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Query: UMLS:C0393754 (HSA)
 2,996 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The use of gene transfer procedures has greatly facilitated the investigation of cell lineage relationships and other developmental processes in a variety of primary tissues. In this report we described the infection and selection of primary human breast epithelial cells using retroviral vectors (Jzen-HSA-NEO and MSCV-HSA.NEO) containing the complete 228 bp coding sequence of a murine cell surface marker (Heat Stable Antigen, HSA) as well as the neomycin resistance (neo(r)) gene. Expression of the transduced HSA gene was detectable using either flow cytometry or immunohistochemistry after staining cells with an anti-murine HSA-specific antibody (M1/69). Expression of the transduced neo(r) gene conferred resistance to G418. In initial experiments with the MCF-7 breast cancer cell line, continued expression of both markers was demonstrated in a high proportion of cells for at least 4 weeks after their infection by positive M1/69 staining of cells that had been selected by prior incubation in G418. Evidence of gene transfer to early stage (< 9 days old) primary cultures of normal human breast epithelial cells (15 experiments with cells from 12 normal individuals) was also obtained using an infection protocol in which these calls were exposed to helper-free viral supernatants (2 incubations, 4 to 6 hr each) after being subcultured for 12 to 18 hr to increase their rate of proliferation. The presence of 5-50% (mean = 26%) HSA+ cells was demonstrated in these experiments within 5 days after their infection and the HSA+ populations included both myoepithelial and luminal phenotypes. The transduced (HSA+) cells within both of these subpopulations could also be separately isolated by FACS and subcultured. These results should provide an important starting point for future studies of genetically modified or marked primary human breast epithelial cell populations.
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PMID:Isolation and analysis of different subpopulations of normal human breast epithelial cells after their infection with a retroviral vector encoding a cell surface marker. 923 74

Previously we designed novel pseudotyped high-titer replication defective human immunodeficiency virus type 1 (HIV-1) vectors to deliver genes into nondividing cells (J. Reiser, G. Harmison, S. Kluepfel-Stahl, R. O. Brady, S. Karlsson, and M. Schubert, Proc. Natl. Acad. Sci. USA 93:15266-15271, 1996). Since then we have made several improvements with respect to the safety, flexibility, and efficiency of the vector system. A three-plasmid expression system is used to generate pseudotyped HIV-1 particles by transient transfection of human embryonic kidney 293T cells with a defective packaging construct, a plasmid coding for a heterologous envelope (Env) protein, and a vector construct harboring a reporter gene such as neo, ShlacZ (encoding a phleomycin resistance/beta-galactosidase fusion protein), HSA (encoding mouse heat-stable antigen), or EGFP (encoding enhanced green fluorescent protein). The packaging constructs lack functional Vif, Vpr, and Vpu proteins and/or a large portion of the Env coding region as well as the 5' and 3' long terminal repeats, the Nef function, and the presumed packaging signal. Using G418 selection, we routinely obtained vector particles pseudotyped with the vesicular stomatitis virus G glycoprotein (VSV-G) with titers of up to 8 x 10(7) CFU/microgram of p24, provided that a functional Tat coding region was present in the vector. Vector constructs lacking a functional Tat protein yielded titers of around 4 x 10(6) to 8 x 10(6) CFU/microgram of p24. Packaging constructs with a mutation within the integrase (IN) core domain profoundly affected colony formation and expression of the reporter genes, indicating that a functional IN protein is required for efficient transduction. We explored the abilities of other Env proteins to allow formation of pseudotyped HIV-1 particles. The rabies virus and Mokola virus G proteins yielded high-titer infectious pseudotypes, while the human foamy virus Env protein did not. Using the improved vector system, we successfully transduced contact-inhibited primary human skin fibroblasts and postmitotic rat cerebellar neurons and cardiac myocytes, a process not affected by the lack of the accessory proteins.
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PMID:High-titer human immunodeficiency virus type 1-based vector systems for gene delivery into nondividing cells. 976 32