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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

The examination of purification parameters on the concentration efficiency of the lentivirus.(a) Representative images (left) and summary graph (right) of the flow cytometric analysis of the HEK293T cells transduced with the GFP-expressing lentivirus purified with RCF. The HEK293T cells were trypsinized 36 hours after the transduction for the flow cytometric analysis. (b) Representative fluorescent images showing the HEK293T cells transduced with either a raw or concentrated GFP-expressing lentivirus. The viruses were enriched at a 1:50 ratio and added to the HEK293T cells at the volume indicated in the lower panels. Scale bar: 80 μm. (c) Summary graph of RT-PCR measurements of GFP-expressing lentivirus titer enriched with various RCFs at a 1:50 ratio. (d) Summary graphs of percentages and mean fluorescent intensities of the GFP-positive HEK293T cells transduced with either a raw or concentrated lentivirus enriched with 10,000 g centrifugation for 4 hours and 90,000 g for 1.5 hours, respectively. (e) Silver staining of equivalent amounts of the purified virus (1 × 105 TU) prepared using either the RCF 10,000 g or ultracentrifugation (Commercial & RCF 90,000  g). (f) Representative images (left) and summary graph (right) of the flow cytometric analysis of the HEK293T cells transduced with the GFP-expressing lentivirus enriched with 10,000 g centrifugation for various durations. (g,h) Summary graph of the flow cytometric analysis of the HEK293T cells transduced with the GFP-expressing lentivirus enriched with 10,000 g with sucrose concentrations ranging from 0% to 50% (g) and 7.5% to 20.0% (h) in the centrifugation buffer. All summary graphs show mean ± SEM; n = 3 independent experiments (***p < 0.001).
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f1: The examination of purification parameters on the concentration efficiency of the lentivirus.(a) Representative images (left) and summary graph (right) of the flow cytometric analysis of the HEK293T cells transduced with the GFP-expressing lentivirus purified with RCF. The HEK293T cells were trypsinized 36 hours after the transduction for the flow cytometric analysis. (b) Representative fluorescent images showing the HEK293T cells transduced with either a raw or concentrated GFP-expressing lentivirus. The viruses were enriched at a 1:50 ratio and added to the HEK293T cells at the volume indicated in the lower panels. Scale bar: 80 μm. (c) Summary graph of RT-PCR measurements of GFP-expressing lentivirus titer enriched with various RCFs at a 1:50 ratio. (d) Summary graphs of percentages and mean fluorescent intensities of the GFP-positive HEK293T cells transduced with either a raw or concentrated lentivirus enriched with 10,000 g centrifugation for 4 hours and 90,000 g for 1.5 hours, respectively. (e) Silver staining of equivalent amounts of the purified virus (1 × 105 TU) prepared using either the RCF 10,000 g or ultracentrifugation (Commercial & RCF 90,000  g). (f) Representative images (left) and summary graph (right) of the flow cytometric analysis of the HEK293T cells transduced with the GFP-expressing lentivirus enriched with 10,000 g centrifugation for various durations. (g,h) Summary graph of the flow cytometric analysis of the HEK293T cells transduced with the GFP-expressing lentivirus enriched with 10,000 g with sucrose concentrations ranging from 0% to 50% (g) and 7.5% to 20.0% (h) in the centrifugation buffer. All summary graphs show mean ± SEM; n = 3 independent experiments (***p < 0.001).

Mentions: To examine the lower limit of RCF, which could produce a reasonable yield of the high-titer lentivirus, the effect of RCF (1,000–10,000 g) on the concentration efficiency was examined. The HEK293T cells were transfected with a packaging vector expressing a green fluorescent protein (GFP) along with transfer and envelope vectors, and the virus-containing medium being collected at 48 hours after transfection was used as the starting materials for the concentration. To measure the titer of the functional virus, the concentrated virus was added to the HEK293T cells, and the percentage and relative intensity of the GFP-positive cells were measured with a flow cytometer (Fig. 1a). We used a 20% sucrose gradient centrifugation for 4 hours, which is most commonly used in published protocols, as the starting point. The concentration of functional virion particles, reflected as the percentage of the cells infected, was increased in a linear relationship with RCF in the range of 2,000–10,000 g (Fig. 1a,b). The virus purified with 1,000 g centrifugation showed almost no significant transduction ability in the HEK293T cells. The fluorescent intensity of the transduced HEK293T cells showed no significant differences in any of the concentrated viruses purified with various RCF tests. The concentration of viral genomes of the purified lentivirus was also measured using the RT-PCR method. Consistently, sucrose gradient centrifugation with a relatively low speed (≤10,000 g) robustly produces a high-titer virus in a RCF-dependent manner (Fig. 1c). The recovery yield of 10,000 g centrifugation was 85.6 ± 0.07% (suppl. Fig. 1). Intriguingly, 10,000 g centrifugation showed a 185.8 ± 23.7% transduction efficiency compared with the ultracentrifuge at the speed of 90,000 g for 90 minutes with otherwise identical settings (Fig. 1d). To test whether the different transduction efficiency was due to the different centrifugation time, the different speed, or both, a set of experiments were performed to compare the concentration efficiencies among the lentiviruses purified at various RCFs for various durations. The results show that the transduction efficiency had a linear relationship with the RCF in the range of 0–10000 g, and remained stable in the range of 10000–90000 g centrifugation for either 1.5 or 4 hours; however, the virus purified with centrifugation for 1.5 hrs always showed significantly less transduction efficiency compared with 4 hrs at all of the RCFs tested (suppl. Fig. 2). The impurity of the lentivirus preparations is an important issue, especially in in vivo applications. Thus, silver staining experiments were performed to examine the lentivirus purity. As shown in Fig. 1e, the virus purified using the low-speed centrifugation method contained as few contaminating proteins as the equivalent amounts of the lentivirus purchased commercially.


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)

The examination of purification parameters on the concentration efficiency of the lentivirus.(a) Representative images (left) and summary graph (right) of the flow cytometric analysis of the HEK293T cells transduced with the GFP-expressing lentivirus purified with RCF. The HEK293T cells were trypsinized 36 hours after the transduction for the flow cytometric analysis. (b) Representative fluorescent images showing the HEK293T cells transduced with either a raw or concentrated GFP-expressing lentivirus. The viruses were enriched at a 1:50 ratio and added to the HEK293T cells at the volume indicated in the lower panels. Scale bar: 80 μm. (c) Summary graph of RT-PCR measurements of GFP-expressing lentivirus titer enriched with various RCFs at a 1:50 ratio. (d) Summary graphs of percentages and mean fluorescent intensities of the GFP-positive HEK293T cells transduced with either a raw or concentrated lentivirus enriched with 10,000 g centrifugation for 4 hours and 90,000 g for 1.5 hours, respectively. (e) Silver staining of equivalent amounts of the purified virus (1 × 105 TU) prepared using either the RCF 10,000 g or ultracentrifugation (Commercial & RCF 90,000  g). (f) Representative images (left) and summary graph (right) of the flow cytometric analysis of the HEK293T cells transduced with the GFP-expressing lentivirus enriched with 10,000 g centrifugation for various durations. (g,h) Summary graph of the flow cytometric analysis of the HEK293T cells transduced with the GFP-expressing lentivirus enriched with 10,000 g with sucrose concentrations ranging from 0% to 50% (g) and 7.5% to 20.0% (h) in the centrifugation buffer. All summary graphs show mean ± SEM; n = 3 independent experiments (***p < 0.001).
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Related In: Results  -  Collection

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f1: The examination of purification parameters on the concentration efficiency of the lentivirus.(a) Representative images (left) and summary graph (right) of the flow cytometric analysis of the HEK293T cells transduced with the GFP-expressing lentivirus purified with RCF. The HEK293T cells were trypsinized 36 hours after the transduction for the flow cytometric analysis. (b) Representative fluorescent images showing the HEK293T cells transduced with either a raw or concentrated GFP-expressing lentivirus. The viruses were enriched at a 1:50 ratio and added to the HEK293T cells at the volume indicated in the lower panels. Scale bar: 80 μm. (c) Summary graph of RT-PCR measurements of GFP-expressing lentivirus titer enriched with various RCFs at a 1:50 ratio. (d) Summary graphs of percentages and mean fluorescent intensities of the GFP-positive HEK293T cells transduced with either a raw or concentrated lentivirus enriched with 10,000 g centrifugation for 4 hours and 90,000 g for 1.5 hours, respectively. (e) Silver staining of equivalent amounts of the purified virus (1 × 105 TU) prepared using either the RCF 10,000 g or ultracentrifugation (Commercial & RCF 90,000  g). (f) Representative images (left) and summary graph (right) of the flow cytometric analysis of the HEK293T cells transduced with the GFP-expressing lentivirus enriched with 10,000 g centrifugation for various durations. (g,h) Summary graph of the flow cytometric analysis of the HEK293T cells transduced with the GFP-expressing lentivirus enriched with 10,000 g with sucrose concentrations ranging from 0% to 50% (g) and 7.5% to 20.0% (h) in the centrifugation buffer. All summary graphs show mean ± SEM; n = 3 independent experiments (***p < 0.001).
Mentions: To examine the lower limit of RCF, which could produce a reasonable yield of the high-titer lentivirus, the effect of RCF (1,000–10,000 g) on the concentration efficiency was examined. The HEK293T cells were transfected with a packaging vector expressing a green fluorescent protein (GFP) along with transfer and envelope vectors, and the virus-containing medium being collected at 48 hours after transfection was used as the starting materials for the concentration. To measure the titer of the functional virus, the concentrated virus was added to the HEK293T cells, and the percentage and relative intensity of the GFP-positive cells were measured with a flow cytometer (Fig. 1a). We used a 20% sucrose gradient centrifugation for 4 hours, which is most commonly used in published protocols, as the starting point. The concentration of functional virion particles, reflected as the percentage of the cells infected, was increased in a linear relationship with RCF in the range of 2,000–10,000 g (Fig. 1a,b). The virus purified with 1,000 g centrifugation showed almost no significant transduction ability in the HEK293T cells. The fluorescent intensity of the transduced HEK293T cells showed no significant differences in any of the concentrated viruses purified with various RCF tests. The concentration of viral genomes of the purified lentivirus was also measured using the RT-PCR method. Consistently, sucrose gradient centrifugation with a relatively low speed (≤10,000 g) robustly produces a high-titer virus in a RCF-dependent manner (Fig. 1c). The recovery yield of 10,000 g centrifugation was 85.6 ± 0.07% (suppl. Fig. 1). Intriguingly, 10,000 g centrifugation showed a 185.8 ± 23.7% transduction efficiency compared with the ultracentrifuge at the speed of 90,000 g for 90 minutes with otherwise identical settings (Fig. 1d). To test whether the different transduction efficiency was due to the different centrifugation time, the different speed, or both, a set of experiments were performed to compare the concentration efficiencies among the lentiviruses purified at various RCFs for various durations. The results show that the transduction efficiency had a linear relationship with the RCF in the range of 0–10000 g, and remained stable in the range of 10000–90000 g centrifugation for either 1.5 or 4 hours; however, the virus purified with centrifugation for 1.5 hrs always showed significantly less transduction efficiency compared with 4 hrs at all of the RCFs tested (suppl. Fig. 2). The impurity of the lentivirus preparations is an important issue, especially in in vivo applications. Thus, silver staining experiments were performed to examine the lentivirus purity. As shown in Fig. 1e, the virus purified using the low-speed centrifugation method contained as few contaminating proteins as the equivalent amounts of the lentivirus purchased commercially.

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