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.