Methods and compositions for transforming cells
First Claim
1. A method of causing site-specific recombination in a mammalian cell comprising contacting a recombinase with (a) an acceptor vector which is integrated into the genome of said mammalian cell, said acceptor vector comprising two lox sequences, L1 and L2, which can not recombine with one another, and (b) a donor vector which is not integrated into the genome of said mammalian cell, said donor vector being present in an amount in excess of said integrated acceptor vector, wherein said donor vector comprises a selected DNA flanked by the same L1 and L2 sequences contained in the acceptor vector, or sequences which can recombine with these L1 and L2 sequences, thereby causing transfer of the selected DNA from the donor vector into the acceptor vector by recombination at the L1 and L2 sequences.
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Abstract
Methods and compositions for transforming cells, resulting in efficient and stable site-specific integration of transgenes, are disclosed. Transformation is achieved by introducing into a cell an acceptor vector, preferably a retroviral vector, which integrates into the genome of the cell. The acceptor vector comprises two incompatible lox sequences, L1 and L2. A donor vector is then introduced into the cell comprising a transgene flanked by the same L1 and L2 sequences. Stable gene transfer is initiated by contacting the lox L1 and L2 sequences with Cre recombinase.
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Citations
24 Claims
- 1. A method of causing site-specific recombination in a mammalian cell comprising contacting a recombinase with (a) an acceptor vector which is integrated into the genome of said mammalian cell, said acceptor vector comprising two lox sequences, L1 and L2, which can not recombine with one another, and (b) a donor vector which is not integrated into the genome of said mammalian cell, said donor vector being present in an amount in excess of said integrated acceptor vector, wherein said donor vector comprises a selected DNA flanked by the same L1 and L2 sequences contained in the acceptor vector, or sequences which can recombine with these L1 and L2 sequences, thereby causing transfer of the selected DNA from the donor vector into the acceptor vector by recombination at the L1 and L2 sequences.
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6. A method of transforming a cell with a selected DNA comprising, in any order, the steps of:
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(a) introducing into the cell an acceptor vector which integrates into the genome of the cell, the acceptor vector comprising two incompatible lox sequences, L1 and L2, which can not recombine with one another; (b) introducing into the cell a donor vector comprising the selected DNA flanked by the same L1 and L2 sequences contained in the acceptor vector or sequences which can recombine with these L1 and L2 sequences, wherein said donor vector is introduced in an amount in excess of said acceptor vector; and (c) contacting L1 and L2 with Cre, thereby causing transfer of the selected DNA from the donor vector into the acceptor vector. - View Dependent Claims (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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21. A Cre/lox mediated gene transfer system comprising:
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(a) an acceptor vector which can integrate into the genome of a host cell, the acceptor vector comprising two lox sequences, L1 and L2, which can not recombine with one another; (b) a donor vector comprising a transgene flanked by the same L1 and L2 sequences contained in the acceptor vector, or sequences which can recombine with these L1 and L2 sequences; and (c) a transient expression vector encoding a recombinase which can catalyze recombination at the L1 and L2 sequences. - View Dependent Claims (22, 23, 24)
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Specification