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.
