129;B6-Otx2^tm3Tlam/J

Stock number: 034472
Product name: Otx2^OG
Strain synonyms: Otx2-GFP, Otx2^Otx2-GFP
Research areas: Neurobiology Research, Research Tools

Description

Otx2^OG knock-in mice (also called Otx2-GFP) have the endogenous orthodenticle homeobox 2 (Otx2) promoter/enhancer sequences directing expression of green fluorescent protein in Otx2-expressing cells and may be useful for studying neural development and medulloblastoma.

Detailed description

The transcription factor orthodenticle homeobox 2 (Otx2) plays a role in the development and organization of the central nervous system (CNS) and visual system, midbrain dopaminergic and serotonergic cell proliferation, and is overexpressed in human medulloblastomas (Di et al. 2005; Vernay et al. 2005). The Otx2^OG allele carries a Green Fluorescent Protein (GFP) inserted into the 3' UTR and is reported to express the Otx2-GFP fusion protein at a level comparable to the endogenous Otx2 protein. However, the Otx2-GFP protein may have functional differences as described in Fossat et al. 2007.

GFP fluorescence is detectable by direct visualization (without the need of antibody staining) and is dynamically expressed in the CNS throughout gestation and adulthood in a manner consistent with endogenous Otx2 expression. While GFP expression is comparable between hetertozygous and homozygous Otx2^OG animals, the donating investigator reports that homozygotes are observed in low ratios (~4%), due to embryonically lethal neurological abnormalities not observed in heterozygous animals, and surviving homozygotes may develop normally but often show mild diencephalic and mesencephalic maturation defects.

Development

The Otx2^OG allele was created in the laboratory of Dr. Thomas Lamonerie at Ecole Normale Supérieure de Lyon. A plasmid containing the full 3' UTR and 1.5kb downstream sequence of the orthodenticle homeobox 2 (Otx2) gene, a Green Fluorescent Protein variant (GFP.RN3; Zernicka-Goetz et al. 1996), and an frt-flanked PGK-neo cassette was electroporated into 129/Sv-derived ENS ES cells. ES cells were transiently transfected with a FLPe-recombinase expression vector to remove the neo selection cassette. Otx2^OG positive ES cells were then injected into recipient blastocysts. Chimeric animals were then bred to C57BL/6N mice for germline transmission. The Otx2^OG strain was subsequently backcrossed to 129S2/SvPas for approximately 30 generations and maintained on the 129S2/SvPas background prior to being donated to The Jackson Laboratory in 2019. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize 129S1/SvImJ oocytes (Stock No. 002448).

Of note, the donating investigator reports that the Y chromosome may not have been fixed to the 129S2/SvPas background during backcrossing.

In 2021, a 48 SNP (single nucleotide polymorphism) panel analysis, with 43 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the rederived living colony at The Jackson Laboratory Repository. Seven of the 43 markers throughout the genome were segregating with B6, suggesting an incomplete backcross.

Allele

Symbol: Otx2^tm3Tlam
Name: targeted mutation 3, Thomas Lamonerie
MGI accession ID: MGI:3774322
Generation method: Targeted
Attributes: Reporter; Hypomorph
Synonyms: Otx2^OG; Otx2^Otx2-GFP
Marker symbol: Otx2
Marker name: orthodenticle homeobox 2
Marker synonyms: CPHD6; E130306E05Rik; MCOPS5
Chromosome: 14
Strain of origin: 129/Sv
Molecular note: A cassette containing a GFP and frt-flanked neo cassette was inserted after the last coding region. The neo cassette was removed by flp-mediated recombination. Expression of the GFP fusion was confirmed by western blot analysis on E9.5 tail and head extracts. Authors state that this allele is hypomorphic.