Efficient expression of exogenous genes in primary vascular cells using IRES-based retroviral vectors.
Analysis of gene function in primary vascular cells has been particularly limited by low transfection efficiencies. Using internal ribosomal entry site (IRES)-based retroviral vectors, we demonstrate efficient infection (range of 45%-95%) of primary human endothelial and smooth muscle cells with genes varying in size from 1.3 to 4.5 kb. Because IRES vectors are designed to allow the expression of two genes from a single mRNA, we can show excellent correlation between the expression of a reporter gene and an inserted gene of interest. Reporter gene expression allows rapid (24-48 h) and unambiguous identification of transduced cells. Additionally, reporter gene expression can be used to isolate subpopulations of cells that express distinct levels of cistron 1 genes by flow cytometry, and sorted cells maintain relative levels of gene expression over multiple passages in culture. Two examples of the usefulness of these vectors to characterize gene function in primary vascular cells include (i) the inhibition of endothelial cell inflammatory responses in a polyclonal population by the expression of a dominant negative inhibitor of nuclear factor-kappaB and (ii) monitoring the in vitro evolution of smooth muscle cells provided with a selective growth advantage by transduction with telomerase. Potential applications of retroviral expression strategies in vascular biology are also discussed.
Garton
Kyle J
KJ
University of Washington School of Medicine, Seattle, USA.
Ferri
Nicola
N
Raines
Elaine W
EW
D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D015870:Gene Expression; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D016328:NF-kappa B / Q000037:antagonists & inhibitors; D012190:Retroviridae / Q000235:genetics*; D012270:Ribosomes / Q000378:metabolism*
D008070:Lipopolysaccharides; D008164:Luminescent Proteins; D016328:NF-kappa B; D049452:Green Fluorescent Proteins