STOCK Lmna^tm1.1Yxz Lmnb2^{tm1c(KOMP)Wtsi} Lmnb1^{tm1c(EUCOMM)Wtsi}/YxzJ

Stock number: 041640
Product name: Lmna, Lmnb1, Lmnb2 triple flox
Research areas: Cell Biology Research, Developmental Biology Research, Mouse/Human Gene Homologs, Neurobiology Research, Research Tools

Description

The Lmna, Lmnb1, Lmnb2 triple flox strain was created by combining the following conditional knock-out alleles: Lmna^flox Stock No. 026284, Lmnb1^flox, and Lmnb2^flox.

Lmna^flox mice possess loxP sites flanking exon 2 of the Lmna gene. Lmnb1^flox mice possess loxP sites flanking exon 2 of the Lmnb2 gene. Lmnb2^flox mice possess loxP sites flanking exons 5-9 of the Lmnb1 gene. This strain may be useful for generating conditional mutations in all three Lamin genes, making them useful for studying the role of Lamins in specific cell types, cellular proliferation and differentiation, and their influences on the progression of human diseases.

Detailed description

Lmna, Lmnb1, Lmnb2 triple flox strain was created by combining the following alleles: Lmna^flox Stock No. 026284, Lmnb1^flox, and Lmnb2^flox.

Lmna^flox mutant mice possess loxP sites flanking exon 2 of the lamin A (Lmna) gene. Lamin-A is part of the nuclear lamina, seen in increasing levels of expression as development progresses. It is required for the normal development of the peripheral nervous system and skeletal muscle and plays an important role in nuclear assembly, chromatin organization, nuclear membrane, and telomere dynamics. Mice that are homozygous for this allele are viable and fertile. When bred to mice that express tissue-specific Cre recombinase, the resulting offspring will have exon 2 deleted in the cre-expressing tissues. Since different Lamin-A isoforms exist, this strain was developed to produce offspring that lack the majority of Lamin-A isoforms.

Lmnb1^flox mice possess loxP sites on either side of the exon 2 of the targeted gene. The targeted Lmnb1 gene encodes a B-type lamin protein that comprises part of the nuclear lamina. Mutations in this gene have been associated with adult-onset, autosomal dominant leukodystrophy. A paralog of this gene is Lmnb2. Mice homozygous for this allele are viable and fertile. When these mutant mice are bred to mice that express Cre recombinase, the resulting offspring will have exon 2 deleted in the cre-expressing tissues. Removal of the floxed sequence creates a null allele.

Lmnb2^flox mice possess loxP sites on either side of the exons 5-9 of the targeted gene. The targeted Lmnb1 gene encodes a B-type lamin protein that comprises part of the nuclear lamina. Mutations in this gene have been associated with acquired partial lipodystrophy and progressive myoclonic epilepsy-9. A paralog of this gene is Lmnb1. Mice homozygous for this allele are viable and fertile. When these mutant mice are bred to mice that express Cre recombinase, the resulting offspring will have exons 5-9 deleted in the cre-expressing tissues. Removal of the floxed sequence creates a null allele.

Mice homozygous for all three alleles are viable and fertile. This line can be used to generate any of the single deletions or single floxed lines for the lamin family genes in the mouse that can serve as a platform for studying the role of Lamins in specific cell types, cellular proliferation, and differentiation, and their influences on the progression of human diseases.

Development

This strain was created by combining the following conditional knock-out alleles: Lmna^flox Stock No. 026284, Lmnb1^flox, and Lmnb2^flox.

A targeting vector was designed to insert a loxP site upstream of the exon 2, and a frt-flanked puromycin resistance (puro) cassette followed by a second loxP site, downstream of exon 2 of the lamin A (Lmna) gene on Chromosome 3. 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, and resulting chimeric males were bred with CD1 females. Offspring were bred to B6;SJL-Tg(ACTFLPe)9205Dym/J transgenic mice (Stock No. 003800) to delete the puro cassette, and progeny were bred together to generate homozygous mice without the Flp-expressing transgene. The resulting Lmna^flox/flox colony was maintained on a mixed background.

The L1L2_gt2 cassette was inserted at position 56,861,051 of Chromosome 18 upstream of the exon 2 of the lamin B1 (Lmnb1) gene (Build GRCm39). The cassette is composed of an FRT-flanked β-galactosidase/neomycin resistance gene fusion (lacZ/neo), and a polyadenylation site (SV40 polyA). This was followed by a loxP site. An additional loxP site is inserted downstream of the targeted exon 2 at position 56,861,914. The critical exon 2 is flanked by loxP sites. The mutation was generated in C57BL/6N-derived JM8.N4 embryonic stem (ES) cells, and correctly targeted ES cells were injected into blastocysts. The resulting chimeric mice were bred to ACTFLPe mice (Stock No. 005703) to remove the lacZ/neo cassette, and then subsequently, the mice were bred together to generate homozygous mice without the Flp-expressing transgene. These Lmnb1^flox/flox mice were backcrossed to C57BL/6 mice for at least 6 generations by the donating laboratory.

The L1L2_Bact_P cassette was inserted at position 80,739,635 of Chromosome 10 upstream of exons 5 of the lamin B2 (Lmnb2) gene (Build GRCm39). The cassette is composed of an FRT site followed by lacZ sequence and a loxP site. This first loxP site is followed by a neomycin resistance (neo) cassette, a second FRT site, and a second loxP site. A third loxP site was inserted downstream of exons 9 at position 80,741,471. The critical exons 5-9 are flanked by loxP sites. The mutation was generated in C57BL/6N-derived JM8.N4 embryonic stem (ES) cells, and correctly targeted ES cells were injected into blastocysts. The resulting chimeric mice were bred to ACTFLPe mice (Stock No. 005703) to remove the lacZ/neo cassette, and then subsequently, the mice were bred together to generate homozygous mice without the Flp-expressing transgene. Resulting Lmnb2^flox/flox mice were bred to C57BL/6 mice for at least 6 generations by the donating laboratory.

Three lines containing Lmna^flox/flox, Lmnb1^flox/flox, and Lmnb2^flox/flox were crossed to create triple flox mice as follows. First, homozygous Lmnb1^flox/flox and Lmnb2^flox/flox mice were crossed to generate double homozygous mice on the C57BL/6 genetic background. Then they were mated to Lmna^flox/flox later to create triple heterozygous mice on a mixed genetic background. Finally, the triple heterozygous mice on a mixed genetic background were intercrossed to generate a triple homozygous line containing Lmna^flox/flox, Lmnb1^flox/flox, and Lmnb2^flox/flox. These mice are maintained on a mixed genetic background by the donating laboratory. Upon arrival at The Jackson Laboratory, triple homozygous Lmna, Lmnb1, and Lmnb2 flox females and males were used to generate embryos, and they were cryopreserved.

Allele

Symbol: Lmnb2^{tm1c(KOMP)Wtsi}
Name: targeted mutation 1c, Wellcome Trust Sanger Institute
MGI accession ID: MGI:6363075
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed)
Marker symbol: Lmnb2
Marker name: lamin B2
Marker synonyms: EPM9; LAMB2; lamin B3; LMN2; MCPH27; RGD1563803
Chromosome: 10
Strain of origin: C57BL/6N
Molecular note: The L1L2_Bact_P cassette was inserted at position 80739635 of Chromosome 10 upstream of the critical exon(s) (Build GRCm39). The cassette is composed of an FRT site followed by lacZ sequence and a loxP site. This first loxP site is followed by a neomycin resistance gene under the control of the human beta-actin promoter, SV40 polyA, a second FRT site and a second loxP site. A third loxP site is inserted downstream of the targeted exon(s) at position 80741471. The critical exon(s) is/are thus flanked by loxP sites. A "conditional ready" (floxed) allele was created by flp recombinase expression in mice carrying this allele to remove the lacZ sequence and neo selection cassette, leaving loxP sites flanking the critical exon(s). Further information on targeting strategies used for this and other IKMC alleles can be found at http://www.informatics.jax.org/mgihome/nomen/IKMC_schematics.shtml

Allele

Symbol: Lmna^tm1.1Yxz
Name: targeted mutation 1.1, Yixian Zheng
MGI accession ID: MGI:5575529
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); No functional change
Synonyms: Lmna^flox
Marker symbol: Lmna
Marker name: lamin A
Marker synonyms: CDCD1; CDDC; CMD1A; CMT2B1; Dhe; EMD2; FPL; FPLD; FPLD2; HGPS; IDC; lamin A/C; LDP1; LFP; LGMD1B; LMN1; LMNC; LMNL1; MADA; PRO1
Chromosome: 3
Strain of origin: (C57BL/6 x 129S4/SvJae)F1
Molecular note: A loxP site was inserted upstream of exon 2. An FRT-flanked puromycin resistance cassette with a 3' loxP site was inserted downstream of exon 2. Flp-mediated recombination removed the selection cassette and left exon 2 floxed.

Allele

Symbol: Lmnb1^{tm1c(EUCOMM)Wtsi}
Name: targeted mutation 1c, Wellcome Trust Sanger Institute
MGI accession ID: MGI:5767330
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed)
Marker symbol: Lmnb1
Marker name: lamin B1
Marker synonyms: ADLD; LMN; LMN2; LMNB; MCPH26
Chromosome: 18
Strain of origin: C57BL/6N
Molecular note: Flp-mediated excision of the parental Lmnb1^{tm1a(EUCOMM)Wtsi} allele resulted in the removal of the promoter-driven neomycin selection cassette and the inserted lacZ reporter sequence, leaving behind the loxP-flanked critical exon(s). Further information on targeting strategies used for this and other IKMC alleles can be found at http://www.informatics.jax.org/mgihome/nomen/IKMC_schematics.shtml.