Format

Send to

Choose Destination
Genesis. 2014 Jan;52(1):68-77. doi: 10.1002/dvg.22727. Epub 2013 Dec 3.

Generation of precise point mutation mice by footprintless genome modification.

Author information

1
Institute for Genetic Medicine, Hokkaido University, Sapporo, Hokkaido, Japan.

Abstract

Point mutation mice are a key tool in the study of biological functions of genomic DNA sequences and the creation of human disease models. These mice are produced by homologous recombination combined with site-specific recombinase, which allows removal of drug selection cassettes. However, the methods currently available leave ectopic sequences in the "inactive" intron region of the targeted genome in addition to the desired mutation. Since recent research suggests that the intron region may actually have some functionality, these sequences could potentially interfere with neighboring gene expression and, as a result, affect the mouse phenotype. To completely avoid this issue, we used the PiggyBac transposon to remove selection cassettes and achieve precise genome modification without leaving behind a footprint. This PiggyBac system allowed us to successfully generate mice carrying an artificially introduced W(v) point mutation in the Kit gene, and these mice were confirmed to have phenotypes identical to spontaneous W(v) mutation mice. Generation of W(v) -mutation corrected mice was also possible, and phenotypes were completely restored. Our footprintless genome modification technology can generate precise point mutation mice with only the desired mutation, and they reflect an accurate phenotype that makes these mice a reliable and "worry-free" research resource.

KEYWORDS:

BAC recombineering; PiggyBac transposon; Wv mutation

PMID:
24265262
DOI:
10.1002/dvg.22727
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for Wiley
Loading ...
Support Center