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US 12,325,865 B2Granted
Methods for increasing efficiency of gene editing in cells
AssigneePresident and Fellows of Harvard College
InventorsGeorge M. Church, Luhan Yang
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Abstract
The present disclosure provides methods of improving nuclease-mediated gene targeting frequencies in cells using PFTα or bFGF.
Claims8
A method of making a population of PERV-free porcine cells, the method comprising:
- (a) genetically modifying a primary porcine cell which comprises porcine endogenous retrovirus (PERV) genes by providing a vector comprising a nucleic acid sequence which encodes a nuclease and three sgRNAs to the primary porcine cell, wherein the nuclease is expressed and cuts the PERV genes, thereby producing a genetically modified, PERV-free, primary porcine cell,
- (b) providing an exogenous pifithrin α (PFTα) and a basic fibroblast growth factor (bFGF) to the primary porcine cell concomitant with step (a),
- (c) contacting the primary porcine cell with a large SV40 T antigen to increase targeting efficiency,
- (d) isolating the genetically modified, PERV-free primary porcine cell, and expanding the genetically modified, PERV-free porcine cell to a population of PERV-free porcine cells, wherein the population of PERV-free porcine cells exhibits a lack of PERV transmission as evidenced by co-culturing a cell with the population of PERV-free porcine cells.
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Dependencies are inferred from the claim text.