The SMN2;SMNRT;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;SMNRT;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.
Christian L Lorson, University of Missouri
Genetic Background | Generation |
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Allele Type | Gene Symbol | Gene Name |
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Targeted (Reporter, Null/Knockout) | Smn1 | survival motor neuron 1 |
Allele Type |
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Transgenic (Hypomorph, Inserted expressed sequence, Humanized sequence) |
Allele Type |
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Transgenic (Inserted expressed sequence, Humanized sequence) |
The SMN2;SMNRT;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 (Smn1tm1Msd; also called Smn1-). The SMN read-through transgene (SMNRT) 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 SMNRT 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 SMNRT 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 SMNRT transgene, and homozygous for the Smn1 null mutation (SMN2+/+;SMNRT;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+/+;SMNRT;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+/+;SMNRT/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+/+;SMNRT;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+/+;SMNRT;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).
These SMN2;SMNRT;Smn- mice harbor several mutations/transgenes, as described below.
The creation of the Tg(SMN2)89 transgene (SMN2) and Smn1 null targeted mutation (Smn1tm1Msd; 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 SMNRT 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 (SMNRT; see details below), and the 3' UTR from the human SMN1 gene. To create the SMNRT 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 (SMNRT). The complete 5.2 kbp SMNRT 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. SMNRT founder male 16 was bred to FVB.SMN2;Smn- females (Stock No. 005024) to generate the SMN2;SMNRT;Smn- colony.
In 2014, SMN2;SMNRT;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;SMNRT;Smn- triple mutant strain was maintained by breeding mice homozygous for the two transgenes and either heterozygous for Smn1tm1Msd or wildtype at the Smn1 locus.
Expressed Gene | lacZ, beta-galactosidase, E. coli |
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Site of Expression | The expression of the lacZ gene in tissues where Smn is normally expressed was noted. |
Expressed Gene | SMN2, survival of motor neuron 2, centromeric, human |
Site of Expression | Grm7Tg(SMN2)89Ahmb |
Expressed Gene | 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. |
Allele Name | targeted mutation 1, Michael Sendtner |
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Allele Type | Targeted (Reporter, Null/Knockout) |
Allele Synonym(s) | SMN- |
Gene Symbol and Name | Smn1, survival motor neuron 1 |
Gene Synonym(s) | |
Expressed Gene | lacZ, beta-galactosidase, E. coli |
Site of Expression | The expression of the lacZ gene in tissues where Smn is normally expressed was noted. |
Strain of Origin | 129P2/OlaHsd |
Chromosome | 13 |
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. |
Mutations Made By | Michael Sendtner |
Allele Name | transgene insertion 89, Arthur H M Burghes |
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Allele Type | Transgenic (Hypomorph, Inserted expressed sequence, Humanized sequence) |
Allele Synonym(s) | SMN2; Tg(SMN2)89Ahmb |
Gene Symbol and Name | Grm7, glutamate receptor, metabotropic 7 |
Gene Synonym(s) | |
Promoter | SMN2, survival of motor neuron 2, centromeric, human |
Expressed Gene | SMN2, survival of motor neuron 2, centromeric, human |
Site of Expression | Grm7Tg(SMN2)89Ahmb |
Strain of Origin | FVB/N |
Chromosome | 6 |
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. |
Mutations Made By | Arthur Burghes, The Ohio State University |
Allele Name | transgene insertion 16, Christian L Lorson |
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Allele Type | Transgenic (Inserted expressed sequence, Humanized sequence) |
Allele Synonym(s) | SMNRT |
Gene Symbol and Name | Tg(SMN1-SMN2*)16Cll, transgene insertion 16, Christian L Lorson |
Gene Synonym(s) | |
Promoter | SMN1, survival of motor neuron 1, telomeric, human |
Expressed Gene | 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. |
Strain of Origin | FVB/N |
Chromosome | UN |
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. |
The Tg(SMN2)89 transgene on chromosome 6, the Smn1 null targeted mutation (Smn1tm1Msd) on chromosome 13, and the randomly inserted SMNRT transgene (Tg(SMN1-SMN2*)16Cll) are not linked and will segregate independently. Breeding pairs offered by The Jackson Laboratory Repository are homozygous for the two transgenes and heterozygous for Smn1tm1Msd. These breeding pairs are phenotypically normal and do not exhibit symptoms of neuropathology. Offspring resulting from the mating of breeder pairs can posses the following genotypes:
1. Homozygous for both transgenes and homozygous for the targeted mutation (25%)
2. Homozygous for both transgenes and heterozygous for the targeted mutation (50%)
3. Homozygous for both transgenes and wildtype at the Smn1 locus (25%)
Mice that are homozygous for both transgenes and homozygous for the targeted mutation will display the SMA-like phenotype. Mice homozygous for both transgenes and heterozygous for the targeted mutation will not display the SMA-like phenotype but can be mated with each other to generate additional affected mice. Mice homozygous for both transgenes and wildtype at the Smn1 locus will also not exhibit an SMA-like phenotype but can be employed as control mice depending on the nature of the experiment.
When using the SMN2;SMNRT;Smn- mouse strain in a publication, please cite the originating article(s) and include JAX stock #018916 in your Materials and Methods section.
Facility Barrier Level Descriptions
Service/Product | Description | Price |
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Homozygous for Tg(SMN2)89Ahmb ,Heterozygous or Wildtype for Smn1<tm1Msd> , Hemizygous for Tg(SMN1-SMN2*)16Cll |
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The Jackson Laboratory has rigorous genetic quality control and mutant gene genotyping programs to ensure the genetic background of JAX® Mice strains as well as the genotypes of strains with identified molecular mutations. JAX® Mice strains are only made available to researchers after meeting our standards. However, the phenotype of each strain may not be fully characterized and/or captured in the strain data sheets. Therefore, we cannot guarantee a strain's phenotype will meet all expectations. To ensure that JAX® Mice will meet the needs of individual research projects or when requesting a strain that is new to your research, we suggest ordering and performing tests on a small number of mice to determine suitability for your particular project. We do not guarantee breeding performance and therefore suggest that investigators order more than one breeding pair to avoid delays in their research.
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