Highly efficient genetic transduction of primary human synoviocytes with concentrated retroviral supernatant.
We are developing retroviral-mediated gene transfer to human fibroblast-like synovial cells (FLS) as one approach to characterizing genetic pathways involved in synoviocyte pathophysiology. Prior work has suggested that FLS are relatively refractory to infection by Moloney murine leukemia virus based vectors. To determine if viral titer influenced the transduction efficiency of FLS, we optimized a rapid, efficient, and inexpensive centrifugation method to concentrate recombinant retroviral supernatant. The technique was evaluated by measurement of the expression of a viral enhanced green fluorescent protein transgene in transduced cells, and by analysis of viral RNA in retroviral supernatant. Concentration (100-fold) was achieved by centrifugation of viral supernatant for four hours, with 100% recovery of viral particles. The transduction of FLS increased from approximately 15% with unconcentrated supernatant, to nearly 50% using concentrated supernatant. This protocol will be useful for investigators with applications that require efficient, stable, high level transgene expression in primary FLS.
Yang
Jianmin
J
Department of Internal Medicine, University of Michigan Medical School, Ann Arbor 48109-0640, USA.
Friedman
Michael S
MS
Bian
Huimin
H
Crofford
Leslie J
LJ
Roessler
Blake
B
McDonagh
Kevin T
KT
D016475:3T3 Cells; D000818:Animals; D001172:Arthritis, Rheumatoid / Q000378:metabolism / Q000473:pathology; D005240:Feasibility Studies; D005347:Fibroblasts / Q000378:metabolism / Q000473:pathology* / Q000821:virology; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D012367:RNA, Viral / Q000032:analysis; D012190:Retroviridae / Q000235:genetics*; D013582:Synovial Fluid / Q000166:cytology / Q000378:metabolism / Q000821:virology; D013583:Synovial Membrane / Q000378:metabolism / Q000473:pathology* / Q000821:virology; D014161:Transduction, Genetic
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D012367:RNA, Viral; D049452:Green Fluorescent Proteins