Efficient removal of loxP-flanked DNA sequences in a gene-targeted locus by transient expression of Cre recombinase in fertilized eggs.

摘要

Transient expression of the Cre recombinase gene directly introduced into fertilized eggs by pronuclear injection has been reported to provide an efficient method of transgene modulation in fertilized eggs. The object of this study was to determinethe efficacy of this method in removing loxP-flanked DNA sequences in a gene-targeted locus in fertilized mouse eggs. Using gene targeting, a part of the T-cell receptor gamma (TCR Vgamma) locus in mouse embryonic stem (ES) cells was replaced with homologous sequences containing a loxP-flanked neo gene. The resulting ES cell clones containing the mutant allele (VgammaLNL) were used to generate chimaeric mice by blastocyst injection. Eight male chimeras were mated with superovulated wild-type female mice. 176 fertilized eggs were collected and subjected to pronuclear injection of the Cre expression plasmid, pCAGGS-Cre, of a covalently closed circular form. Three of 11 pups inherited the targeted Vgamma locus. The inherited targeted allele of these 3 mice was shown to have undergone Cre-mediated recombination, resulting in the deletion of the loxP-flanked sequence, Vgammadelta, as shown by Southern blot analysis of DNA from tail biopsies. All 3 founder mutant mice were capable of transmitting the Vgammadelta locus to their offspring. The other 8 pups carried only wild-type alleles. There were no pups carrying the unrecombined VgammaLNL locus. Thus, the frequency of Cre-mediated recombination was 100% (3/3) with this method. In contrast, when closed circular pCAGGS-Cre plasmid was introduced into ES cells by electroporation, the recombination frequency of the VgammaLNL locus was 9.6%. The method described is fast and efficient for generating mutant mice with desired deletions or translocations in target genes.