STOCK Mir34a^tm1.1Lhe Mirc21^tm1.1Lhe/J

Stock number: 025705
Product name: mir-34a; mir-34b/c DKO
Research areas: Cancer Research, Cell Biology Research, Research Tools

Description

This Mir34a Mirc21 (mir-34a; mir-34b/c ) double knockout strain is deficient for all 3 members of the mir-34 microRNA family. 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 microRNA precursor family has 3 members: Mir34a, Mir34b and Mir34c, which all share the same seed sequence. As Mir34b & Mir34c share a primary transcript, they are known as Mirc21, microRNA cluster 21, including Mir34c through Mir34b. All 3 mir-34 members are transcriptional targets of p53. Mir34a is an important regulator of tumor suppression via the p53 pathway in a positive feedback loop. These mice carry knock out alleles for Mir34a and Mirc21.

Mice that are homozygous for the targeted mutations are viable and fertile. No Mir34 microRNA is detected by real-time PCR analysis of culture stressed MEFs from double knock out mice. Heterozygotes have an intermediate phenotype of expression. MEFs derived from double homozygotes exhibit an increase in iPSC (induced pluripotent stem cell) generation.

Development

To generate the Mir34a^tm1.1Lhe allele, a targeting vector containing a Kozak sequence, lacZ gene and a FRT & loxP site flanked NEO cassette, was used to disrupt Mir34a pre-microRNA and 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 the 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 Mirc21^tm1.1Lhe (mir-34b/c) allele, a targeting vector containing a FRT site flanked NEO cassette with a downstream loxP site, was inserted downstream of Mirc21. A second loxP site was inserted upstream of Mirc21. The construct was electroporated into (C57BL/6 x 129S4/SvJae)F1 derived v6.5 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 the C57BL/6 background, to excise the loxP flanked region containing the Mirc21 pre-microRNA cluster and 200bp of flanking sequence.

The single mutant lines were intercrossed to generate this double targeted mutation strain. Upon arrival at The Jackson Laboratory, the mice were crossed to C57BL/6J (Stock No. 000664) at least once to establish the colony.

Allele

Symbol: Mirc21^tm1.1Lhe
Name: targeted mutation 1.1, Lin He
MGI accession ID: MGI:5308794
Generation method: Targeted
Attributes: Null/Knockout
Marker symbol: Mirc21
Marker name: microRNA cluster 21, including Mir34c through Mir34b
Chromosome: 9
Strain of origin: (C57BL/6 x 129S4/SvJae)F1
Molecular note: A loxP site was inserted upstream of Mir34b and an FRT-flanked neo cassette with a 3' loxP site was inserted downstream of Mir34c. Cre-mediated recombination removed the neo cassette and both microRNAs. Real-time PCR confirmed the absence of transcript expression in mouse embryonic fibroblasts.

Allele

Symbol: Mir34a^tm1.1Lhe
Name: targeted mutation 1.1, Lin He
MGI accession ID: MGI:5308792
Generation method: Targeted
Attributes: Null/Knockout
Marker symbol: Mir34a
Marker name: microRNA 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 neo and FRT sites. Flp-mediated recombination removed the neo cassette. Real-time PCR confirmed the absence of transcript expression in mouse embryonic fibroblasts.