Type and position of promoter elements in retroviral vectors have substantial effects on the expression level of an enhanced green fluorescent protein reporter gene.
OBJECTIVE
Although gene transfer with retroviral vectors has already been applied to patients as part of clinical protocols, low expression of transgenes in target cells still remains a problem. Therefore, we compared various retroviral vectors using different promoters and backbones for expression of the enhanced green fluorescent protein (EGFP) reporter gene in fibroblasts and CD34+ cells.


METHODS
The N2A retroviral vector was used to test expression from the herpes simplex virus thymidine kinase promoter (vector N2A-TK-EGFP), a human phosphoglycerate kinase promoter (vector N2A-PGK-EGFP), and the SV40 promoter (vector N2A-SV-EGFP). Additional constructs used the spleen focus-forming virus (SFFV) long terminal repeat (LTR) as promoter and expressed EGFP alone (vector SFbeta1-EGFP) or EGFP and a downstream (vector SFbeta1-EGFP-IRES) or upstream (vector SFbeta1-IRES-EGFP) internal ribosomal entry site.


RESULTS
For NIH 3T3 cells the fluorescence-activated cell sorting analysis revealed that the most active internal promoter was the SV40 promoter in the vector N2A-SV-EGFP (mean fluorescence intensity, MFI, 66.7 +/- 0.4), followed by N2A-PGK-EGFP (26.3 +/- 1.8 MFI), and N2A-TK-EGFP (4.8 +/- 0.1 MFI). Expression from the SFbeta1-EGFP vector (82.6 +/- 6.7 MFI) and the SFbeta1-EGFP-IRES vector (102.8 +/- 6.2 MFI) was higher than from SFbeta1-IRES-EGFP vector (15.5 +/- 1.8 MFI). In human CD34-positive cells, the EGFP expression from all vectors was considerably lower than in fibroblasts with the SFbeta1-EGFP vector still being four- to fivefold more active than the internal promoters tested.


CONCLUSIONS
The SFFV LTR seems to allow a high expression of transgenes, as long as the transgene is not expressed downstream of an internal ribosomal entry site. Internal promoters may be useful for targeted gene expression in specific cell types, but the reduced level of expression from some internal promoters has to be taken into consideration.
Department of Internal Medicine (Cancer Research), West German Cancer Center, University of Essen Medical School, Germany. michael.flasshove@uni-essen.de
M
M
Flasshove
W
W
Bardenheuer
A
A
Schneider
G
G
Hirsch
P
P
Bach
C
C
Bury
T
T
Moritz
S
S
Seeber
B
B
Opalka
D016475:3T3 Cells; D000818:Animals; D018952:Antigens, CD34 / Q000378:metabolism; D015536:Down-Regulation; D005347:Fibroblasts / Q000276:immunology / Q000378:metabolism; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D017930:Genes, Reporter; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins; D006412:Hematopoietic Stem Cells; D006801:Humans; D007202:Indicators and Reagents; D008164:Luminescent Proteins / Q000235:genetics*; D051379:Mice; D009369:Neoplasms / Q000235:genetics / Q000628:therapy; D010735:Phosphoglycerate Kinase / Q000235:genetics; D011401:Promoter Regions, Genetic; D012190:Retroviridae / Q000235:genetics*; D013539:Simian virus 40 / Q000235:genetics; D018139:Simplexvirus / Q000201:enzymology; D013937:Thymidine Kinase / Q000235:genetics; D014161:Transduction, Genetic
D018952:Antigens, CD34; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D013937:Thymidine Kinase; D010735:Phosphoglycerate Kinase