Naslov
Use of VSV pseudotyped retrovectors to target murine osteoprogenitor cells

Autori
Kalajzić, Ivo ; Višnjić, Dora ; Liu, Peng ; Stover, Mary Louise ; Hurley, M ; Rowe, W David ; Lichtler, C Alex

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
Bone 23:5 (Suppl), S532, SA018, 1998 / - , 1998

Skup
Second Joint Meeting of ASBM and IBMR

Mjesto i datum
San Francisco (CA), Sjedinjene Američke Države, 01.12.1998. - 06.12.1998

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
retrovector; osteoprogenitor

Sažetak
Retrovector packaging cell lines that provide the env gene that serves as the ligand for receptor mediated uptake of the virus notoriously have low titers and restricted spectrum of susceptible cells. Because few cells are infected by these vectors, prolonged G418 selection is required to obtain a large population of genetically modified cells. Vesicular stoamatitis virus (VSV) pseudotyped retrovectors can obtain high titers of stable viral particles capable of gaining entry to all cell types because the G protein interacts with the phospholipid of the cells membrane. We have examines the 293 GPG cell line developed in Mulligen's laboratory in which the G protein is under control of the tet gene (Ory, D. et al. PNAS 93:11400, 1996). two different rectrovectors encoding b-galactosidase adn eGFP as marker gene were used for transduction of stromal cell. Titers of retroviral supernatants were determined on NIH 3T3 cells and were 5x105/ml for eGFP and 5x107/ml for b-gal encoding vector. Murine marrow stromal fibroblasts were infected by the viral supernatants. To asses the best protocol for transduction of MSF we tested different condition, with usage of several facilitating agents, and performing infections either once or repetitively at day 3, 4, or 5 of primary culture. We have founfd that protamine sulfate and repetitive infection on dayxs 3 and 4 transduces approximately 50-60% of stromal cells. Primary murine MSF yield b-gal positive nodules with infrequent staining of internodalar cells (mostly macrophages). To enchance the progenitor proliferation and to inhibit differentiation we treated the culture with fibroblast growth factor and noggin. We found that both FGF and noggin are inhibiting alkaline phosphatase expression in primary cultures under continuoes treatment. Noggin inhibits mRNA levels of BSP and OC and does not affect the nodule formation ; indeed most of the nodules are not expressing alkaline phosphatase. Currently, the ability of transduced cells to progress to osteogenesis after continous treatment with noggin and FGF is being examined. It does appear that the VSV pseudotyped retrovectors are capable of delivering genes to bone stem cells in a manner that does not require prolonged selection.

Izvorni jezik
Engleski

Znanstvena područja
Temeljne medicinske znanosti

POVEZANOST RADA