STOCK Grm7^{Tg(SMN2)89Ahmb} Smn1^{tm3(SMN2/Smn1)Mrph} Tg(CAG-cre/Esr1*)5Amc Tg(SMN2*delta7)4299Ahmb/J

Stock number: 008783
Product name: SMN2;Δ7;Cre-ER;Smn^Res , SMN2;Δ7;Cre-ER;Smn^COIN , Delta 7 mouse with hybrid rescue allele and tamoxifen inducible Cre
Research areas: Cancer Research, Developmental Biology Research, Neurobiology Research, Research Tools

Description

The severely affected "rescuable" SMA model mice, SMN2;Δ7;Cre-ER;Smn^Res/Res, allow tamoxifen-inducible rescue of Type II (moderate) proximal spinal muscular atrophy.

Detailed description

These SMN2;Δ7;Cre-ER;Smn^Res mice harbor multiple mutations/transgenes.

The Smn rescue allele (Smn^Res; also called Smn1 conditional inversion or Smn1 COIN) is a functional null in the non-recombined state. Prior to Cre recombination, no full-length SMN transcript is detected in somatic tissue by RT-PCR. No spontaneous inversion of the allele is reported in the absence of Cre recombinase. The Smn^Res allele is designed to revert to a fully functional SMN upon exposure to Cre recombinase.

Mice homozygous for the Tg(SMN2)89 transgene (SMN2+/+), homozygous for the Tg(SMNΔ7)4299 transgene (SMNΔ7+/+), and homozygous for the Smn1^{tm3(SMN2/Smn1)Mrph} mutation (Smn^Res/Res) are a moderate Type II SMA mouse model with similar phenotype as Stock No. 005025. Those SMN2+/+;SMNΔ7+/+;Smn^Res/Res mice exhibit greatly reduced SMN protein in P4 spinal cord tissue, are born small with neuromuscular defects by 4-5 days old that progress to abnormal gait, hind limb weakness/immobility, and eventually death at approximately 13-17 days of age.

When also harboring a tamoxifen-inducible Cre recombinase (Cre-ER) under control of a ubiquitous promoter [Tg(CAG-CreER)5], mice with SMN2+/+;SMNΔ7+/+; Cre-ER+/-;Smn^Res/Res genotype are the severely affected "rescuable" SMA model (SMN2;Δ7;Cre-ER;Smn^Res/Res), and exhibit the same moderate Type II SMA phenotype in the absence of tamoxifen. Addition of tamoxifen results in widespread Cre recombinase expression and inversion of the Smn^Res allele to the "rescue" conformation. Specifically, Cre recombinase irreversibly inverts the fragment bordered by the lox71 and lox66 sites, and the resulting allele is "rescued" into a format that contains mouse Smn1 exons 1-7 and human SMN2 exon 8. Because the mouse Smn1 exon 7 is efficiently spliced, the majority of the mRNA from the Cre recombinase-induced rescue allele contains mouse Smn1 exon 7. When 4 day old SMN2+/+;SMNΔ7+/+;Cre-ER+/-;Smn^Res/Res mice are administered a single tamoxifen dose (75 mg/kg), they gradually gain strength, perform better in a righting reflex assay, and become significantly larger by 17 days old, and have significantly improved lifespan. Such improvements are progressively diminished when administering tamoxifen at later timepoints.

Mice heterozygous for the Smn1^{tm3(SMN2/Smn1)Mrph} mutation (Smn^Res/+) and homozygous for the other mutations/transgenes are viable and fertile with no reported abnormalities or symptoms of neuropathology. As such, The Jackson Laboratory Repository Stock No. 008783 is maintained as SMN2+/+;SMNΔ7+/+;Cre-ER+/-;Smn^Res/+. A researcher must breed SMN2+/+;SMNΔ7+/+;Cre-ER+/-;Smn^Res/+ mice together to generate the severely affected "rescuable" SMA model (SMN2;Δ7;Cre-ER;Smn^Res/Res).

Development

The SMN2;Δ7;Cre-ER;Smn^Res mice harbor several mutations/transgenes, as described below.

The creation of the Smn rescue allele (Smn^Res; also called Smn1 conditional inversion or Smn1 COIN) is described as Stock No. 007249. These mice were maintained upon mixed B6J;129 genetic background prior to being used for breeding as below.

The Tg(SMN2)89 transgene (SMN2) and Tg(SMNΔ7)4299 (Δ7) transgenes were created by Dr. Arthur Burghes (The Ohio State University) as described for the moderate type II SMA mouse model; Stock No. 005025. Note the Tg(SMN2)89 transgene insertion site in on chromosome 6. These mice were bred to and maintained upon an FVB/N-congenic background prior to being used for breeding as below.

The pCAGGCre-ER^TM transgene [Tg(CAG-CreER)5] was designed by Drs. Shigemi Hayashi and Andrew P. McMahon (while at Harvard University) to have the CMV-IE enhancer/chicken β-actin/rabbit β-globin hybrid promoter (CAG; originally from the pCAGGS vector) and CreERTM fusion gene (CreER^TM; Cre recombinase fused to a G525R mutant form of the mouse estrogen receptor ligand binding domain) - as described for Stock No. 004682. These mice were bred to and maintained upon a C57BL/6J-congenic background prior to being used for breeding as below.

To generate the SMN2;Δ7;Cre-ER;Smn^Res animals, Smn^Res mice were bred to moderate type II SMA mice (Tg(SMN2)89;Smn1^+/-;Tg(SMNΔ7)4299). The SMN2;Δ7;Smn^Res/+ progeny (not retaining the Smn1^- knockout mutation) were identified, and then bred to Tg(CAG-CreER)5 mice. The resulting SMN2;Δ7;Cre-ER;Smn^Res/+ offspring (on a mixed C57BL/6;FVB/N;129 genetic background) were then bred together at The Jackson Laboratory Repository as Stock No. 008783.

The Jackson Laboratory Repository Stock No. 008783 colony is maintained as heterozygous for the Smn1^{tm3(SMN2/Smn1)Mrph} mutation (Smn^Res/+), homozygous for the Tg(SMNΔ7)4299 transgene (SMNΔ7+/+), homozygous for the Tg(SMN2)89 transgene (SMN2+/+), and hemizygous for the Tg(CAG-CreER)5 transgene (Cre-ER). That is, Smn^Res/+;SMNΔ7+/+;SMN2+/+;Cre-ER+/-. A researcher must breed Smn^Res/+;SMNΔ7+/+;SMN2+/+;Cre-ER+/- mice together to generate the severely affected "rescuable" SMA model (SMN2;Δ7;Cre-ER;Smn^Res/Res).

In 2022, the Tg(CAG-cre/Esr1*)5Amc transgene creator's laboratory provided the cDNA sequence of the Cre‐ERTM portion - available here. Currently, this transgene is present in the following available Stock Nos. 004682, 004453, 008783, 008813, 017595, 019102, 034795, 034859.

Allele

Symbol: Tg(CAG-cre/Esr1*)5Amc
Name: transgene insertion 5, Andrew P McMahon
MGI accession ID: MGI:2182767
Generation method: Transgenic
Attributes: Recombinase-expressing; Inducible
Marker symbol: Tg(CAG-cre/Esr1*)5Amc
Marker name: transgene insertion 5, Andrew P McMahon
Chromosome: UN
Strain of origin: (C57BL/6 x CBA)F1
Expressed genes: cre/Esr1,Cre recombinase and estrogen receptor 1 fusion gene,
Site of expression: tamoxifen-inducible cre; widespread pattern of expression; tamoxifen administration will also induce Cre recombination in developing embryos of treated mothers and in cultured cells derived from transgenic mice
Molecular note: This transgene expresses a fusion protein consisting of Cre recombinase joined to the ligand-binding domain of a mouse estrogen receptor modified to bind to 4-hydroxytamoxifen, but not to endogenous estrogen. The CAG promoter, containing a chicken beta actin promoter/enhancer coupled with the cytomegalovirus immediate-early (CMV-IE) enhancer, drives high levels of expression in most tissues. In the presence of tamoxifen, the fusion protein is transported into the nucleus, where cre can excise loxP-flanked segments from conditionally modified genes.
General note: Homozygous transgenic mice are not viable or fertile. Hemizygous transgenic mice are viable, fertile, normal in size and do not display any gross physical or behavioral abnormalities.

In transgenic mice the mutant mouse estrogen receptor does not bind natural ligand at physiological concentrations but will bind the synthetic ligand, 4-hydroxytamoxifen. Restricted to the cytoplasm, the Cre/Esr1 fusion protein can only gain access to the nuclear compartment after exposure to tamoxifen. When crossed with a strain containing loxP sites flanking a sequence of interest, tamoxifen induced, Cre-mediated targeted deletions are generated in the offspring. Tamoxifen administration will induce Cre recombination in developing embryos of treated mothers and in cultured cells derived from transgenic mice.

Note that this transgene does not contain the cre/ERT2 fusion but was mistakenly referred to by the synonym CAG-CreERT2 in publication. J:136965

Note that this transgene does not contain the cre/ERT2 fusion but was mistakenly referred to by the synonym CAGG-CreERT2 in publication. J:130851

Allele

Symbol: Tg(SMN2*delta7)4299Ahmb
Name: transgene insertion 4299, Arthur H M Burghes
MGI accession ID: MGI:3056918
Generation method: Transgenic
Attributes: Inserted expressed sequence; Humanized sequence
Marker symbol: Tg(SMN2*delta7)4299Ahmb
Marker name: transgene insertion 4299, Arthur H M Burghes
Chromosome: UN
Strain of origin: FVB/N
Expressed genes: SMN2,survival of motor neuron 2, centromeric,human
Site of expression: Tg(SMN2*delta7)4299Ahmb
Molecular note: The transgene contains a human SMN2 promoter and a human SMN2 cDNA (SMNdelta7) that lacks exon 7. There are 14 copies of this allele integrated into the genome.

Allele

Symbol: Smn1^{tm3(SMN2/Smn1)Mrph}
Name: targeted mutation 3, Andrew Murphy
MGI accession ID: MGI:3794199
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Humanized sequence; No functional change
Marker symbol: Smn1
Marker name: survival motor neuron 1
Chromosome: 13
Strain of origin: (129S6/SvEvTac x C57BL/6NTac)F1
Site of expression: Following Cre-mediated irreversible inversion of the fragment bordered by the lox71 and lox66 sites, the allele is "rescued" into a format that contains mouse Smn1 exons 1 through 7 and human SMN2 exon 8 and the resulting SMN protein is produced in motor neurons.
Molecular note: Exons 7 and 8 of the mouse Smn1 (survival motor neuron 1) gene and a several hundred base pairs of flanking sequence were replaced with a fragment containing, in order: 1) an inverted lox71 site; 2) exon 7 from the human SMN2 (survival of motor neuron 2, centromeric) gene flanked by several hundred base pairs of intron sequence; 3) an inverted copy of mouse Smn1 exon 7 flanked by several hundred base pairs of intron sequence; 4) a lox66 site; 5) an FRT site remnant from a deleted selection cassette; and 6) human SMN2 exon 8 including the 3'UTR and polyA signal with several hundred base pairs of flanking sequence. The engineered allele expresses a hybrid Smn1 gene containing mouse Smn1 exons 1 through 6 and human SMN2 exons 7 and 8. The human SMN2 exon 7 is skipped in the majority of mRNA derived from the hybrid gene due to a single base pair difference in human SMN2 exon 7, compared to human SMN1 exon 7. Following Cre-mediated irreversible inversion of the fragment bordered by the lox71 and lox66 sites, the allele is "rescued" into a format that contains mouse Smn1 exons 1 through 7 and human SMN2 exon 8. Because the mouse Smn1 exon 8 is efficiently spliced, the majority of the mRNA from the rescue allele after Cre-mediated inversion contains mouse Smn1 exon 7.

Allele

Symbol: Grm7^{Tg(SMN2)89Ahmb}
Name: transgene insertion 89, Arthur H M Burghes
MGI accession ID: MGI:2448989
Generation method: Transgenic
Attributes: Hypomorph; Inserted expressed sequence; Humanized sequence
Marker symbol: Grm7
Marker name: glutamate receptor, metabotropic 7
Chromosome: 6
Strain of origin: FVB/N
Expressed genes: SMN2,survival of motor neuron 2, centromeric,human
Site of expression: Human SMN2 protein is expressed in the tissues examined (brain, liver, spinal cord) [Stock No. 005024].
Molecular note: A 35.5 kb genomic fragment containing the human survival motor neuron 2 (SMN2) gene and promoter was used for the transgene. The transgene is ubiquitously expressed in all tissues examined by Northern blot analysis. Line 89 carries 1 copy of the transgene integrated into intron 4 of the gene. RT-PCR confirmed reduced expression of the gene the transgene is integrated into.