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An optimized method for high-titer lentivirus preparations without ultracentrifugation.

Jiang W, Hua R, Wei M, Li C, Qiu Z, Yang X, Zhang C - Sci Rep (2015)

Bottom Line: Lentiviral technology has proven to be a powerful tool to express exogenous genes in dividing and non-dividing cells.Currently, most protocols for generating high-titer lentivirus require ultracentrifugation, which can be an instrumental barrier for routine operations in a laboratory.In summary, we describe an efficient and easy-to-handle protocol for high-titer lentivirus purification.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Membrane Biology, School of Life Sciences; PKU-IDG/McGovern Institute for Brain Research, Peking University, Beijing 100871, China.

ABSTRACT
Lentiviral technology has proven to be a powerful tool to express exogenous genes in dividing and non-dividing cells. Currently, most protocols for generating high-titer lentivirus require ultracentrifugation, which can be an instrumental barrier for routine operations in a laboratory. In this study, the effect of relative centrifugal force (RCF) on the concentration efficiency of the lentivirus was systematically explored, and it was found that sucrose gradient centrifugation with a relatively low speed (≤10,000 g) robustly produces a high-titer virus (up to 2×10(8) TU/ml). The optimal sucrose concentration is 10%, and the recovery rate of the functional virus is greater than 80%. The infection efficiency of both concentrated and un-concentrated lentivirus decreases rapidly when the viruses are stored at 4 °C (τ≈1.3 days) or subjected to multiple freeze-thaw cycles (τ=1.1 rounds). In summary, we describe an efficient and easy-to-handle protocol for high-titer lentivirus purification.

No MeSH data available.


Related in: MedlinePlus

Optimization of lentivirus concentration/storage conditions.(a) Summary graphs of transduction efficiency as a function of storage duration at 4 °C. The percentages (black) and mean intensities (red) of the transduced cells were plotted to reflect the effectiveness of the raw (left) or concentrated (right) lentivirus. (b) Summary graphs of percentages (black) and mean intensities (red) of the HEK293T cells infected with the virus subject to multiple freeze-thaw cycles. (c) Representative images (left) and summary graph (right) of the flow cytometric analysis of the HEK293T cells transduced with the GFP-expressing lentivirus purified from virus-containing medium collected from the first 48 hours or 48–72 hours after transfection. All summary graphs show mean ± SEM; n = 3 independent experiments (***p < 0.001).
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f2: Optimization of lentivirus concentration/storage conditions.(a) Summary graphs of transduction efficiency as a function of storage duration at 4 °C. The percentages (black) and mean intensities (red) of the transduced cells were plotted to reflect the effectiveness of the raw (left) or concentrated (right) lentivirus. (b) Summary graphs of percentages (black) and mean intensities (red) of the HEK293T cells infected with the virus subject to multiple freeze-thaw cycles. (c) Representative images (left) and summary graph (right) of the flow cytometric analysis of the HEK293T cells transduced with the GFP-expressing lentivirus purified from virus-containing medium collected from the first 48 hours or 48–72 hours after transfection. All summary graphs show mean ± SEM; n = 3 independent experiments (***p < 0.001).

Mentions: After concentration, the purified virus is typically either used for a short period of time or aliquoted and stored at −80 °C for long-term usage. Thus, the change in the virus titer upon prolonged storage of the lentivirus at 4 °C was tested. As shown in Fig. 2a, the infection efficiency of the un-concentrated and concentrated viruses was reduced exponentially (τ = 1.4 days and 1.3 days, respectively). The stability of the commercial-purchased virus was also measured, and it was found that the infection efficiency was reduced exponentially (τ = 1.9 days), which is similar to the un-concentrated virus and the concentrated virus purified by 10000 g purification (suppl. Fig. 4). Next, the effect of multiple freeze-thaw cycles on the infection efficiency of the virus was tested. During the test, the frozen virus was thawed at 4 °C for 20 minutes and added to the cells immediately at a 1:50 ratio. The results showed that the infection efficiency after one round of freeze and thaw was reduced by 33.3 ± 6.4% (τ = 1.1 rounds, Fig. 2b). To increase the yield of the lentivirus production, experimenters sometimes collect virus-containing medium from transfected HEK293T cells twice, once during the first 48 hours and again 48–72 hours after transfection. The results in this study consistently showed no significant difference in the infection efficiency or the fluorescent intensity of the cells infected with the viruses produced between 0 and 48 hours and between 48 and 72 hours after transfection (Fig. 2c).


An optimized method for high-titer lentivirus preparations without ultracentrifugation.

Jiang W, Hua R, Wei M, Li C, Qiu Z, Yang X, Zhang C - Sci Rep (2015)

Optimization of lentivirus concentration/storage conditions.(a) Summary graphs of transduction efficiency as a function of storage duration at 4 °C. The percentages (black) and mean intensities (red) of the transduced cells were plotted to reflect the effectiveness of the raw (left) or concentrated (right) lentivirus. (b) Summary graphs of percentages (black) and mean intensities (red) of the HEK293T cells infected with the virus subject to multiple freeze-thaw cycles. (c) Representative images (left) and summary graph (right) of the flow cytometric analysis of the HEK293T cells transduced with the GFP-expressing lentivirus purified from virus-containing medium collected from the first 48 hours or 48–72 hours after transfection. All summary graphs show mean ± SEM; n = 3 independent experiments (***p < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4562269&req=5

f2: Optimization of lentivirus concentration/storage conditions.(a) Summary graphs of transduction efficiency as a function of storage duration at 4 °C. The percentages (black) and mean intensities (red) of the transduced cells were plotted to reflect the effectiveness of the raw (left) or concentrated (right) lentivirus. (b) Summary graphs of percentages (black) and mean intensities (red) of the HEK293T cells infected with the virus subject to multiple freeze-thaw cycles. (c) Representative images (left) and summary graph (right) of the flow cytometric analysis of the HEK293T cells transduced with the GFP-expressing lentivirus purified from virus-containing medium collected from the first 48 hours or 48–72 hours after transfection. All summary graphs show mean ± SEM; n = 3 independent experiments (***p < 0.001).
Mentions: After concentration, the purified virus is typically either used for a short period of time or aliquoted and stored at −80 °C for long-term usage. Thus, the change in the virus titer upon prolonged storage of the lentivirus at 4 °C was tested. As shown in Fig. 2a, the infection efficiency of the un-concentrated and concentrated viruses was reduced exponentially (τ = 1.4 days and 1.3 days, respectively). The stability of the commercial-purchased virus was also measured, and it was found that the infection efficiency was reduced exponentially (τ = 1.9 days), which is similar to the un-concentrated virus and the concentrated virus purified by 10000 g purification (suppl. Fig. 4). Next, the effect of multiple freeze-thaw cycles on the infection efficiency of the virus was tested. During the test, the frozen virus was thawed at 4 °C for 20 minutes and added to the cells immediately at a 1:50 ratio. The results showed that the infection efficiency after one round of freeze and thaw was reduced by 33.3 ± 6.4% (τ = 1.1 rounds, Fig. 2b). To increase the yield of the lentivirus production, experimenters sometimes collect virus-containing medium from transfected HEK293T cells twice, once during the first 48 hours and again 48–72 hours after transfection. The results in this study consistently showed no significant difference in the infection efficiency or the fluorescent intensity of the cells infected with the viruses produced between 0 and 48 hours and between 48 and 72 hours after transfection (Fig. 2c).

Bottom Line: Lentiviral technology has proven to be a powerful tool to express exogenous genes in dividing and non-dividing cells.Currently, most protocols for generating high-titer lentivirus require ultracentrifugation, which can be an instrumental barrier for routine operations in a laboratory.In summary, we describe an efficient and easy-to-handle protocol for high-titer lentivirus purification.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Membrane Biology, School of Life Sciences; PKU-IDG/McGovern Institute for Brain Research, Peking University, Beijing 100871, China.

ABSTRACT
Lentiviral technology has proven to be a powerful tool to express exogenous genes in dividing and non-dividing cells. Currently, most protocols for generating high-titer lentivirus require ultracentrifugation, which can be an instrumental barrier for routine operations in a laboratory. In this study, the effect of relative centrifugal force (RCF) on the concentration efficiency of the lentivirus was systematically explored, and it was found that sucrose gradient centrifugation with a relatively low speed (≤10,000 g) robustly produces a high-titer virus (up to 2×10(8) TU/ml). The optimal sucrose concentration is 10%, and the recovery rate of the functional virus is greater than 80%. The infection efficiency of both concentrated and un-concentrated lentivirus decreases rapidly when the viruses are stored at 4 °C (τ≈1.3 days) or subjected to multiple freeze-thaw cycles (τ=1.1 rounds). In summary, we describe an efficient and easy-to-handle protocol for high-titer lentivirus purification.

No MeSH data available.


Related in: MedlinePlus