Methods and compositions for homologous recombination in human cells
AssigneeThe General Hospital Corporation
InventorsNiels Geijsen, Christa Buecker
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Abstract
The methods and compositions described herein are based, in part, on the discovery of a stem cell state in human cells that resembles the morphology observed in murine-derived stem cells. Induction of such a state in human stem cells permits an increase in the efficiency of homologous recombination. Thus, the methods and compositions described herein relate to cells and methods for increasing the efficiency of homologous recombination in human stem cells.
Claims24
A composition comprising:
- (a) a human pluripotent stem cell in admixture with a GSK3β inhibitor and a protein kinase A (PKA) inhibitor, and
- (b) a homologous recombination cassette.
A stem cell composition comprising:
- (a) a human pluripotent stem cell clone isolated from a population of pluripotent stem cells treated with a GSK3β inhibitor and a protein kinase A (PKA) inhibitor having the following characteristics:
- (i) survives propagation from a single cell,
- (ii) an efficiency of homologous recombination at least 1-fold higher than the efficiency of homologous recombination in a corresponding human pluripotent stem cell not treated with the GSK3β inhibitor,
- (iii) a doubling time at least 2 hours faster than the growth rate of a corresponding human pluripotent stem cell not treated with the GSK3β inhibitor,
- (iv) survives passaging as a single cell using trypsin digest, and
- (b) a homologous recombination cassette.
A kit for enhancing the efficiency of homologous recombination in a human stem cell, the kit comprising:
- (a) a GSK3β inhibitor,
- (b) a protein kinase A (PKA) inhibitor,
- (c) a homologous recombination cassette, and
- (d) instructions and packaging thereof.
Dependencies are inferred from the claim text.