Methods for producing transgenic rodents having recombinant immunoglobulin loci
The invention provides methods for the production of transgenic animals comprising a recombinant Ig locus, as well as transgenic antibodies derived therefrom. The methods involve meganuclease cleavage-stimulated homologous recombination in mammalian embryos.
1. A method for integrating an exogenous nucleic acid sequence into an endogenous immunoglobulin (Ig) gene of a rodent embryo, comprising
introducing a meganuclease into said rodent embryo, wherein said meganuclease causes a chromosomal break in genomic DNA of said embryo at a cleavage site located within 1.5 kb of said Ig gene, and
introducing into said rodent embryo an exogenous nucleic acid sequence, wherein said exogenous nucleic acid sequence comprises an insertion sequence flanked by two homology arms such that said insertion sequence is integrated into said genomic DNA at said endogenous Ig gene by homologous recombination.
2. The method according to claim 1 , wherein said rodent embryo is a single cell fertilized oocyte.
3. The method according to claim 1 , wherein said insertion sequence is homologous to an exon of said endogenous Ig gene.
4. The method according to claim 3 , wherein said insertion sequence comprises a human Ig exon.
5. The method according to claim 3 , wherein said endogenous Ig gene comprises an Ig gene segment selected from the group consisting of a rodent variable (V) gene segment, a rodent diversity (D) gene segment, a rodent joining (J) gene segment, and a rodent constant (C) gene segment.
6. The method according to claim 1 , wherein said insertion sequence comprises a nucleotide sequence selected from the group consisting of a human variable (V) Ig segment, a human diversity (D) Ig segment, a human joining (J) Ig segment, a human constant (C) Ig segment, and portions thereof.
7. The method according to claim 6 , wherein said insertion sequence comprises a human V gene segment.
8. The method according to claim 1 , wherein said insertion sequence introduces a deletion into said endogenous Ig gene.
9. The method according to claim 1 , wherein said insertion sequence introduces a stop codon into said endogenous Ig gene.
10. The method according to claim 1 , wherein said insertion sequence disrupts said endogenous Ig gene.