US 11,299,754 B2 · Gene targeting method

Abstract

Provided are a novel two-step gene targeting method and a nucleotide construct for gene targeting. The method can improve the gene targeting efficiency and accurately identify a target gene knock-out mutant. The method of the present invention comprises: firstly, efficiently replacing a target gene in a genome with a targeting box by homologous recombination, the targeting box consisting of a target gene activity variant, a marker gene and site-specific recombination sites; and secondly, resecting the targeting box by recombinase, leaving a site-specific recombination site on the target gene to generate a target gene knock-out mutant, and removing a recombinase expression vector from the knock-out mutant by using a counter selection marker in the recombinase expression vector.

Claims17

Dependent claims

A plasmid construct for altering a genome by gene targeting, comprising the following structure:

5′-A1-B1-C1-C2-X-B2-A2-3′;
5′-A1-B1-X-C1-C2-B2-A2-3′;
5′-A1-B1-C1-X-B2-C2-A2-3′;
or
5′-A1-B1-X-C1-B2-C2-A2-3′
wherein
B1 and B2 are site-specific recombination sites;
C1 and C2 are marker genes and cannot be absent at the same time;
X is an active variant of a target gene in the genome;
the active variant possesses a functional activity of the target gene;
A1 is a sequence that is homologous to a region upstream of the target gene in the genome;
and
A2 is a sequence that is homologous to a region downstream of the target gene in the genome.

10 more elements

Dependencies are inferred from the claim text.

Gene targeting method

Life of this patent

Gene targeting method

Life of this patent

Abstract

Claims17