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Efficient removal of loxP-flanked DNA sequences in a gene-targeted locus by transient expression of Cre recombinase in fertilized eggs.
Mol Reprod Dev. 1997 Feb; 46(2):109-13.MR

Abstract

The bacteriophage P1 Cre/loxP site-specific recombination system is a useful tool for engineering chromosomal changes in animal cells. 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. In the present study, we examined the efficacy of this method to remove loxP-flanked DNA sequences in a gene-targeted locus in fertilized eggs. We replaced a part of the T-cell receptor gamma (TCR V gamma) locus with homologous sequences containing a loxP-flanked neogene in mouse embryonic stem (ES) cells by gene-targeting technique. The resulting ES cell clones containing the mutant allele (V gamma LNL) were used to generate chimeric mice by blastocyst injection. Eight male chimeras were bred with superovulated wild-type female mice. One hundred and seventy-six fertilized eggs were collected, and subjected to pronuclear injection of the Cre expression plasmid, pCAGGS-Cre, of a covalently closed circular form. Three out of 11 pups inherited the targeted V gamma locus. The inherited targeted allele of these 3 mice was shown to have undergone Cre-mediated recombination, resulting in a deletion of the loxP-flanked sequences (V gamma delta) as shown by Southern blot analysis of DNA from tail biopsies. All 3 founder mutant mice were capable of transmitting the V gamma delta locus to their offspring. The other 8 pups carried only wild-type alleles. There were no pups carrying the unrecombined V gamma LNL 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 V gamma LNL locus was 9.6%. These results indicated that our system based on transient expression of the Cre recombinase gene directly introduced into fertilized eggs by pronuclear injection provides a fast and efficient method for generating mutant mice with desired deletions or translocations in target genes.

Authors+Show Affiliations

Department of Disease-Related Gene Regulation Research (Sandoz), Tokyo, Japan.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

9021742

Citation

Sunaga, S, et al. "Efficient Removal of loxP-flanked DNA Sequences in a Gene-targeted Locus By Transient Expression of Cre Recombinase in Fertilized Eggs." Molecular Reproduction and Development, vol. 46, no. 2, 1997, pp. 109-13.
Sunaga S, Maki K, Komagata Y, et al. Efficient removal of loxP-flanked DNA sequences in a gene-targeted locus by transient expression of Cre recombinase in fertilized eggs. Mol Reprod Dev. 1997;46(2):109-13.
Sunaga, S., Maki, K., Komagata, Y., Ikuta, K., & Miyazaki, J. I. (1997). Efficient removal of loxP-flanked DNA sequences in a gene-targeted locus by transient expression of Cre recombinase in fertilized eggs. Molecular Reproduction and Development, 46(2), 109-13.
Sunaga S, et al. Efficient Removal of loxP-flanked DNA Sequences in a Gene-targeted Locus By Transient Expression of Cre Recombinase in Fertilized Eggs. Mol Reprod Dev. 1997;46(2):109-13. PubMed PMID: 9021742.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Efficient removal of loxP-flanked DNA sequences in a gene-targeted locus by transient expression of Cre recombinase in fertilized eggs. AU - Sunaga,S, AU - Maki,K, AU - Komagata,Y, AU - Ikuta,K, AU - Miyazaki,J I, PY - 1997/2/1/pubmed PY - 2000/6/20/medline PY - 1997/2/1/entrez SP - 109 EP - 13 JF - Molecular reproduction and development JO - Mol Reprod Dev VL - 46 IS - 2 N2 - The bacteriophage P1 Cre/loxP site-specific recombination system is a useful tool for engineering chromosomal changes in animal cells. 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. In the present study, we examined the efficacy of this method to remove loxP-flanked DNA sequences in a gene-targeted locus in fertilized eggs. We replaced a part of the T-cell receptor gamma (TCR V gamma) locus with homologous sequences containing a loxP-flanked neogene in mouse embryonic stem (ES) cells by gene-targeting technique. The resulting ES cell clones containing the mutant allele (V gamma LNL) were used to generate chimeric mice by blastocyst injection. Eight male chimeras were bred with superovulated wild-type female mice. One hundred and seventy-six fertilized eggs were collected, and subjected to pronuclear injection of the Cre expression plasmid, pCAGGS-Cre, of a covalently closed circular form. Three out of 11 pups inherited the targeted V gamma locus. The inherited targeted allele of these 3 mice was shown to have undergone Cre-mediated recombination, resulting in a deletion of the loxP-flanked sequences (V gamma delta) as shown by Southern blot analysis of DNA from tail biopsies. All 3 founder mutant mice were capable of transmitting the V gamma delta locus to their offspring. The other 8 pups carried only wild-type alleles. There were no pups carrying the unrecombined V gamma LNL 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 V gamma LNL locus was 9.6%. These results indicated that our system based on transient expression of the Cre recombinase gene directly introduced into fertilized eggs by pronuclear injection provides a fast and efficient method for generating mutant mice with desired deletions or translocations in target genes. SN - 1040-452X UR - https://www.unboundmedicine.com/medline/citation/9021742/Efficient_removal_of_loxP_flanked_DNA_sequences_in_a_gene_targeted_locus_by_transient_expression_of_Cre_recombinase_in_fertilized_eggs_ L2 - https://doi.org/10.1002/(SICI)1098-2795(199702)46:2<109::AID-MRD1>3.0.CO;2-U DB - PRIME DP - Unbound Medicine ER -