Immortalization and disimmortalization of cells
AssigneeCalifornia Institute of Technology
InventorDavid J. Anderson
Life of this patent
Estimating the term…
Abstract
Methods and compositions for the conditional immortalization of cells are provided.
Claims39
An immortalized mammalian cell that contains in the genome of said cell exogenous nucleic acid comprising at least a first and a second recombinase target site flanking an immortalization gene, wherein said first and second target sites mediate excision of said immortalization gene when said first and second target sites are contacted with a recombinase.
A method for creating an immortalized cell that contains recombinase target sites flanking an immortalization gene in the genome of said immortalized cell, said method comprising transforming a cell with exogeneous nucleic acid comprising
- a) a first recombinase site;
- b) an immortalization gene;
- and
- c) a second recombinase site;
- such that in the absence of a recombinase said exogeneous nucleic acid is incorporated into the genome of said cell.
A method for the disimmortalization of an immortalized cell that contains exogeneous nucleic acid comprising recombinase target sites flanking an immortalization gene in the genome of said immortalized cell, said method comprising contacting said recombinase target sites with a recombinase which recognizes said recombinase target sites, wherein said recombinase is produced by expression of a recombinase nucleic acid within said cell.
A method for the disimmortalization of an immortalized cell comprising:
- a) incorporating exogeneous nucleic acid comprising:
- i) a first recombinase target site;
- ii) an immortalization gene;
- iii) a negative selection marker gene;
- and
- iv) a second recombinase target site;
- into the genome of a cell to produce an immortalized cell which contains an excisable immortalization gene in an orientation such that excision of the sequence between the recombinase target sites excises the immortalization gene and the negative selection marker;
- and
- b) contacting said recombinase target sites with a recombinase which recognizes said recombinase target sites such that said immortalization gene and said negative selection marker gene are excised, wherein said recombinase is produced by expression of a recombinase gene within said cell.
A method for the disimmortalization of an immortalized cell comprising:
- a) incorporating exogeneous nucleic acid comprising:
- i) a first recombinase target site;
- ii) an immortalization gene;
- iii) a selection marker gene;
- and
- iv) a second recombinase target site;
- into the genome of a cell to produce an immortalized cell which contains an excisable immortalization gene in an orientation such that excision of the sequence between the recombinase target sites excises the immortalization gene, resulting in the expression of the selection marker;
- and
- b) contacting said recombinase target sites with a recombinase which recognizes said recombinase target sites such that said immortalization gene is excised, wherein said recombinase is produced by expression of a recombinase gene within said cell.
A method for the disimmortalization of an immortalized cell comprising:
- a) incorporating exogeneous nucleic acid comprising:
- i) a first recombinase target site;
- ii) an immortalization gene;
- iii) a first selection marker gene;
- iv) a second recombinase target site;
- and
- v) a second selection marker gene;
- into the genome of a cell to produce an immortalized cell which contains an excisable immortalization gene in an orientation such that when the immortalization gene is expressed, the first selection marker gene is also expressed and the second selection marker gene is not expressed and when said immortalization gene is excised said second selection marker is expressed;
- and
- b) contacting said recombinase target sites with a recombinase which recognizes said recombinase target sites such that said immortalization gene is excised, wherein said recombinase is produced by expression of a recombinase gene within said cell.
Dependencies are inferred from the claim text.