B6.Cg-En1^{tm1.1(dre)Pjen}/J

Stock number: 033953
Product name: En1^Dre KI
Strain synonyms: B6.Cg-En1^{tm1.1(dreo)Pjen}/J
Research areas: Research Tools

Description

En1^Dre knock-in mice express dreo (mammalian codon-optimized bacteriophage D6 site-specific) recombinase from the endogenous En1 locus targeting Dre recombinase activity during neural development in En1 expressing cells.

Detailed description

En1^Dre knock-in mice express dreo (mammalian codon-optimized bacteriophage D6 site-specific) recombinase from the endogenous En1 locus, mimicking the endogenous En1 gene expression pattern. Insertion of dreo at the 3' end of the gene maintains expression of En1 and dreo. The targeted En1 gene encodes a homeodomain protein important in embryogenesis and neural development. Mutations in this gene have been associated with Sudden Infant Death Syndrome and Eccrine Acrospiroma. Mice that are homozygous for the targeted mutation are viable and fertile. Dre mRNA was observed in midbrain/hindbrain junction of E9.5 En1^Dre embryos and in midbrain/hindbrain junction, spinal cord, somites, and limb buds of E11.0 embryos. When En1^Dre mice were bred to RC::RLTG or RC::RG reporter mice, the double mutant mice exhibited reporter activity in the midbrain/hindbrain, first pharyngeal arch, somites, and limb buds at E10.5; and in midbrain and anterior hindbrain, including cerebellum, of adults.

Development

A targeting vector containing 2A peptide sequence, cDNA encoding dreo (mammalian codon-optimized bacteriophage D6 site-specific) recombinase, SV40 poly(A) signal, and an attB/attP-flanked PGK/Em7-Neo cassette was used to disrupt exon 2 including the stop codon and 41 bp of the 3’ UTR while retaining correct coding sequence of the endogenous En1 gene. The construct was electroporated into (129S6/SvEvTac x C57BL/6NCrl)F1 derived G4 embryonic stem (ES) cells which were transiently transfected with the PhiC31 recombinase plasmid, pPGKPhiC31obpa, to remove the selection cassette. ES cells that had successfully undergone PhiC31-mediated recombination and no longer retained the selection cassette were injected into B6(Cg)-Tyr^c-2J/J blastocysts. The resulting male chimeric animals were bred to C57BL/6J females to achieve germline transmission and mice were subsequently bred to C57BL/6J mice for 19 generations. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

Allele

Symbol: En1^{tm1.1(dre)Pjen}
Name: targeted mutation 1.1, Patricia Jensen
MGI accession ID: MGI:5776371
Generation method: Targeted
Attributes: Recombinase-expressing
Synonyms: En1^Dre; En1^{tm1.1(dreo)Pjen}
Marker symbol: En1
Marker name: engrailed 1
Marker synonyms: En-1; ENDOVESLB; engrailed-1; Mo-en.1
Chromosome: 1
Strain of origin: (129S6/SvEvTac x C57BL/6NCrl)F1
Site of expression: Brain
Molecular note: The mammalian codon-optimized bacteriophage D6 site-specific recombinase gene (dreo) with a 5' a viral 2A oligopeptide (T2A) that mediates ribosomal skipping replaced the stop codon and 41 bp of the 3' UTR in exon 2 without altering the coding sequence. phiC31-mediated recombination removed the neomycin resistance cassette inserted downstream of the recombinase gene.