B6;DBA-Celf4^{em1(icre/ERT2)Zyliu}/J

Stock number: 034391
Product name: Celf4-iCreER
Research areas: Neurobiology Research, Research Tools

Description

Celf4-iCreER mice have a CRISPR/Cas9-generated Celf4-3xHA-P2A-iCreER-T2A-EGFP cassette inserted immediately before the stop codon of the CUGBP, Elav-like family member 4 (Celf4) gene. These mice are useful for visualizing inner ear cochlear spiral ganglion neurons (SGNs) and as a tool for studying SGN developmental and regenerative studies.

Detailed description

Celf4-iCreER mice have a CRISPR/Cas9-generated Celf4-3xHA-P2A-iCreER-T2A-EGFP cassette inserted immediately before the stop codon of the CUGBP, Elav-like family member 4 (Celf4) gene. Celf4 encodes an RNA-binding protein involved in mRNA stability, protein translation and seizure occurrence. These mice allow for visualizing the Celf4 protein with an HA antibody and Cefl4 mRNA expression can be detected via EGFP. In these mice, Celf4 is detected by EGFP immunohistochemistry in inner ear cochlear spiral ganglion neuron (SGN) areas in all three cochlea turns and in vestibular neuron (VN) regions at E15.5 and is further increased at P1.

Restricted to the cytoplasm, cre-ERT2 can only gain access to the nuclear compartment after exposure to tamoxifen. When Celf4-iCreER mice are bred with mice containing loxP-flanked sequences, tamoxifen-inducible Cre-mediated recombination will result in deletion of the floxed sequences in the Cre-expressing cells of the offspring. With this line Cre recombination occurs specifically in SGN regions at different ages such as P1, P8 and P30. Some expression is also seen in neuroblasts (SGN progenitors) beginning at E10.5, and is also expressed in other inner ear cells such as mesenchymal cells. The donating lab did not check endogenous Celf4 expression in these mice. Homozygous mice are viable and fertile.

Development

Plasmids encoding a sgRNA (5’-TACGGGCGATTGGCGTCTTT-3’), were designed to introduce a 3 x hemagglutinin tag (HA) followed by a viral P2A oligopeptide (P2A) (that mediates ribosomal skipping) fused to a codon improved Cre recombinase and estrogen receptor 1 (human) fusion gene (cre/ERT2). This cassette, followed by a viral T2A oligopeptide (T2A) (that mediates ribosomal skipping) fused to an enhanced green fluorescent protein sequence (EGFP), was inserted immediately before the stop codon of the CUGBP, Elav-like family member 4 (Celf4) gene, and the cas9 nuclease were introduced into the cytoplasm of C57BL/6/DBA-derived fertilized eggs with well recognized pronuclei. Correctly targeted embryos were transferred to pseudopregnant females and pups were identified by PCR. These Celf4-iCreER mice were further bred to C57BL/6NCrl mice to develop the colony. Southern-blot analysis showed that there were no random insertions of donor DNA in the genome. Upon arrival at The Jackson Laboratory, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6NJ (Stock No. 005304) oocytes.

In 2020, 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. Three of the 5 markers that determine C57BL/6J from C57BL/6N, on Chromosomes 1, 8, and 19, were found to be segregating. These include the rd8 recessive mutation associated with retinal degeneration on Chromosome 1 within the Crb1 locus. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed genetic background.

Allele

Symbol: Celf4^{em1(icre/ERT2)Zyliu}
Name: endonuclease-mediated mutation 1, Zhiyong Liu
MGI accession ID: MGI:6368146
Generation method: Endonuclease-mediated
Attributes: Recombinase-expressing; Reporter; Inducible
Synonyms: Celf4-3xHA-P2A-iCreER-T2A-EGFP
Marker symbol: Celf4
Marker name: CUGBP, Elav-like family member 4
Marker synonyms: A230070D14Rik; Brul4; BRUNOL4; BRUNOL-4; C130060B05Rik; CELF-4
Chromosome: 18
Strain of origin: C57BL/6
Site of expression: Inner ear cochlear spiral ganglion neurons (SGNs).
Molecular note: Using CRISPR/Cas9 with single guide RNAs and a targeting vector, the following elements were inserted immediately upstream of the stop codon: a 3x hemagglutinin tag (HA) followed by sequence for self-cleaving oligopeptide P2A fused to a codon improved Cre recombinase and estrogen receptor 1 (human) fusion gene, and sequence for self-cleaving oligopeptide T2A fused to an enhanced green fluorescent protein sequence (EGFP) gene. This allele thus expresses the endogenous peptide, Cre and EGFP under the control of the endogenous promoter.