Method of deleting an IgM gene in an isolated rat cell

摘要

Disclosed herein are methods and compositions for genome editing of one or more loci in a rat, using fusion proteins comprising a zinc-finger protein and a cleavage domain or cleavage half-domain. Polynucleotides encoding said fusion proteins are also provided, as are cells comprising said polynucleotides and fusion proteins.

主权项

1. A method for making a deletion in an endogenous IgM gene, the method comprising:
introducing mRNA into an isolated rat cell, wherein the mRNA comprises (i) mRNAs encoding a first zinc finger nuclease (ZFN) that binds to a first target site in an endogenous rat IgM gene wherein the first ZFN comprises a cleavage domain and a zinc finger protein, wherein the first zinc finger protein-comprises the following recognition helices in the following order:
DRSHLTR (SEQ ID NO:41);RSDALTQ (SEQ ID NO:40);DRSDLSR (SEQ ID NO:28);RSDALAR (SEQ ID NO:39);RSDSLSA (SEQ ID NO:38); andTSSNRKT (SEQ ID NO:37), and (ii) mRNA encoding a second ZFN that binds to a second target site in the endogenous rat IgM gene, wherein the second ZFN comprises a cleavage domain and a zinc finger protein comprising the following recognition helices in the following order:
NKVGLIE (SEQ ID NO:46);TSSDLSR (SEQ ID NO:45);RSDHLSR (SEQ ID NO:44);RSDNLSE (SEQ ID NO:43); andQNAHRKT(SEQ ID NO:42), such that the first and second ZFNs are expressed, dimerize and cleave the endogenous rat IgM gene and non-homologous end joining (NHEJ) occurs causing the cell to have a deletion in the endogenous rat IgM gene.