STOCK Mir34a^tm1.1Lhe Mirc34^tm1.1Mkss/LheJ

Stock number: 040320
Product name: mir-34a/mir-449 DKO
Research areas: Cancer Research, Cell Biology Research, Research Tools

Description

This Mir34a Mirc34 (mir-34a; mir-449) double knock-out strain is deficient for mir-34a (encoding miR-34a) and mir-449 (encoding all members of the mir-449 microRNA family: miR-449a, miR-449b and miR-449c). These mice may be suitable for use in studies related to somatic cell reprogramming, stem cell differentiation, spermatogenesis, neuronal development, aging, and tumor suppression.

Detailed description

The mir-34/449 microRNA family consists of six homologous miRNAs at three genomic loci: Mir34a, Mir34b/34c, and Mirc34 (mir-449). All six mature miRNAs from the mir-34/449 family share the same seed sequence. mir-34a and mir-34b/c are induced by p53 activation, and mediate aspects of the p53 tumor suppressor response. Mirc34 (mir-449) encodes three homologous miRNAs, miR-449a, miR-449b, and miR-449c. All 6 mir-34/449 members are highly enriched in mucociliary epithelia, particularly those encoded by mir-34b/34c and mir-449. These mir-34a/mir-449 double knock-out (DKO) mice carry deletion alleles for mir-34a and mir-449. Mice that are homozygous for the targeted mutations are viable and fertile. The Donating investigator has reported that mir-34a/mir-449 DKO mice crossed with the mir-34a; mir-34b/c DKO (Stock No. 025705) mice produce mir-34/449 triple knock-out (mir-34a, mir-34b/34c and mir-449 deficient) mice that develop functional defects in multicilia and show respiratory, fertility and brain phenotypes.

Of note, in addition to these mice, the donating investigator provided The Jackson Laboratory Repository with mir-34a; mir-34b/c DKO (Stock No. 025705), mir-34b/c^flNeo (Stock No. 026010), and Mir34a^- (Stock No. 018279).

Development

To generate the Mir34a^tm1.1Lhe allele, a targeting vector containing a Kozak sequence, lacZ gene, and an FRT and loxP site-flanked neo cassette was used to disrupt Mir34a pre-microRNA and a 20 bp flanking sequence. The construct was electroporated into C57BL/6 (B6.Cg-Thy1^a) derived Bruce 4 embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts. The resulting chimeric animals were tested for germline transmission. The mice were crossed with an Actin-FLP line on a C57BL/6 background to excise the FRT site-flanked neo cassette. The mice were then backcrossed to C57BL/6 for approximately 6 generations.

To generate the Mirc34^tm1.1Mkss (mir-449) allele, a targeting vector containing an FRT site-flanked neo cassette with a downstream loxP site was inserted downstream of Mir449a. A second loxP site was inserted upstream of Mir449c located in intron 2 of the Cdc20b locus containing the 3 cluster Members Mir449c, Mir449b, Mir449a (Chr13:113172517-113174158 bp). All mir-449 miRNAs are within intron 2 of their host gene, Cdc20b. The deletion of mir-449 is predicted to have minimal impact on Cdc20b. The construct was electroporated into 129-derived mouse embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts. The resulting chimeric animals were tested for germline transmission. The mice were then crossed to a transgenic strain expressing Cre recombinase under the control of the beta actin promoter (ACTB-Cre) on a C57BL/6 background to excise the loxP-flanked region containing the Mirc34 pre-microRNA cluster and flanking sequence.

mir-449 knock-out mice on a mixed C57BL/6 and 129 genetic background were crossed to mir-34a-/- mice on a C57BL/6 background for at least 2 generations to generate mir-34a/mir449 double knock-out (DKO) mice. These DKO mice were then crossed to mir-34b/c-/- mice on a C57BL/6 background for another two generations to generate mir-34a-/-, mir-34b/c+/- and mir-449-/- mice. Resultant mice were backcrossed to the C57BL/6 for at least 4 generations by the donating investigator before sending animals to The Jackson Laboratory. Upon arrival at The Jackson Laboratory, sperm were cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J (Stock No. 000664) oocytes.

Allele

Symbol: Mir34a^tm1.1Lhe
Name: targeted mutation 1.1, Lin He
MGI accession ID: MGI:5308792
Generation method: Targeted
Attributes: Null/Knockout
Synonyms: Mir34a^tm1.1Ghan
Marker symbol: Mir34a
Marker name: microRNA 34a
Marker synonyms: mir 34a; mir-34; mir-34a; Mirn34a; miRNA34A; mmu-mir-34a
Chromosome: 4
Strain of origin: B6.Cg-Thy1^a
Molecular note: The microRNA was replaced with a lacZ gene and neo cassette flanked by loxP and FRT sites. Flp-mediated recombination removed the neo cassette. Real-time PCR confirmed the absence of transcript expression in mouse embryonic fibroblasts.

Allele

Symbol: Mirc34^tm1.1Mkss
Name: targeted mutation 1.1, Michael Kessel
MGI accession ID: MGI:5705617
Generation method: Targeted
Attributes: Null/Knockout
Synonyms: mir-449^-
Marker symbol: Mirc34
Marker name: microRNA cluster 34, including Mir449a through Mir449c
Chromosome: 13
Strain of origin: Not Specified
Molecular note: A loxP site was inserted upstream of Mir449c. A FRT flanked neomycin resistance cassette and a 3' loxP site were inserted downstream of Mir449a. Cre-mediated recombination removed Mir449a, Mir449b, Mir449c and the selection cassette.