Retrovirus infection: effect of time and target cell number

Morgan, Jeffrey R.; LeDoux, Joseph M.; Snow, Richard; Tompkins, Ronald G.; Yarmush, Martin L.

doi:10.1128/jvi.69.11.6994-7000.1995

Cited by 85 publications

(40 citation statements)

References 47 publications

(57 reference statements)

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“…5B). Interestingly, previous studies showed that prolonging the incubation time for up to 24 h led to increased retrovirus transduction efficiency (Morgan et al, 1995), but those studies reported that virus adsorption ceased after 2 h. In this study, Q-PCR confirmed that the increase in incubation time linearly increased the number of viral genes entering the cells, suggesting that adsorption continued for at least 8 h. The discrepancy between the previous and present results may be due to the varying mechanisms of virus adsorption into different cells because various factors and receptors (e.g., electrostatic interactions, phospholipids) involved in virus binding and entry have been proposed (Duisit et al, 1999;Tani et al, 2001). Although the current protocol successfully increased transduction efficiency and overall transgene expression, it is noteworthy that this approach may require modification when being applied to different cells, because cells of various origins respond differently under different incubation conditions (surrounding solutions, incubation time and temperatures, etc.).…”

Section: Discussionmentioning

confidence: 99%

Highly efficient baculovirus‐mediated gene transfer into rat chondrocytes

Chen

Wang

et al. 2004

Biotech & Bioengineering

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To explore the potential of baculovirus serving as a gene delivery vector in tissue engineering of articular cartilage, the efficiencies of baculovirus-mediated gene delivery into primary rat chondrocytes were evaluated and the transduction protocol commonly employed by others (using concentrated virus at multiplicity of infection [MOI] 200 for 1 h) was found to be ineffective (<1%). Therefore, a modified protocol was adopted, which markedly enhanced the efficiency (68%). Optimization of the transduction parameters, such as incubation time (8 h), temperature (25 degrees C), and surrounding solutions (PBS), further increased the efficiency to 88% and prolonged the duration of expression to 21 days, suggesting that the cells previously considered nonpermissive to baculovirus transduction may be reexamined for their permissiveness using alternative transduction protocols. The elevated efficiency correlated well with increased virus uptake upon extended incubation time, as demonstrated by quantitative real-time polymerase chain reaction (Q-PCR). The Q-PCR also revealed the degradation of viral DNA over culture time. Although the virus transduction somewhat hindered the cell proliferation, growth rate could be restored in the long-term culture. More importantly, transduced cells could secrete articular cartilage-specific type II collagen and glycosaminoglycan as well as mock-transduced cells, confirming that normal differentiation state of rat chondrocytes is retained upon baculovirus transduction. Taken together, these data indicate that baculovirus is a safe and highly efficient gene delivery vehicle into rat chondrocytes.

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Section: Discussionmentioning

confidence: 99%

Highly efficient baculovirus‐mediated gene transfer into rat chondrocytes

Chen

Wang

et al. 2004

Biotech & Bioengineering

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“…Similar experiments were conducted in the absence of Polybrene, except the cells were incubated with virus for 4 h instead of 2 h. After incubation with the virus, the cells were washed two times with 100 µL/well of fresh medium. The posttreated cells were then incubated for 2 d at 37°C with 100 µL of fresh medium and brought to either 20 µg/mL CSB or 10 µg/mL CSPG until confluent, and then they were tested for -galactosidase activity with the transduction efficiency assay as previously described (18,19). All other cells (control, pretreated, and cotreated) were incubated for 2 d at 37°C with 100 µL of fresh medium until confluent and then tested for -galactosidase activity.…”

Section: Methodsmentioning

confidence: 99%

“…The amphotropic packaging cell line ψ -CRIP, producing the R-SGC-lacZ virus, was kindly provided by L. K. Cohen of Somatix Therapy Corp., Alameda, CA (17). LacZ virus-containing medium was harvested from confluent cultures of the virus-producing cell line (overnight incubation of 10 mL of culture medium in a 10-cm tissue culture dish), filter sterilized (0.45 µm), and then stored at -85°C for later use (18).…”

Section: Introductionmentioning

confidence: 99%

Differential Inhibition of Retrovirus Transduction by Proteoglycans and Free Glycosaminoglycans

1999

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We have previously shown that the efficiency of retrovirus-mediated gene transfer is limited in part due to the presence of chondroitin sulfate proteoglycans in virus stocks. In this study, we have used a model recombinant retrovirus encoding the Escherichia coli lacZ gene, bovine aorta chondroitin sulfate proteoglycan (CSPG), various free glycosaminoglycan chains (GAGs), and quantitative assays for retrovirus transduction to explore the mechanism by which proteoglycans and glycosaminoglycans inhibit retroviruses. We found that CSPG and GAGs block an early step in virus-cell interactions but do not act by inactivating viruses or by reducing the growth rate of the target cells. CSPG and most of the GAGs tested (chondroitin sulfate A, chondroitin sulfate B, heparin, heparan sulfate, and hyaluronic acid) inhibited transduction, but with widely varying degrees of activity. The chemical structure of GAGs was found to be an important determinant of their inhibitory activity, which suggests that GAGs do not inhibit transduction simply because they are highly negatively charged polymers. When GAGs were used in combination with a cationic polymer (Polybrene), however, their inhibitory activity was neutralized, and interestingly, at optimal doses of GAG and Polybrene, transduction efficiency was actually enhanced by as much as 72%. In contrast, the inhibitory activity of CSPG, due to the influence of its core protein, was not substantially reduced by Polybrene. The importance of these findings to our understanding of retrovirus-cell interactions and to the development of more efficient retrovirus gene transfer protocols is discussed.

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“…Also, in the absence of cell lysate, the number of transduced cells increased with target cell concentration, whereas the transduction efficiency remained constant except at the lowest concentration of target cells (1.0 Â 10 5 cells/mL). Decreased transduction efficiency at low target cell concentration has previously been reported but this phenomenon remains unexplained (Hanenberg et al, 1997;Morgan et al, 1995). To probe whether this was possibly an artifact of the low cell concentration or potentially delayed expression, the cells were expanded for an additional 2 days and transduction efficiency was measured a second time.…”

Section: Effect Of Target Cell Concentrationmentioning

confidence: 99%

Effect of cell lysates on retroviral transduction efficiency of cells in suspension culture

Beauchesne

Bruce

Bowen

et al. 2010

Biotech & Bioengineering

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Recombinant retroviruses are effective vectors able to integrate transgenes into the target cell's genome to achieve longer-term expression. This study investigates the effect of cell lysis products, a common cell culture by-product, on the transduction of suspension cells by gammaretroviral vectors. Cell lysates derived from human and murine suspension cell lines significantly increased the transduction of human TF-1 and K-562 cell lines by gibbon ape leukemia virus-pseudotyped retroviral vectors without altering tropism. The transduction efficiency of TF-1 cells increased as a function of lysate concentration and decreased with increasing target cell concentrations. This was adequately predicted using a saturation equation based on the lysed-to-target cell concentration ratio, R, where: Fold increase = 1+Fold_(Max) (R/(K_(L)+R)). Lysate completely masked the effects of fibronectin when the two were added in combination. With protamine sulfate, the transduction efficiency was increased by lysate to 58% from 20% for protamine sulfate alone. Overall, the presence of cell lysate significantly influenced the outcome of the transduction process, either alone or in the presence of protamine sulfate or fibronectin.

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Retrovirus infection: effect of time and target cell number

Cited by 85 publications

References 47 publications

Highly efficient baculovirus‐mediated gene transfer into rat chondrocytes

Highly efficient baculovirus‐mediated gene transfer into rat chondrocytes

Differential Inhibition of Retrovirus Transduction by Proteoglycans and Free Glycosaminoglycans

Effect of cell lysates on retroviral transduction efficiency of cells in suspension culture

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