NUCLEASE-MEDIATED TARGETING WITH LARGE TARGETING VECTORS Russian patent published in 2018 - IPC C12N15/85 C12N15/90 C07K14/715 

FIELD: biotechnology.

SUBSTANCE: invention relates to genetic engineering, in particular to a method for modification of a target genomic locus by homologous recombination in a mouse embryonic stem (ES) cell. To perform this method, zinc-finger-nuclease (ZFN) is first introduced into the mouse ES cell, which performs a single- or double-chain break in the target genomic locus or near it, and a large targeting vector (LTVEC). The target mouse ES cell containing the nucleotide insert in the target genomic locus is then selected. At that, integration leads to deletion of the endogenous nucleotide sequence at the target genomic locus and replacement of the nucleotide sequence. The said LTVEC comprises a nucleotide insert flanked by an overlying homologous arm and an underlying homologous arm, wherein the nucleotide insert is from about 5 kb up to about 30 kb and the total length of the overlying homologous arm and the underlying homologous arm is at least 10 kb.

EFFECT: invention allows to increase targeting efficiency by applying LTVEC to ZFN compared to application of LTVEC only.

36 cl, 1 dwg, 3 tbl, 1 ex