STOCK Pax6^{em8(PAX6*R240X)Ems}/1Mmjax

Stock number: 039574
Product name: B6.Pax6^R240X [~N2 on B6]
Research areas: Cell Biology Research, Developmental Biology Research, Sensorineural Research

Description

B6.Pax6^R240X [~N2 on B6] knock-in (KI) mice have a humanized exon 9 sequence, containing the causative human mutation (c.718C>T (p.Arg240Ter)), replacing mouse exon 9 of the Pax6 gene. These KI mice, on a C57BL/6 background, also have a FLAG-tagged ATG in exon 4. These KI mice, on a C57BL/6J background, may be useful for studying the role of Pax6 in eye and brain development.

Of note, the donating laboratory has made this knock-in allele available on a 129S background (Stock No. 071761), showing a less severe phenotype as compared to this B6-Pax6^R240X KI mouse line.

Detailed description

Pax6, a member of the Pax family, encodes a homeobox and paired domain-containing protein that functions as a transcription factor and is expressed in pancreatic endocrine cells as well as the eye, nose and central nervous system. These B6.Pax6^R240X [~N2 on B6] knock-in (KI) mice have a humanized exon 9 sequence, which also contains the causative human mutation (c.718C>T (p.Arg240Ter)), replacing mouse exon 9 of the Pax6 gene as well as a FLAG-tagged ATG in exon 4. Heterozygous mice are viable and fertile but do demonstrate severe microphthalmia, central corneal clouding, and corneal vascularization. Homozygous mice are perinatal lethal. These mice may be useful for studying the role of Pax6 in eye and brain development.

Of note, the donating laboratory has made this knock-in allele available on a 129S background (Stock No. 071761), showing a less severe phenotype as compared to this B6-Pax6^R240X KI mouse line.

In 2024, 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 males sent to The Jackson Laboratory Repository. More than 10 markers were segregating for C57BL/6 and 129S; these data are consistent with N2 mice being sent to The Jackson Laboratory Repository (and used for the frozen sperm in 2024) as reported by the donating investigator.

Development

The B6.Pax6^R240X [~N2 on B6] knock-in (KI) allele has a point mutation homologous to the human variant c.718 c>t (R240X) inserted into exon 9 of the paired box 6 (Pax6) gene on chromosome 2. An ESC line was made from a homozygous B6129F1-Pax6^em3Ems mouse line (MGI:6423490; the Pax6^em3Ems allele was derived via retargeting of B6.Cg-Pax6^Sey/EmsMmmh to insert a triple FLAG tag at the ATG translation initiation sequence in exon 4). A megamer and two guide RNAs were electroporated into the homozygous B6129F1-Pax6^em3Ems ESCs to replace mouse exon 9 with human exon 9 sequence that contains the human mutation (c.718C>T (p.Arg240Ter)). Pax6 transcript Pax6-204 (Ensembl ID ENSMUST00000111083.10) was used as reference for the exon number and the guide sequences. Correctly targeted ES cells were injected into C57BL/6J-albino blastocysts. The chimeric mice produced were identified by coat color, PCR and sequencing. The donating lab reports the resulting mice were bred to C57BL/6J mice for at least 2 generations (to reduce off-target events) prior to sending males to The Jackson Laboratory in 2024 (see SNP results). To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes.

In 2024, 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 males sent to The Jackson Laboratory Repository. More than 10 markers were segregating for C57BL/6 and 129S; these data are consistent with N2 mice being sent to The Jackson Laboratory Repository (and used for the frozen sperm in 2024) as reported by the donating investigator.

Allele

Symbol: Pax6^{em8(PAX6*R240X)Ems}
Name: endonuclease-mediated mutation 8, Elizabeth M Simpson
MGI accession ID: MGI:7579265
Generation method: Endonuclease-mediated
Attributes: Inserted expressed sequence; Humanized sequence; Epitope tag
Synonyms: Pax6^{em8(FLAG/PAX6*R240X)Ems}; Pax6^FHe9-
Marker symbol: Pax6
Marker name: paired box 6
Marker synonyms: 1500038E17Rik; AEY11; AN; AN1; AN2; ASGD5; D11S812E; Dey; Dickie's small eye; FVH1; Gsfaey11; MGDA; Pax-6; WAGR
Chromosome: 2
Strain of origin: B6.Cg-Pax6^Sey/EmsMmmh
Molecular note: CRISPR-targeting of zygotes carrying Pax6^em3Ems allele harbors a triple FLAG tag at the ATG translation initiation sequence in exon 4 was used to replace mouse exon 9 with human exon 9 sequence that contains the human mutation (c.718C>T (p.Arg240Ter)).