Recombinant retroviruses with amphotropic and ecotropic host ranges
AssigneeWhitehead Institue For Biomedical Research
InventorsOlivier Danos, Richard C. Mulligan
Life of this patent
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Abstract
Packaging cell lines useful for the generation of helper-free recombinant retroviruses with amphotropic or ecotropic host ranges, methods of constructing such packaging cell lines and methods of using the recombinant retroviruses to introduce DNA of interest into eukaryotic cells, both in vitro and in vivo.
Claims28
A packaging cell line comprising packaging cells capable of generating helper-free recombinant retrovirus with an amphotropic host range, wherein said packaging cells' genome comprises:
- a) a first retroviral construct that codes for gag/pol and env genes wherein one of the genes selected from the group consisting of:
- gag/pol or env possesses a frameshift mutation;
- b) a second retroviral construct that codes for gag/pol and env genes wherein the gene selected from the group consisting of:
- gag/pol or env that does not possess a frameshift mutation in the first retroviral construct, possesses a frameshift mutation in the second retroviral construct;
- wherein the first and second retroviral constructs were sequentially introduced into the packaging cells' genome.
A packaging cell line comprising packaging cells capable of generating helper-free recombinant retrovirus with an ecotropic host range, wherein said packaging cells' genome comprises:
- a) a first retroviral construct that codes for gag/pol and env genes wherein one of the genes selected from the group consisting of:
- gag/pol or env possesses a frameshift mutation;
- b) a second retroviral construct that codes for gag/pol and env genes wherein the gene selected from the group consisting of:
- gag/pol or env that does not possess a frameshift mutation in the first retroviral construct, possesses a frameshift mutation in the second retroviral construct;
- wherein the first and second retroviral constructs were sequentially introduced into the packaging cells' genome.
A method of making packaging cell lines capable of generating helper-free recombinant retrovirus with either an amphotropic or an ecotropic host range, comprising the steps of:
- a) transfecting mammalian host cells with
- 1) a first retroviral construct that codes for gag/pol and env genes wherein one of the genes selected from the group consisting of:
- gag/pol or env possesses a frameshift mutation;
- and
- 2) a plasmid containing a gene which serves as a first selectable marker;
- b) culturing the transfected cells of step a) under conditions which select for the first selectable marker;
- c) transfecting the cells selected in step b) with
- 1) a second retroviral construct that codes for gag/pol and env genes wherein the gene selected from the group consisting of:
- gag/pol or env that does not possess a frameshift mutation in the first retroviral construct, possesses a frameshift mutation in the second retroviral construct;
- and
- 2) a plasmid containing a gene which serves as a second selectable marker;
- and
- d) culturing the transfected cells of step c) under conditions which select for the second selectable marker.
Dependencies are inferred from the claim text.