STOCK Grm7^{Tg(SMN2)89Ahmb} Tg(SMN2-SMN1*Q282A)2Ahmb Smn1^tm1Msd/J

Stock number: 031907
Product name: SMN2 Q282A; SMN2; Smn KO
Research areas: Neurobiology Research

Description

SMN Q282A+/+; SMN2+/+; KO+/+ are triple mutant mice harboring the Grm7^{Tg(SMN2)89Ahmb} Smn1^tm1Msd alleles described for the severe type I spinal muscular atrophy (SMA) mouse model, as well as the Tg(SMN2-SMN1*Q282A)2Ahmb transgene containing the Q282A missense mutation in exon 7 of the human SMN1 gene - resulting in amelioration of the SMA phenotype of FVB.Cg-Grm7^{Tg(SMN2)89Ahmb} Smn1^tm1Msd/J mice (Stock No. 005024).

Of note, for other mice carrying the same Tg(SMN2-SMN1*Q282A)2Ahmb transgene (Stock No. 031909), it was discovered that Tg(SMN2-SMN1*Q282A)2Ahmb integrated on chromosome 11 (70055009-70372513; mouse mm10) and resulted in a 317 kb genomic region duplication that includes several loci (Asgr1, Asgr2, Mgl2, Clec10a, Slc16a11, Slc16a13, Bcl6b, Mir497b, Rnasek, Alox12 and Alox15). Therefore, Tg(SMN2-SMN1*Q282A)2Ahmb mice and can be expected to have overexpression of those duplicated loci in hemizygotes, and to a greater extent in homozygotes.

Detailed description

These triple mutant mice carry the Grm7^{Tg(SMN2)89Ahmb} (or Tg(SMN2)89Ahmb) transgene, the Smn1^tm1Msd knock-out null allele and the Tg(SMN2-SMN1*Q282A)2Ahmb missense mutant transgene.

The Tg(SMN2)89Ahmb transgene expresses the entire human SMN2 gene, whereas no endogenous Smn1 expression is observed from the Smn1^tm1Msd knock-out null allele.

The Tg(SMN2-SMN1*Q282A)2Ahmb transgene contains the Q282A missense mutation in exon 7. Tg(SMN2-SMN1*Q282A)2Ahmb transgene mRNA expression is detected by RT-PCR analysis of brain and spinal cord tissue and western blot analysis of spinal cord tissue. Tg(SMN2-SMN1*Q282A)2Ahmb transgene expression has been examined in brain and spinal cord, but not in any other tissues. The donating investigator reported that mice hemizygous for the Tg(SMN2-SMN1*Q282A)2Ahmb transgene carry 4 copies and mice homozygous for the Tg(SMN2-SMN1*Q282A)2Ahmb transgene carry 8 copies. Additionally, they reported that the Tg(SMN2-SMN1*Q282A)2Ahmb transgene copy number is stable. See below for transgene insertion information.

Mice homozygous for the Tg(SMN2)89 transgene, homozygous for Smn1 null and homozygous for the Tg(SMN2-SMN1*Q282A)2Ahmb transgene (SMN I116F+/+; SMN2++; KO+/+) survive beyond 200 days, exhibit a 3-fold increase in human SMN mRNA expression when compared to Δ7SMA mice (Stock No. 005025) as detected by RT-PCR of spinal cord tissue, and have increased snRNP activity levels in spinal cord compared to Δ7SMA mice. The Tg(SMN2-SMN1*Q282A)2Ahmb transgene ameliorates the severe phenotype of FVB.Cg-Grm7^{Tg(SMN2)89Ahmb} Smn1^tm1Msd/J mice (Stock No. 005024).

Tg(SMN2-SMN1*Q282A)2Ahmb transgene insertion information: For other mice carrying the same Tg(SMN2-SMN1*Q282A)2Ahmb transgene (Stock No. 031909), in October 2019, sequencing performed by The Jackson Laboratory discovered that the Tg(SMN2-SMN1*Q282A)2Ahmb transgene integrated on chromosome 11 (70055009-70372513; mouse mm10) and resulted in a duplication of the 317 kb genomic region in between the two identified fusion points. This duplicated region includes several loci (Asgr1, Asgr2, Mgl2, Clec10a, Slc16a11, Slc16a13, Bcl6b, Mir497b, Rnasek, Alox12 and Alox15). Therefore, Tg(SMN2-SMN1*Q282A)2Ahmb mice and can be expected to have overexpression of those duplicated loci in hemizygotes, and to a greater extent in homozygotes. The transgene copy number for homozygous mice was estimated to be greater than eight.

Development

These SMN Q282A+/+; SMN2+/+; KO+/+ mice harbor several mutations/transgenes, as described below.

The Smn1 null allele (Smn1^tm1Msd) was created in the laboratory of Dr. Michael Sendtner at the University of Wurzburg, Germany. Exon 2 of the endogenous mouse Smn gene was disrupted by employing a targeting vector encoding a neomycin cassette and a lacZ gene fused to the first 40 nucleotides of the disrupted exon to permit expression of the lacZ gene in tissues where Smn is normally expressed. The construct was electroporated into 129P2/OlaHsd-derived E14Tg2a-IV embryonic stem (ES) cells. Correctly targeted ES cells were injected into C57BL/6 blastocysts and chimeric animals obtained. Chimeric animals were crossed to C57BL/6 for several (unspecified number) of generations.

Both transgenes were created in the laboratory of Dr. Arthur Burghes at Ohio State University. The SMN2 transgene was designed with a 35.5 kbp BamHI genomic fragment encoding the human SMN2 promoter and gene (derived from genomic clone PAC215P15). This transgene was injected into fertilized FVB/N mouse oocytes, and founder animal 89 obtained (Tg(SMN2)89). Note the Tg(SMN2)89 transgene insertion site is in intron 4 of the Grm7 (glutamate receptor, metabotropic 7) gene on chromosome 6.

The Tg(SMN2-SMN1*Q282A)2Ahmb transgene was designed with a human SMN1 cDNA carrying the Q282A missense mutation in exon 7 under the control of the 4.1 kb SMN promoter derived from the human SMN2 gene. This transgene was microinjected into fertilized FVB/N oocytes. The donating investigator reported that founder line 2, bearing 4 copies of the transgene (as hemizygote), was obtained. They also reported that the Tg(SMN2-SMN1*Q282A)2Ahmb transgene copy number is stable. See below for transgene insertion information.

The triple mutant line was maintained on the FVB genetic background by the donating lab (see SNP note below). Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize oocytes from FVB.Cg-Grm7^{Tg(SMN2)89Ahmb} Smn1^tm1Msd/J mice (Stock No. 005024). Triple homozygous embryos were later cryopreserved.

A 48 SNP (single nucleotide polymorphism) panel analysis, with 43 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the rederived living colony at The Jackson Laboratory Repository. Six of the 43 markers throughout the genome were segregating suggesting an incomplete backcross.

Tg(SMN2-SMN1*Q282A)2Ahmb transgene insertion information: For other mice carrying the same Tg(SMN2-SMN1*Q282A)2Ahmb transgene (Stock No. 031909), in October 2019, sequencing performed by The Jackson Laboratory discovered that the Tg(SMN2-SMN1*Q282A)2Ahmb transgene integrated on chromosome 11 (70055009-70372513; mouse mm10) and resulted in a duplication of the 317 kb genomic region in between the two identified fusion points. This duplicated region includes several loci (Asgr1, Asgr2, Mgl2, Clec10a, Slc16a11, Slc16a13, Bcl6b, Mir497b, Rnasek, Alox12 and Alox15). Therefore, Tg(SMN2-SMN1*Q282A)2Ahmb mice and can be expected to have overexpression of those duplicated loci in hemizygotes, and to a greater extent in homozygotes. The transgene copy number for homozygous mice was estimated to be greater than eight.

Allele

Symbol: Smn1^tm1Msd
Name: targeted mutation 1, Michael Sendtner
MGI accession ID: MGI:2183946
Generation method: Targeted
Attributes: Reporter; Null/Knockout
Synonyms: SMN^-
Marker symbol: Smn1
Marker name: survival motor neuron 1
Marker synonyms: BCD541; C-BCD541; GEMIN1; SMA; SMA@; SMA1; SMA2; SMA3; SMA4; Smn; SMNC; SMNT; T-BCD541; TDRD16A; TDRD16B
Chromosome: 13
Strain of origin: 129P2/OlaHsd
Expressed genes: lacZ,beta-galactosidase,E. coli
Site of expression: The expression of the lacZ gene in tissues where Smn is normally expressed was noted.
Molecular note: A lacZ-neo cassette was inserted into exon 2 by homologous recombination resulting in an in-frame fusion of lacZ to exon 2. Homozygous mutant embryos were identified up to 80 hours post coitum. The expression of the lacZ gene in tissues where Smn is normally expressed was noted.

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
Synonyms: SMN2; Tg(SMN2)89Ahmb
Marker symbol: Grm7
Marker name: glutamate receptor, metabotropic 7
Marker synonyms: 6330570A01Rik; E130018M02Rik; GLUR7; Gpr1g; GPRC1G; MGLU7; mGlu7a receptor; mGluR7; NEDSHBA; PPP1R87
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.

Allele

Symbol: Tg(SMN2-SMN1*Q282A)2Ahmb
Name: transgene insertion 2, Arthur H M Burghes
MGI accession ID: MGI:6189119
Generation method: Transgenic
Attributes: Inserted expressed sequence; Humanized sequence
Synonyms: SMNQ282A
Marker symbol: Tg(SMN2-SMN1*Q282A)2Ahmb
Marker name: transgene insertion 2, Arthur H M Burghes
Chromosome: 11
Strain of origin: FVB/N
Molecular note: The transgenic construct contains a human SMN1 cDNA carrying the p.Gln282Ala missense mutation in exon 7 under the control of a 4.1 kb SMN promoter derived from the SMN2 gene. Founder line 2 carries eight copies of the transgene. The transgene integrated on chromosome 11 (70055009-70372513; mouse mm10]) resulting in a duplication of the 317 kb genomic region between the two identified fusion points. This duplicated region includes several loci (Asgr1, Asgr2, Mgl2, Clec10a, Slc16a11, Slc16a13, Bcl6b, Mir497b, Rnasek, Alox12 and Alox15).