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Concentration of viral vectors by co-precipitation with calcium phosphate
L Pham1, H Ye, F L Cosset
1Molecular Medicine Program, Mayo Foundation, Rochester, MN 55905, USA.
Background:
The envelope glycoproteins, surface unit (SU) and transmembrane (TM) of the murine leukemia virus (MLV) are not covalently linked and tend to dissociate upon high-speed centrifugation, leading to loss of vector infectivity. This study describes a gentle and simple method to concentrate MLV vectors or HIV vectors pseudotyped with MLV envelopes. Having a fast and inexpensive method to concentrate large volumes of vector supernatant will facilitate in vivo experiments and clinical trials that require high titer vector stocks.
Methods:
The methods employed in the study were co-precipitation of viral supernatant with calcium phosphate, low-speed centrifugation, dialysis, and infection assays with Lac-Z transducing vectors.
Results:
Murine leukemia virus vectors and HIV vectors pseudotyped with vesicular stomatitis virus glycoprotein (VSV.G) or MLV envelopes were concentrated successfully using the calcium phosphate co-precipitation method. Parameters that influence virus yield and the reproducibility of the method were investigated. The optimized protocol involves virus harvest in serum-free media, co-precipitation using 60mM calcium chloride, pelleting at 2,000 g, resuspending the pellet in a small volume of 0.1M EDTA-saline, and dialysis against saline to remove EDTA. Volumes were decreased from 300 ml to 10 ml, with 50-100% recovery, and titers can be concentrated up to 1,000-fold.
Conclusions:
The calcium phosphate co-precipitation method to concentrate virus is applicable to retrovirus and lentivirus preparations. It uses simple techniques and does not require expensive equipment. Multiple rounds of co-precipitation can be carried out if required.
Insights
A new calcium phosphate co-precipitation method gently concentrates retroviral and lentiviral vectors. This simple, inexpensive technique enhances vector infectivity for in vivo studies and clinical trials by increasing titers up to 1,000-fold.
Area of Science:
- Biotechnology
- Molecular Biology
- Virology
Background:
- Murine leukemia virus (MLV) envelope glycoproteins dissociate during high-speed centrifugation, reducing vector infectivity.
- Concentrating MLV or MLV-pseudotyped HIV vectors is crucial for high-titer stocks needed for in vivo experiments and clinical trials.
Purpose of the Study:
- To develop a gentle, simple, and inexpensive method for concentrating retroviral and lentiviral vectors.
- To improve vector infectivity and facilitate large-volume concentration for research and clinical applications.
Main Methods:
- Co-precipitation of viral supernatant with calcium phosphate.
- Low-speed centrifugation and dialysis steps.
- Infection assays using Lac-Z transducing vectors to assess viral titer and infectivity.
Main Results:
- Calcium phosphate co-precipitation successfully concentrated MLV vectors and HIV vectors with MLV or VSV.G envelopes.
- Optimized protocol achieved 50-100% recovery, concentrating volumes up to 1,000-fold.
- Method parameters for virus yield and reproducibility were investigated.
Conclusions:
- The calcium phosphate co-precipitation method is effective for concentrating retroviruses and lentiviruses.
- This technique is simple, cost-effective, and does not require specialized equipment.
- Multiple rounds of co-precipitation can be performed to achieve higher concentrations if necessary.