B6;129S4-Nfib^tm2.1Rmg Nfia^{tm1c(EUCOMM)Hmgu} Nfix^tm1.2Rmg/RmgJ

Stock number: 040481
Product name: Nfia Nfib Nfix triple conditional KO
Research areas: Cell Biology Research, Developmental Biology Research, Neurobiology Research, Research Tools

Description

The Nfia Nfib Nfix triple conditional knock-out (cKO) mouse line harbors 3 alleles; each allele has a loxP-flanked exon 2 for the respective gene. These Nfia Nfib Nfix triple cKO mice may be useful for studying brain, lung, and muscle development as well as hematopoietic development.

Detailed description

Nfia, Nfib, Nfic and Nfix code for Nuclear Factor I transcription factors and are highly expressed in embryonic stem cells as well as progenitor cells of the central nervous system (CNS), lung and skeletomuscular tissues. These Nfia Nfib Nfix triple conditional knock-out (cKO) mice have loxP sites flanking exon 2 of each of the respective genes.

Mice homozygous for all three alleles are viable and fertile with no overt phenotype. When bred to mice that express tissue-specific Cre recombinase, resulting offspring will have exon 2 of each gene deleted in the cre-expressing tissues, resulting in complete loss of NFIA, NFIB and NFIX in Cre-expressing tissues. Nfia Nfib Nfix triple cKO mice may be useful for studying brain, lung and skeletomuscular development.

Development

This Nfia Nfib Nfix triple conditional knock-out (cKO) mouse line harbors 3 conditional alleles as described below. Note that the Nfia and Nfib genes are 5cM apart on chromosome 4.

The Nfia^{tm1c(EUCOMM)Hmgu} allele was generated using C57BL/6N-derived JM8A3.N1 embryonic stem (ES) cells bearing the Nfia^{tm1c(EUCOMM)Hmgu} targeted allele obtained from The European Conditional Mouse Mutagenesis Program (EUCOMM). The targeting cassette is composed of an FRT site followed by lacZ sequence and an initial loxP site upstream of exon 2 of the nuclear factor I/A (Nfia) gene on chromosome 4 (97660971-98007111 (GRCm39)). The 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 exon 2. Targeted ES cells were injected into recipient blastocysts. The resulting chimeric mice were bred with FLP-Recombinase mice (Stock No. 009086) to excise the FRT site flanked lacZ-loxP-neo cassette. The Nfia^{tm1c(EUCOMM)Hmgu} mice, with neomycin cassette removed and floxed exon 2 intact, were maintained on a C57BL/6 genetic background before being bred with the Nfib^tm2.1Rmg mouse line by the donating laboratory.

The Nfib^tm2.1Rmg allele was designed to express a loxP-flanked exon 2 of the nuclear factor I/B (Nfib) gene on chromosome 4 (82208410-82424988 (GRCm39)). The targeting construct consisted of a 4.4kb 5' homology arm containing all of exon 2 of Nfib and 300bp of intron 2, with a loxP site inserted 363bp 5' to the start of exon 2. The 5' arm was followed by an FRT-flanked PGK-neomycin expression cassette in the opposite transcriptional orientation, a 3' loxP site, the contiguous 3.6kb 3' homology arm and a PGK-diphtheria toxin A chain cassette in the opposite transcriptional orientation. The target construct was electroporated into 129S4/SvJae-derived J1 embryonic stem (ES) cells and correctly targeted ES cells were injected into C57BL/6 blastocysts. Chimeric mice were bred to C57BL/6 to confirm germline transmission and crossed to a FLP-Recombinase mouse line (Stock No. 009086) to remove the FRT-flanked neomycin cassette. The Nfib^tm2.1Rmg mice, with neomycin cassette removed and floxed exon 2 intact, were backcrossed to C57BL/6 for at least 18 generations before being bred with the Nfia^{tm1c(EUCOMM)Hmgu} mouse line by the donating laboratory.

The Nfix^tm1.2Rmg allele was designed to express a loxP-flanked exon 2 of the nuclear factor I/X (Nfix) gene on chromosome 8 (85431341-85527086 (GRCm39)). The targeting construct consisted of a 4.2kb 5' homology arm containing all of exon 2 and 633bp of intron 2, with a loxP site inserted 406bp 5' to the start of exon 2. The 5' arm was followed by an FRT-flanked PGK-neomycin expression cassette in the opposite transcriptional orientation, a 3' loxP site, the contiguous 2.7kb 3' homology arm and a PGK-diphtheria toxin A chain cassette in the opposite transcriptional orientation. The target construct was electroporated into 129S4/SvJae-derived J1 embryonic stem (ES) cells and correctly targeted ES cells were injected into C57Bl/6 blastocysts. Chimeric mice were bred to C57BL/6 to confirm germline transmission and crossed to a FLP-Recombinase mouse line (Stock No. 009086) to remove the FRT-flanked neomycin cassette. The Nfix^tm1.2Rmg mice, with neomycin cassette removed and floxed exon 2 intact, were backcrossed to C57BL/6 for 2-5 generations before being bred with the double Nfia^{tm1c(EUCOMM)Hmgu}; Nfib^tm2.1Rmg mutant line to generate this Nfia Nfib Nfix triple cKO line.

Donor mice provided to The Jackson Laboratory were heterozygous for all 3 conditional alleles (Nfia, Nfib and Nfix), as well as a being heterozygous for a SunF^GFP floxed allele. The SunF^GFP allele was removed through breeding to C57BL/6J (Stock No. 000664) inbred mice for at least one generation. After the unwanted SunF^GFP was removed, sperm was cryopreserved carrying only the Nfia^{tm1c(EUCOMM)Hmgu}, Nfib^tm2.1Rmg and Nfix^tm1.2Rmg conditional alleles.

Allele

Symbol: Nfib^tm2.1Rmg
Name: targeted mutation 2.1, Richard M Gronostajski
MGI accession ID: MGI:6368821
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed)
Marker symbol: Nfib
Marker name: nuclear factor I/B
Marker synonyms: 6720429L07Rik; CTF; E030026I10Rik; HMGIC/NFIB; MACID; NF1-B; NF-I/B; NFI-B; NFIB2; NFIB3; NFI-RED
Chromosome: 4
Strain of origin: 129S4/SvJae
Molecular note: A loxP site was inserted upstream of exon 2. An FRT-flanked neo cassette with a 3' loxP site was inserted downstream of exon 2. The FRT-flanked neo cassette was removed via Flp-mediated recombination in the germline.

Allele

Symbol: Nfia^{tm1c(EUCOMM)Hmgu}
Name: targeted mutation 1c, Helmholtz Zentrum Muenchen GmbH
MGI accession ID: MGI:6280648
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed)
Synonyms: NFIA^fl; Nfia^tm2Rmg
Marker symbol: Nfia
Marker name: nuclear factor I/A
Marker synonyms: 1110047K16Rik; 9430022M17Rik; BRMUTD; C1DELp32p31; CTF; DEL1P32P31; NF1A; NF1-A; NF-I/A; NFI-A; NFI-L
Chromosome: 4
Strain of origin: C57BL/6N-A^tm1Brd
Molecular note: The L1L2_Bact_P cassette was inserted at position 97670780 of Chromosome 4 upstream of exon 2 (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 2 at position 97672189. Exon 2 is 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 exon 2.

Allele

Symbol: Nfix^tm1.2Rmg
Name: targeted mutation 1, Richard M Gronostajski
MGI accession ID: MGI:8192697
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed)
Marker symbol: Nfix
Marker name: nuclear factor I/X
Marker synonyms: CTF; MALNS; MRSHSS; NF1A; Nf1x; NF1-X; NF-I/X; SOTOS2
Chromosome: 8
Strain of origin: 129S4/SvJae
Molecular note: A loxP site was inserted upstream of exon 2 and an frt-flanked neo cassette with a 3' loxP site was inserted downstream of exon 2. A FLP-recombinase removed the FRT-flanked neomycin cassette.