STOCK Grm7^{Tg(SMN2)89Ahmb} Smn1^tm1Msd Tg(SMN1-SMN2*)16Cll/CllJ

Stock number: 018916
Product name: SMN2;SMN^RT;Smn-
Research areas: Developmental Biology Research, Neurobiology Research, Research Tools

Description

The SMN2;SMN^RT;Smn- triple mutant mouse expresses a modified SMNdelta7 cDNA with a single nucleotide change that allows read-through of the exon 8 stop codon; resulting in increased stability and a modestly more functional SMN protein. These SMN2;SMN^RT;Smn- mice are an intermediate SMN2-based model for studying proximal spinal muscular atrophy (SMA); having a longer lifespan than the SMNdelta7 model (Stock No. 005025), but with a phenotype severe enough for relatively rapid analysis of therapeutics.

Detailed description

The SMN2;SMN^RT;Smn- triple mutant mouse harbors two transgenes and a single targeted mutation. The Tg(SMN2)89Ahmb transgene (SMN2) expresses the entire human SMN2 gene, whereas no endogenous Smn1 expression is observed from the Smn1 null targeted mutation (Smn1^tm1Msd; also called Smn1^-). The SMN read-through transgene (SMN^RT) has an identical SMN cDNA as the SMNdelta7 cDNA except that a single nucleotide change in exon 8 converts the SMNdelta7 stop codon to tyrosine, allowing read-through and addition of four more amino acids to the expressed SMN protein; resulting in increased stability and a modestly more functional SMN protein. Regarding expression levels, SMN protein levels in hemizygous SMN^RT transgenic line 16 mice are similar to hemizygous SMNdelta7 mice in brain, spinal cord and skeletal muscle, and the SMN protein levels in both animals are significantly reduced compared to unaffected animals. The SMN^RT transgene in line 16 has a Mendelian inheritance pattern indicative of a single integration event. The donating investigator also reports the transgene copy number was stable via qPCR.

Mice that are homozygous for the Tg(SMN2)89 transgene, hemizygous for the SMN^RT transgene, and homozygous for the Smn1 null mutation (SMN2+/+;SMN^RT;Smn-/-) have mean lifespan of 34 days and begin showing signs of necrosis on the ears, tail and/or eye at approximately 40-50 days of age. Nearly 25% of the SMN2+/+;SMN^RT;Smn-/- animals live beyond 40 days, with several animals living beyond 70 days. Mice that are homozygous for both transgenes and homozygous for the Smn1 null mutation (SMN2+/+;SMN^RT/RT;Smn-/-) also exhibit mild necrosis and have similar lifespan.

Compared to the moderate type II SMA mouse model of similar genotype (SMN2+/+;SMNdelta7+/-;Smn-/- mice; Stock No. 005025), these SMN2+/+;SMN^RT;Smn-/- mice have extended lifespan, increased weight gain, enhanced strength and physical capabilities, significant improvement in skeletal muscle, intermediate NMJ pathology, and milder cardiac pathology. More specifically, the mean lifespan of SMN2+/+;SMN^RT;Smn-/- is 34 days; significantly longer than the severe type I SMA mouse model (SMN2+/+;Smn-/- mice; Stock No. 005024) (7 days) and the moderate type II SMA mouse model (SMN2+/+;SMNdelta7+/-;Smn-/- mice; Stock No. 005025) (13 days).

Development

These SMN2;SMN^RT;Smn- mice harbor several mutations/transgenes, as described below.

The creation of the Tg(SMN2)89 transgene (SMN2) and Smn1 null targeted mutation (Smn1^tm1Msd; also called Smn1^-) are described for the severe type I SMA mouse model on an FVB/NJ background; Stock No. 005024. Note the Tg(SMN2)89 transgene insertion site is on chromosome 6. These mice were bred to and maintained upon an FVB/NJ background prior to being used for breeding as below.

The SMN^RT transgene was designed by Dr. Christian L. Lorson (University of Missouri) with a 3.4 kbp promoter sequence from the human SMN1 gene, 5' UTR from the human SMN1 gene, a CDNA sequence encoding the SMN read-through protein (SMN^RT; see details below), and the 3' UTR from the human SMN1 gene. To create the SMN^RT CDNA sequence, the complete coding sequence of SMNdelta7 was obtained from Dr. Arthur H.M. Burghes (The Ohio State University Medical Center; see 005025). The SMNdelta7 cDNA sequence, already containing the silent C-to-T transition in exon 7 that results in the alternatively spliced transcript without exon 7, was further modified by Dr. Lorson to change the first stop codon in exon 8 from TAG (STOP) to TAT (tyrosine). After the tyrosine, four additional amino acids (serine, serine, threonine and lysine) are incorporated prior to recognizing the next stop codon. The resulting SMN cDNA sequence, modified to encode a modestly more functional protein, is referred to as SMN read-through (SMN^RT). The complete 5.2 kbp SMN^RT transgene was microinjected into the pronucleus of FVB/N embryos. Transgenic founder animals were bred to non-transgenic cohorts to establish the colony. Mice from founder line 16 had SMN protein expression levels most comparable with SMNdelta7 levels in disease relevant tissues, as well as a Mendelian inheritance pattern indicative of a single integration event. SMN^RT founder male 16 was bred to FVB.SMN2;Smn- females (Stock No. 005024) to generate the SMN2;SMN^RT;Smn- colony.

In 2014, SMN2;SMN^RT;Smn- animals with white coat color on a predominantly FVB genetic background (some C57BL/6 and 129 contributions from the FVB.SMN2;Smn- breeding) were sent to The Jackson Laboratory Repository as Stock No. 018916. Upon arrival, male mice were bred to non-affected females from the severe type I SMA strain (Stock No. 005024; hemizygous for the Tg(SMN2)89 transgene and wildtype at the Smn1 locus) to establish our live colony. Thereafter, this SMN2;SMN^RT;Smn- triple mutant strain was maintained by breeding mice homozygous for the two transgenes and either heterozygous for Smn1^tm1Msd or wildtype at the Smn1 locus.

Allele

Symbol: Smn1^tm1Msd
Name: targeted mutation 1, Michael Sendtner
MGI accession ID: MGI:2183946
Generation method: Targeted
Attributes: Reporter; Null/Knockout
Marker symbol: Smn1
Marker name: survival motor neuron 1
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: Tg(SMN1-SMN2*)16Cll
Name: transgene insertion 16, Christian L Lorson
MGI accession ID: MGI:5490785
Generation method: Transgenic
Attributes: Inserted expressed sequence; Humanized sequence
Marker symbol: Tg(SMN1-SMN2*)16Cll
Marker name: transgene insertion 16, Christian L Lorson
Chromosome: UN
Strain of origin: FVB/N
Expressed genes: SMN2,survival of motor neuron 2, centromeric,human
Site of expression: Reduced levels of SMN protein are expressed in brain, spinal cord, and skeletal muscle.
Molecular note: This transgene consists of 3.4 kb promoter and 5'-untranslated region from the human SMN1 gene, the complete coding sequence of human SMN2 cDNA containing the silent C to T transition in exon 7 that results in the alternatively spliced transcript without exon 7 (SMNdelta7) and a TAG to TAT alteration in the first stop codon in exon 8 (change from a stop codon to a tyrosine), and the 3'-UTR of SMN1. The TAG to TAT change in exon 8 allows for translational stop codon read-through of the SMNdelta7 and generates an extended SMN2 protein. Thirteen lines were produced.

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.