STOCK Kpnb1^tm1.1Mfz/J

Stock number: 029616
Product name: Importin beta1 3'UTR flox
Research areas: Neurobiology Research, Research Tools

Description

Importin β1 3’ UTR loxP mice possess loxP sites flanking a 1.1 kb region downstream of the stop codon of the karyopherin (importin) beta 1 (Kpnb1) gene. This strain may be useful for studying the regulation of size and growth of neurons by subcellular mRNA localization.

Detailed description

Importin β1 3’ UTR loxP mice possess loxP sites flanking a 1.1 kb region downstream of the stop codon of the karyopherin (importin) beta 1 (Kpnb1) gene. This insertion site flanks a distal segment of the UTR with loxP sites to allow for cre-mediated deletion. Localized upregulation of Importin β1 in injured axons coordinates a retrograde injury-signaling complex to be transported to the neuronal cell body. The mRNA transcribed from the long 3’UTR directs axonal localization of Importin β1.

When these mutant mice are bred to mice that express Cre recombinase, resulting Importin β1 3’ UTR ^-/- offspring express the short 3’ UTR transcript causing subcellular loss of Importin β1 mRNA and protein in injured axons, without affecting cell body levels or nuclear functions in sensory neurons. This attenuates cell body transcriptional responses to nerve injury and delays functional recovery in vivo. However, these mice have significantly higher sensory axon growth rates than wildtype mice in vitro. Mice that are homozygous for this allele are viable and fertile.

Development

A targeting vector was designed to insert a single loxP site into exon 22, downstream of the 3’UTR of the karyopherin (importin) beta 1 (Kpnb1) gene. A frt-flanked neomycin resistance (neo) cassette, followed by a second loxP site and three polyadenylation sites, was placed 1.1 kb downstream at the 3’ end of exon 22. This insertion site is located between the 3’ UTR for the short form and the long form of the Importin β1 3’ UTR, flanking the differential sequence between short and long UTRs with loxP sites to allow for cre-mediated deletion. The construct was electroporated into C57BL/6-derived Bruce4 embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts and resulting chimeric males were bred to C57BL/6-Tyr^c-Brd albino females. Offspring were bred to FLP-expressing mice on a C57BL/6 background to remove the neo cassette. Resulting Importin β1 3’ UTR loxP mice were bred to C57BL/6JHsd mice and the Flp-expressing transgene was selected away during the process (see SNP note below). 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). The Tyr^c-Brd allele was not maintained in this colony.

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. Six of the 43 markers throughout the genome were segregating, suggesting an incomplete backcross. Also, 1 of 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6J ; C57BL/6N genetic background.

Allele

Symbol: Kpnb1^tm1.1Mfz
Name: targeted mutation 1.1, Mike Fainzilber
MGI accession ID: MGI:5694121
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed)
Marker symbol: Kpnb1
Marker name: karyopherin (importin) beta 1
Marker synonyms: IMB1; Impnb; IPO1; IPOB; NTF97
Chromosome: 11
Strain of origin: B6.Cg-Thy1^a
Molecular note: A loxP site was inserted into exon 22 downstream of the STOP codon. An FRT-flanked neomycin resistance cassette with a 3' loxP site was inserted into exon 22 downstream of the 1.1 kb sequence to be deleted from the 3'UTR. Flp-mediated recombination removed the selection cassette.