Concentration of viral vectors by co-precipitation with calcium phosphate

Linh Pham, Hong Ye, Francois Loic Cosset, Stephen J. Russell, Kah Whye Peng

Research output: Contribution to journalArticle

35 Scopus citations

Abstract

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 60 mM calcium chloride, pelleting at 2000g, resuspending the pellet in a small volume of 0.1 M 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 1000-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.

Original languageEnglish (US)
Pages (from-to)188-194
Number of pages7
JournalJournal of Gene Medicine
Volume3
Issue number2
DOIs
StatePublished - Mar 1 2001

Keywords

  • Co-precipitation
  • Concentration
  • Inexpensive production
  • MLV pseudotyped lentivirus
  • Retrovirus

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics
  • Drug Discovery
  • Genetics(clinical)

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