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Highly efficient genetic transduction of primary human synoviocytes with concentrated retroviral supernatant.

Yang J, Friedman MS, Bian H, Crofford LJ, Roessler B, McDonagh KT - Arthritis Res. (2002)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, University of Michigan Medical School, Ann Arbor 48109-0640, USA.

ABSTRACT
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.

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Quantitation of viral RNA by slot blot hybridization analysis after concentration of virus by centrifugation for four hours. Viral supernatant was centrifuged for four hours at the indicated relative centrifugal force (RCF). The viral pellet was resuspended in a hundredth of the original volume. The indicated volumes of (a) unconcentrated supernatant, (b) the resuspended viral pellet, and (c) the post-centrifugation supernatant were loaded onto a nylon membrane in a 48-well slot blot format, hybridized with an enhanced green fluorescent protein probe, and exposed to film. Experiments were repeated three times with similar results.
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Figure 7: Quantitation of viral RNA by slot blot hybridization analysis after concentration of virus by centrifugation for four hours. Viral supernatant was centrifuged for four hours at the indicated relative centrifugal force (RCF). The viral pellet was resuspended in a hundredth of the original volume. The indicated volumes of (a) unconcentrated supernatant, (b) the resuspended viral pellet, and (c) the post-centrifugation supernatant were loaded onto a nylon membrane in a 48-well slot blot format, hybridized with an enhanced green fluorescent protein probe, and exposed to film. Experiments were repeated three times with similar results.

Mentions: To determine if viral titer influenced the transduction efficiency of FLS, we optimized a superspeed centrifugation protocol for concentration of viral supernatant. Prior studies reported improved transduction of primary cells with retro-virus concentrated by centrifugation at 6000 g for 16 hours [9-11]. We systematically evaluated different centrifugation parameters to minimize the time required for maximal concentration while preserving viral infectivity. A virally encoded EGFP transgene [12-14] was used to monitor viral concentration and infectious titer. We concentrated viral supernatant 100-fold in as few as four hours by centrifugation at 20,000 g, with complete recovery of infectious viral particles. This data is presented in the Supplementary Material (Supplementary Figs s1, s2, s3, and s4).


Highly efficient genetic transduction of primary human synoviocytes with concentrated retroviral supernatant.

Yang J, Friedman MS, Bian H, Crofford LJ, Roessler B, McDonagh KT - Arthritis Res. (2002)

Quantitation of viral RNA by slot blot hybridization analysis after concentration of virus by centrifugation for four hours. Viral supernatant was centrifuged for four hours at the indicated relative centrifugal force (RCF). The viral pellet was resuspended in a hundredth of the original volume. The indicated volumes of (a) unconcentrated supernatant, (b) the resuspended viral pellet, and (c) the post-centrifugation supernatant were loaded onto a nylon membrane in a 48-well slot blot format, hybridized with an enhanced green fluorescent protein probe, and exposed to film. Experiments were repeated three times with similar results.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC111025&req=5

Figure 7: Quantitation of viral RNA by slot blot hybridization analysis after concentration of virus by centrifugation for four hours. Viral supernatant was centrifuged for four hours at the indicated relative centrifugal force (RCF). The viral pellet was resuspended in a hundredth of the original volume. The indicated volumes of (a) unconcentrated supernatant, (b) the resuspended viral pellet, and (c) the post-centrifugation supernatant were loaded onto a nylon membrane in a 48-well slot blot format, hybridized with an enhanced green fluorescent protein probe, and exposed to film. Experiments were repeated three times with similar results.
Mentions: To determine if viral titer influenced the transduction efficiency of FLS, we optimized a superspeed centrifugation protocol for concentration of viral supernatant. Prior studies reported improved transduction of primary cells with retro-virus concentrated by centrifugation at 6000 g for 16 hours [9-11]. We systematically evaluated different centrifugation parameters to minimize the time required for maximal concentration while preserving viral infectivity. A virally encoded EGFP transgene [12-14] was used to monitor viral concentration and infectious titer. We concentrated viral supernatant 100-fold in as few as four hours by centrifugation at 20,000 g, with complete recovery of infectious viral particles. This data is presented in the Supplementary Material (Supplementary Figs s1, s2, s3, and s4).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, University of Michigan Medical School, Ann Arbor 48109-0640, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus