Influence of multiplicity of infection and protein stability on retroviral vector-mediated gene expression in hematopoietic cells

Abstract

Using retroviral vectors encoding enhanced green fluorescent protein (EGFP), we addressed to what extent expression of retroviral transgenes in hematopoietic cells depends on the multiplicity of infection (MOI) and on the halflife of the encoded protein. We show that an elevation of the MOI not only elevates the frequency of transduced cells, but also increases transgene expression levels and reduces interanimal variability in vivo (hematopoietic cells of C57BL/6J mice analyzed 13 weeks after transplantation). This suggests that the MOI has to be carefully controlled and should be adapted as desired for clinical studies when evaluating vector performance in preclinical models. The impact of protein stability is demonstrated by comparing vectors expressing EGFP or a destabilized variant with a C-terminal PEST-sequence, d2EGFP. The loss of expression

Oncogenic Vav1 induces Rac-dependent apoptosis via inhibition of Bcl-2 family proteins and collaborates with p53 deficiency to promote hematopoietic progenitor cell proliferation

“…Three hemagglutinin (HA) tandem repeats were fused to the 5 0 end of cDNAs for transgene detection. Retrovirus supernatant was generated in phoenix-gp cells as previously described (Wahlers et al, 2001).…”

Optimization of equine infectious anemia derived vectors for hematopoietic cell lineage gene transfer

“…Due to the extended protein half-life of EGFP, we used a destabilized version of EGFP (dsGFP; 2 h half-life) in order to more accurately compare the relative activity of the various promoters. 23 We compared a 2.3 kb fragment of the elongation factor 1a promoter (EF1a), an 800 bp fragment of the phosphoglycerol kinase gene (PGK), the long terminal repeat of the myeloid proliferative sarcoma virus (MPSV), and the modified murine leukemia virus LTR (MND), with the immediate early CMV promoter. Hematopoietic cells were transduced with the various E-SIN promoter vectors and dsGFP and were analyzed 14 days later by flow cytometry.…”

Self-Inactivating Alpharetroviral Vectors with a Split-Packaging Design

“…Next, we determined to what extent the transduction level of the target cells can be determined by the dose of vector used. As indicated based on studies performed with gammaretroviral vectors, an increase in the viral particleto-cell ratio would not only elevate the percentage of transduced cells but also introduce an increasing number of insertions per cell if Poisson statistics apply to the transduction process (6,16,41). We thus escalated the MOI to transduce, based on EGFP expression determined by flow cytometry, between 10 and 90% of the target cell population.…”