Method of homologous recombination followed by in vivo selection

Details Method of homologous recombination followed by in vivo selection Method of homologous recombination followed by in vivo selection

1997-10-20

1999-07-20

Fredman, Jeffrey

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

435 6, 435 911, 435 912, 435 914, 4351723, 536 221, 536 231, 536 243, C12P 2100, C12P 1934, C12Q 168, C07H 2104

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059255447

ABSTRACT:
A method of amplifying in vivo a DNA sequence B present in a genome of a parent cell, comprising (a) integrating in a genome of said cell a DNA construct comprising the structure C-M-A-D, in which both A and C denote a DNA sequence which is homologous with a genomic DNA fragment either flanking or overlapping the DNA sequence B, the sequence C being located in the opposite end the sequence B as compared to A, D denotes a DNA sequence which is homologous with a genomic DNA fragment located distal for C as compared to B, and M denotes a DNA sequence encoding a selection marker, (b) selecting for cells in which the DNA sequence M has been integrated in the genome, which cells comprise, in any orientation, the structure A-B-C-M-A-D and (c) propagating the cells selected in step (b) under increasing selection pressure to obtain a cell which has obtained an increased number of genomically integrated copies of the DNA sequences B and M.

REFERENCES:
patent: 5733753 (1998-03-01), Jorgensen
Raibaud et al., Gene, vol. 29, pp. 231-241 (1984).
Kallio et al., Appl Microbiol Biotechnol, vol. 27, pp. 64-71 (1987).
Janniere et al., Gene, vol. 40, pp. 47-55 (1985).
Hanson et al, "Analysis of biological selections for high-efficiency gene targeting", Mol. Cell. Biol. 15(1):45-51, Jan. 1995.

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