B6.129X1(Cg)-Shh^tm6Amc/J

Stock number: 008466
Research areas: Developmental Biology Research, Neurobiology Research, Research Tools

Description

These Shh::gfp mice harbor a targeted mutation of the Sonic hedgehog (Shh) locus that secretes, after normal Shh processing, a bioactive GFP-tagged Shh signaling ligand (N-Shh::GFPp). These Shh::gfp may be useful to directly visualize the function of Shh ligand in an in vivo context, such as during neural tube patterning.

Detailed description

While mice heterozygous for the Shh::gfp allele are viable, fertile, and indistinguishable from wild-type littermates, homozygotes are stillborn and show developmental defects consistent with reduced Sonic Hedgehog (Shh) signaling. The Shh::gfp mutation has GFP inserted into the endogenous Shh processing site (and adds a new processing site after the GFP). Thus normal Shh processing leads to secretion of the GFP-tagged Shh signaling ligand (N-Shh::GFPp) instead of wild-type Shh; with N-Shh::GFPp retaining both GFP and lipid modifications post-processing. Biochemical and cellular analysis indicates that Shh::GFP undergoes correct processing to produce active, bi-lipidated signaling peptides. Shh::GFP processing is, however, less efficient and results in reduced levels of Shh::GFP compared with wild-type Shh protein. These Shh::gfp mice produce bioactive, fluorescently labeled Shh from the endogenous Shh locus and may be useful to directly visualize the function of Shh ligand in an in vivo context, such as during neural tube patterning. In addition, these mice may also be useful in conjunction with other Sonic Hedgehog (Shh) mutant strains including Shh knockout mice (Stock No. 003318), Shh-floxed mice (Stock No. 004293), Shh-GFP/cre mice (Stock No. 005622), and Shh-creERT2 mice (Stock No. 005623).

Development

A targeting vector was designed to replace the endogenous intein cleavage-cholesterol attachment site (Shh processing site) in exon 3 of the targeted gene with a Green Fluorescent Protein (GFP) followed by a new intein cleavage-cholesterol attachment site (Shh processing site), as well as insert a frt-flanked PGK-Neo cassette in intron 2. The donating investigator reports that this construct was microinjected into 129X1/SvJ-derived AV3 embryonic stem (ES) cells, correctly targeted ES cells were injected into recipient blastocysts, and chimeric mice were bred to C57BL/6 inbred mice to generate Shh::gfp mice. These Shh::gfp mice were reported to have been bred with C57BL/6 mice for a small number generations prior to sending to The Jackson Laboratory Repository in 2008 (see SNP notes below). Upon arrival, these mice were bred to C57BL/6J inbred mice (Stock No. 000664) to establish the colony. Over the next two years, the colony was backcrossed to C57BL/6J and/or wildtype mice from the colony for several additional generations. By March 2011, the colony was found to be ~96% C57BL/6 genetic background by SNP analysis (see SNP notes below).

In 2009, a 27 SNP (single nucleotide polymorphism) panel analysis performed on the rederived living colony at The Jackson Laboratory Repository. This revealed 16 markers (across several different chromosomes) that were not C57BL/6 allele-type. These data suggest the strain was sent to The Jackson Laboratory Repository with multiple sources of genetic background contamination (more than 129 genetic background).

In 2011, a 32 SNP (single nucleotide polymorphism) panel analysis, with markers covering all 19 chromosomes and the X chromosome, was performed on the colony at The Jackson Laboratory Repository. This revealed one marker on chromosome 1 (~89.1 Mbp) that was not fixed for C57BL/6 allele-type (e.g.: still segregating for 129X1 allele-type).

Allele

Symbol: Shh^tm6Amc
Name: targeted mutation 6, Andrew P McMahon
MGI accession ID: MGI:3775846
Generation method: Targeted
Attributes: Reporter; Null/Knockout
Marker symbol: Shh
Marker name: sonic hedgehog
Chromosome: 5
Strain of origin: 129X1/SvJ
Expressed genes: GFP,Green Fluorescent Protein,
Site of expression: early neuronal precursors
Molecular note: A GFP cassette was inserted into exon 3 and an FRT site flanked neomycin resistance gene cassette was inserted between exon 2 and 3. The presence of the fusion protein and the absence of the endogenous product was confirmed by western blot analysis on E9.5 embryo extracts. This allele produces functional albeit inefficient protein product.