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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 lentivirus purified with 10,000 g centrifugation mediates the GFP expression in mice hippocampus.1 μl virus (0.97 × 108 TU/ml) was injected unilaterally into the hippocampus of new born mice pups (P0), and GFP expression was examined 12 days after injection in the cryostat sections. Low (upper panels in (a,b) Scale bar = 200 μm) and high (lower panels in (a,b) Scale bar = 50 μm) magnification views of brain sections show GFP-expressing neurons in CA1 and CA3 regions of hippocampus.
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f4: The lentivirus purified with 10,000 g centrifugation mediates the GFP expression in mice hippocampus.1 μl virus (0.97 × 108 TU/ml) was injected unilaterally into the hippocampus of new born mice pups (P0), and GFP expression was examined 12 days after injection in the cryostat sections. Low (upper panels in (a,b) Scale bar = 200 μm) and high (lower panels in (a,b) Scale bar = 50 μm) magnification views of brain sections show GFP-expressing neurons in CA1 and CA3 regions of hippocampus.

Mentions: One of the major uses of the lentivirus is to infect non-dividing cells, such as neurons, for both in vitro and in vivo applications1415161718. The virus purified with 10,000 g centrifugation was tested under both circumstances. The hippocampal neuronal cultures (8 × 104 cells per coverslip in a 24-well plate) were infected with 4 μl of the virus (2.04 × 107 TU/ml). As shown in Fig. 3a, almost 100% of the neurons showed GFP fluorescence at days in vitro (DIV) 14 when the concentrated virus was added at DIV 4. To test whether or not the concentrated virus may affect the growth of the neural cells, the miniature excitatory postsynaptic synaptic currents (mEPSCs) and miniature inhibitory postsynaptic synaptic currents (mIPSCs) of the infected neurons were measured and compared with the uninfected neurons. As shown in Fig. 3b, the mEPSCs and mIPSCs were normal both in frequencies and in amplitudes, implying that the addition of the concentrated virus did not cause detectable toxic effects to the neurons. In addition, the miniature synaptic transmission from the neurons infected with purified lentivirus at DIV 10 was measured when the synapses had already formed. The results show that the virus infection had no effect on the synaptic transmission, implying that the neurons in the late development stage (DIV 10) were not affected by the virus infection (Fig. 3c). Using a stereotaxic injection, the concentrated virus was injected into the CA1 and CA3 regions of the hippocampus in newborn mice pups. The GFP-positive CA1 and CA3 neurons were observed at P12, demonstrating the in vivo application of the method (Fig. 4). To further test whether or not the injected virus might result in a significant immune response, we detected the GFAP-positive astrocytes on hippocampal tissue slides from mice injected unilaterally with the lentivirus purified with 10,000 g centrifugations. The results show that no significant aggregation of GFAP-positive astrocytes were observed around the virus-injected sites in CA3 or CA1 region of hippocampus comparing to the uninjected hippocampus (suppl. Fig. 5). Thus, these results showed that the virus enriched with our method is capable of expressing exogenous genes in the non-dividing cells.


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 lentivirus purified with 10,000 g centrifugation mediates the GFP expression in mice hippocampus.1 μl virus (0.97 × 108 TU/ml) was injected unilaterally into the hippocampus of new born mice pups (P0), and GFP expression was examined 12 days after injection in the cryostat sections. Low (upper panels in (a,b) Scale bar = 200 μm) and high (lower panels in (a,b) Scale bar = 50 μm) magnification views of brain sections show GFP-expressing neurons in CA1 and CA3 regions of hippocampus.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: The lentivirus purified with 10,000 g centrifugation mediates the GFP expression in mice hippocampus.1 μl virus (0.97 × 108 TU/ml) was injected unilaterally into the hippocampus of new born mice pups (P0), and GFP expression was examined 12 days after injection in the cryostat sections. Low (upper panels in (a,b) Scale bar = 200 μm) and high (lower panels in (a,b) Scale bar = 50 μm) magnification views of brain sections show GFP-expressing neurons in CA1 and CA3 regions of hippocampus.
Mentions: One of the major uses of the lentivirus is to infect non-dividing cells, such as neurons, for both in vitro and in vivo applications1415161718. The virus purified with 10,000 g centrifugation was tested under both circumstances. The hippocampal neuronal cultures (8 × 104 cells per coverslip in a 24-well plate) were infected with 4 μl of the virus (2.04 × 107 TU/ml). As shown in Fig. 3a, almost 100% of the neurons showed GFP fluorescence at days in vitro (DIV) 14 when the concentrated virus was added at DIV 4. To test whether or not the concentrated virus may affect the growth of the neural cells, the miniature excitatory postsynaptic synaptic currents (mEPSCs) and miniature inhibitory postsynaptic synaptic currents (mIPSCs) of the infected neurons were measured and compared with the uninfected neurons. As shown in Fig. 3b, the mEPSCs and mIPSCs were normal both in frequencies and in amplitudes, implying that the addition of the concentrated virus did not cause detectable toxic effects to the neurons. In addition, the miniature synaptic transmission from the neurons infected with purified lentivirus at DIV 10 was measured when the synapses had already formed. The results show that the virus infection had no effect on the synaptic transmission, implying that the neurons in the late development stage (DIV 10) were not affected by the virus infection (Fig. 3c). Using a stereotaxic injection, the concentrated virus was injected into the CA1 and CA3 regions of the hippocampus in newborn mice pups. The GFP-positive CA1 and CA3 neurons were observed at P12, demonstrating the in vivo application of the method (Fig. 4). To further test whether or not the injected virus might result in a significant immune response, we detected the GFAP-positive astrocytes on hippocampal tissue slides from mice injected unilaterally with the lentivirus purified with 10,000 g centrifugations. The results show that no significant aggregation of GFAP-positive astrocytes were observed around the virus-injected sites in CA3 or CA1 region of hippocampus comparing to the uninjected hippocampus (suppl. Fig. 5). Thus, these results showed that the virus enriched with our method is capable of expressing exogenous genes in the non-dividing cells.

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