Complexation with chondroitin sulfate C and Polybrene rapidly purifies retrovirus from inhibitors of transduction and substantially enhances gene transfer.
Using amphotropic retrovirus stocks produced by TELCeB6-A cells that encode the Escherichia coli lacZ gene, we found that complexation with chondroitin sulfate C (CSC) and Polybrene (PB) is an effective means to purify retrovirus. Virus stocks contained high levels of inhibitory activity that blocked amphotropic, but not ecotropic, retrovirus transduction. When virus stocks were brought to 80 microg/mL each of CSC and PB, complexes of CSC and PB formed. These complexes incorporated more than 70% of the virus particles but less than 0.4% of all other proteins and no detectable inhibitory activity. Purified virus transduced NIH 3T3 murine fibroblasts 21 to 186-fold more efficiently than virus that was not purified. In addition, virus purification significantly altered the dose response of transduction. When virus that had not been purified was used to transduce cells, the relationship between transduction and virus concentration was highly non-linear. In contrast, when purified virus was used, transduction increased monotonically and was linearly proportional to virus concentration, except when high doses of virus were used. Interestingly, when high doses of virus were used gene transfer reached a maximum plateau level, most likely because particle-associated amphotropic envelope proteins had saturated the cellular receptors for the virus. Our findings illustrate that retrovirus purification increases the maximum number of genes that can be transferred, reduces the amount of virus required to achieve a given level of gene transfer, and reduces uncertainties about the relationship between the amount of virus used and the number of genes transferred.
Landázuri
Natalia
N
The Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University, Atlanta, USA.
Le Doux
Joseph M
JM
D000818:Animals; D002809:Chondroitin Sulfates / Q000737:chemistry*; D005822:Genetic Vectors / Q000302:isolation & purification*; D006367:HeLa Cells; D006583:Hexadimethrine Bromide / Q000737:chemistry*; D006801:Humans; D051379:Mice; D008979:Moloney murine leukemia virus / Q000302:isolation & purification*; D041681:NIH 3T3 Cells; D014161:Transduction, Genetic / Q000379:methods
D006583:Hexadimethrine Bromide; D002809:Chondroitin Sulfates