STOCK Gt(ROSA)26Sor^{tm1.1(rtTA,EGFP)Nagy} Grm7^{Tg(SMN2)89Ahmb} Smn1^tm1Msd Tg(SMN2*delta7)4299Ahmb Tg(tetO-SMN2,-luc)#aAhmb/J

Stock number: 017596
Research areas: Developmental Biology Research, Neurobiology Research, Research Tools

Description

These Tg(SMN2)89; Smn1^+/-; Tg(SMNΔ7)4299; ROSA26^rtTA; "old" Luci-TRE-SMN mice allow high levels of both luciferase and full-length human SMN expression to be regulated by the addition/removal of doxycycline. These may be useful as a fluorescent reporter and/or Tet-On/Tet-Off tool strain for doxycycline-inducible rescue of Type II (moderate) proximal spinal muscular atrophy (SMA).

Detailed description

These Tg(SMN2)89; Smn1^+/-; Tg(SMNΔ7)4299; ROSA26^rtTA; "old" Luci-TRE-SMN mice harbor multiple mutations/transgenes.
The moderate Type II SMA mouse model (see Stock No. 005025) is defined by mice homozygous for the Tg(SMN2)89 transgene (SMN2+/+), homozygous for the Smn1^tm1Msd mutation (Smn1^-/-), and homozygous for the Tg(SMNΔ7)4299 transgene (SMNΔ7+/+). At birth, SMN2+/+; Smn1^-/-; SMNΔ7+/+ mice are small with neuromuscular defects by 5 days old that progress to abnormal gait, hind limb weakness/immobility, and eventually death at approximately 13 days of age.
The ROSA26^rtTA targeted mutation results in widespread reverse tetracycline transactivator (rtTA)-expression. The Luci-TRE-SMN transgene has expression of both luciferase and a full-length (exons 1-8) human SMN under the control of a bi-directional tet-responsive promoter. When together in mice, addition of doxycycline (dox) results in widespread and simultaneous expression of luciferase and full-length human SMN expression. When doxycycline (dox) food is introduced to the dams at pup birth, neonatal brain and spinal cord exhibit high levels of luciferase activity beginning at ~48 hours post-induction and increasing to maximum after 10 days on dox. Similarly, full-length SMN is induced at 3 days post-induction and continues to increase to 10 days post-induction (in a similar manner to endogenous SMN protein). Similar results are observed following dox food administration to 28 day old mice. Following 10 days of dox, western blot shows luciferase and human SMN expression in widespread tissues including muscle, brain, liver, heart, spinal cord, heart, kidney, lung, and spleen. In addition, dox induction results in increased SMN expression in motor neurons as well as gems within those motor neurons. Following 28 days of dox administration, the subsequent removal of dox for 10 days results in luciferase and SMN expression levels dropping to non-induced levels.

These Tg(SMN2)89; Smn1^+/-; Tg(SMNΔ7)4299; ROSA26^rtTA; "old" Luci-TRE-SMN mice allow high levels of both luciferase and full-length human SMN expression to be regulated by the addition/removal of dox. Mice heterozygous for the Smn1^tm1Msd mutation (Smn1^+/-) and homozygous for the other mutations/transgenes are viable and fertile with no reported abnormalities or symptoms of neuropathology. The phenotype of mice homozygous for the Tg(SMN2)89 transgene (SMN2+/+), homozygous for the Smn1^tm1Msd mutation (Smn1^-/-), homozygous for the Tg(SMNΔ7)4299 transgene (SMNΔ7+/+), homozygous for the ROSA26^rtTA mutation (ROSA26^rtTA+/+), and hemizygous or homozygous for the Luci-TRE-SMN is described below.
In the absence of dox, the SMN2+/+; Smn1^-/-; SMNΔ7+/+; ROSA26^rtTA+/+; Luci-TRE-SMN mice (called non-induced SMA mice) die on average of 13 days old with no extension of lifespan (no mice live past ~18 days old). As expected, this is the same phenotype as moderate Type II SMA mouse model (see Stock No. 005025).

When pregnant dams are given dox beginning coincident with gestating pup embryonic day (E)13, the high levels of full-length human SMN expression over the early postnatal period in SMN2+/+; Smn1^-/-; SMNΔ7+/+; ROSA26^rtTA+/+; Luci-TRE-SMN mice results in substantial rescue of SMA: the mice have no marked motor deficits, near normal motor neuron electrophysiology/neuromuscular junction (NMJ) function, fully developed NMJs, and average lifespan of 132 days (~30% of mice live over 200 days). Additionally, early postnatal SMN induction followed by removal of dox from 28-58 days of age, result in no morphological or electrophysiological abnormalities at the NMJ and no overt motor phenotype. Later dox administration to nursing dams rescues SMA but to a lesser extent: dox treatment to nursing dams at pup birth/postnatal day 1 (P0/P1) results in average lifespan of 86 days (~15% of mice live over 200 days), while dox treatment to nursing dams at pup postnatal day 2 (P2) leads to average lifespan of 25 days.

The donating investigator reports the following doxycycline approach. For embryonic induction, pregnant females were given water containing 500 mg/ml of doxycycline. At pup birth, doxycycline food (200 mg/kg) was introduced to the cage and the doxycycline water removed. For postnatal induction, high-concentration doxycycline food (6 g/kg) was administered to the mother; thus the pups received doxycycline in the mother's milk. Of note, high-concentration doxycycline food (6 g/kg) was found to severely affect birth rates if administered to pregnant mice.

Development

These Tg(SMN2)89; Smn1^+/-; Tg(SMNΔ7)4299; ROSA26^rtTA; "old" Luci-TRE-SMN mice harbor several mutations/transgenes, as described below.

Mice homozygous for the Tg(SMN2)89 transgene (SMN2+/+), homozygous for the Smn1^tm1Msd mutation (Smn1^-/-), and homozygous for the Tg(SMNΔ7)4299 transgene (SMNΔ7+/+) define the moderate type II SMA mouse model. The Tg(SMN2)89; Smn1^-/-; Tg(SMNΔ7)4299 mice are described as Stock No. 005025. These mice were bred to and maintained upon an FVB/N genetic background prior to being used for breeding as below.

To generate the widespread rtTa-expressing mutation ROSA26^rtTA, mice with the flox-STOP ROSA26-rtTA mutation (ICR;129 background; Stock No. 005572) were bred with Sox2-Cre transgenic mice (FVB;Swiss Webster;C57BL6;CBA background; see Stock No. 004783). The resulting offspring with pan-deletion of the lox-flanked STOP cassette/widespread expression of rtTA were bred to and maintained upon a C57BL/6 genetic background prior to being used as a donor for the Gt(ROSA)26Sor^{tm1.1(rtTA,EGFP)Nagy} allele (see below).

The Luci-TRE-SMN transgene was created by Dr. Arthur HM Burghes (The Ohio State University). The Luci-TRE-SMN transgenic construct used here ["old" pBI-L-SMN] was designed with a luciferase sequence on one side, and a full-length human SMN2 cDNA sequence (encoding exons 1-8) on the other side of the P_bi-1 bi-directional tetracycline-responsive promoter. The 1.37 kb human SMN2 cDNA sequence was derived via RT-PCR performed on total RNA isolated from SMA type 1-derived patient fibroblast cells (Coriell Institute GM 3813; shown to express both full-length and truncated SMN2 transcripts). Compared to the "new" pBI-L-SMN(B) construct, the "old" pBI-L-SMN construct used here retains part of a hemagglutinin epitope tag sequence between the P_bi-1 promoter and 5' end of SMN2 cDNA. The P_bi-1 promoter contains the Tet-responsive element (TRE; seven copies of the 42-bp tet operator sequence [tetO]) placed between two minimal CMV enhancer-less promoters. The "old" Luci-TRE-SMN transgene was microinjected into the male pronucleus of FVB/N embryos. Founder mice were bred with Camk2a-tTA mice (FVB background; see Stock No. 003010) to determine which lines had high dox-inducible levels of luciferase and SMN in brain. The high-expressing "old" Luci-TRE-SMN founder line was identified and used as below.

To generate the Tg(SMN2)89; Smn1^+/-; Tg(SMNΔ7)4299; ROSA26^rtTA; "old" Luci-TRE-SMN animals, mice from the moderate type II SMA colony (Tg(SMN2)89+/+; Smn1^+/-; Tg(SMNΔ7)4299+/+) were bred to ROSA26^rtTA mice. Because the ROSA26^rtTA mutation and Tg(SMN2)89 transgene insertion lie relatively close to each other on chromosome 6, additional crosses were performed to FVB/N wildtype mice; and a single mouse with a recombinant chromosome containing both Tg(SMN2)89 and ROSA26^rtTA was identified. Next, those mice were bred to also harbor the "old" Luci-TRE-SMN transgene. The resulting Tg(SMN2)89; Smn1^+/-; Tg(SMNΔ7)4299; ROSA26^rtTA; "old" Luci-TRE-SMN mice (on a mixed FVB/N and C57BL/6 genetic background) were sent to The Jackson Laboratory Repository as Stock No. 017596. Upon arrival, males were used to cryopreserve sperm. To establish the living mouse colony, an aliquot of the frozen sperm was used to fertilize oocytes from moderate type II SMA females (Stock No. 005025).

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: Tg(tetO-SMN2,-luc)#aAhmb
Name: transgene insertion, Arthur H M Burghes
MGI accession ID: MGI:5286602
Generation method: Transgenic
Attributes: Reporter; Inducible; Inserted expressed sequence; Humanized sequence
Marker symbol: Tg(tetO-SMN2,-luc)#aAhmb
Marker name: transgene insertion, Arthur H M Burghes
Chromosome: UN
Strain of origin: FVB/N
Expressed genes: luc,luciferase,firefly; SMN2,survival of motor neuron 2, centromeric,human
Site of expression: Following 10 days of dox, western blot shows luciferase and human SMN expression in widespread tissues including muscle, brain, liver, heart, spinal cord, heart, kidney, lung, and spleen. In addition, dox induction results in increased SMN expression in motor neurons as well as gems within those motor neurons.
Molecular note: The tetracycline response element with two minimal CMV promoters drives inducible expression of human SMN (exons 1 through 7) and a luciferase gene. Two lines were generated. The pound symbol (#) is used when no line is specified and/or lines are pooled.

Allele

Symbol: Gt(ROSA)26Sor^{tm1.1(rtTA,EGFP)Nagy}
Name: targeted mutation 1.1, Andras Nagy
MGI accession ID: MGI:5286599
Generation method: Targeted
Attributes: Reporter; Transactivator
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Chromosome: 6
Strain of origin: (129X1/SvJ x 129S1/Sv)F1-Kitl⁺
Expressed genes: GFP,Green Fluorescent Protein,
Site of expression: Widespread reverse tetracycline transactivator (rtTA)-expression.
Molecular note: A targeting vector containing a floxed Pgk-neo-pA cassette and a rtTA-IRES-EGFP-pA cassette was inserted into intron 1 of the locus. Cre-mediated recombination removed the floxed neo cassette allowing the ROSA26 promoter to drive expression of rtTA and EGFP. Presence of doxycycline results in the formation of an active transcriptional activator and the activation of the responder transgene.

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: 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.