Cells homozygous for disrupted target loci
AssigneesCell Genesys, Inc., President and Fellows of Harvard College
InventorsJonathan G. Seidman, Aya Jakobovits
Life of this patent
Estimating the term…
Abstract
Homozygotic cells are obtained by employing homologous recombination with a construct comprising a marker gene. The marker gene allows for selection without amplification and by employing elevated levels of the antibiotic to which the marker gene imparts resistance, gene conversion can occur, where in a diploid host, both copies of the target locus will be the same. In this manner, knock-outs of genes can be readily achieved without requiring two steps of homologous recombination.
Claims11
A method for making diploid mammalian cells homozygous for disrupted target loci, said method comprising:
- a. introducing into diploid mammalian cells a construct, said construct comprising a selectable marker gene flanked by regions of sequence homology to said loci, wherein said marker gene is a non-amplifiable gene which confers on said cells a preferential ability to grow in a selective medium;
- b. growing the cells obtained in step (a) in said selective medium at a first level of selective agent and analyzing viable cells to provide a population of cells which have undergone homologous recombination at one or both of the target loci;
- c. subjecting the population of cells obtained in step (b) to a level of selective agent greater that said first level, whereby cells which have undergone homologous recombination at both loci are selected;
- and
- d. isolating said cells obtained in step (c).
A method for making mammalian embryonic stem cells which are homozygous for disrupted target loci, said method comprising:
- a. introducing into mammalian embryonic stem cells a construct, said construct comprising a selectable marker gene flanked by regions of sequence homologous to said loci, wherein said marker gene is a non-amplifiable gene which confers on said cells a preferential ability to grow in a selective medium;
- b. growing the cells obtained in step (a) in said selective medium at a first level of selective agent and analyzing viable cells to provide a population of cells which have undergone homologous recombination at one or both of the target loci;
- c. subjecting the population of cells obtained in step (b) to a level of selective agent greater that said first level, whereby cells which have undergone homologous recombination at both loci are selected;
- and
- d. isolating said cells obtained in step (c).
Dependencies are inferred from the claim text.