at a significant level only during late stages. The MSCV LTR and the GALV LTR exhibited much lower promoter activities at all stages. Interestingly, mouse ES cells transduced with the EF1alpha promoter-containing lentiviral vector lost most of their transgene expression during in vitro differentiation to neural precursors and neuronal cells. Our results demonstrate that different cellular and viral promoters exhibit very distinct and dynamic properties not only in terms of promoter strength but also with respect to differentiation stage-specific activity.
Hong
Sunghoi
S
Molecular Neurobiology Laboratory, McLean Hospital, Harvard Medical School, Belmont, Massachusetts, USA.
Hwang
Dong-Youn
DY
Yoon
Soonsang
S
Isacson
Ole
O
Ramezani
Ali
A
Hawley
Robert G
RG
Kim
Kwang-Soo
KS
D000818:Animals; D002454:Cell Differentiation / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D002470:Cell Survival / Q000235:genetics / Q000502:physiology; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D005434:Flow Cytometry; D005822:Genetic Vectors / Q000235:genetics; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D008856:Microscopy, Fluorescence; D020648:Peptide Elongation Factor 1 / Q000235:genetics; D010735:Phosphoglycerate Kinase / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors
D020648:Peptide Elongation Factor 1; D010735:Phosphoglycerate Kinase