德晋贵宾厅

• Description: SOD1 is critical for detoxifying superoxide radicals in the nervous system. The G93A mutation leads to a toxic gain of function, and the accumulation of misfold SOD1 protein can form aggregates that are thought to be detrimental to neuronal health, resulting in impaired antioxidant defense, mitochondrial dysfunction, glutamate dysregulation, neuroinflammation and finally leading to motor neuron degeneration. Gene therapy targeting the SOD1 gene itself, including CRISPR/Cas9 and antisense oligonucleotides, are being explored for their potential to reduce or eliminate the toxic SOD1*G93A protein.
• Application: this mice can be used for nucleic acids drug screening targeting SOD1 toxic protein.
• Validation: Human SOD1 mRNA was exclusively detectable in transgenic B-Tg(hSOD1*G93A) mice but not in wild-type mice. Misfold human SOD1 protein was exclusively detectable in hippocampus, cortex, cerebellum, spinal cord, heart and muscle

Gene targeting strategy for B-Tg(hSOD1*G93A) mice. The BAC DNA containing the whole human SOD1*G93A genome sequence was randomly inserted into mouse genome in B-Tg(hSOD1*G93A) mice.

Strain specific analysis of SOD1 mRNA expression in wild-type C57BL/6 mice and B-Tg(hSOD1*G93A) mice by RT-PCR. Brain RNA were isolated from wild-type C57BL/6 mice (+/+) and transgenic B-Tg(hSOD1*G93A) mice (TG/+), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human SOD1 primers. Mouse Sod1 mRNA were detectable in wild-type C57BL/6 mice and transgenic mice. Human SOD1 mRNA was exclusively detectable in transgenic B-Tg(hSOD1*G93A) mice but not in wild-type mice.

Western blot analysis of SOD1 protein expression in transgenic B-Tg(hSOD1*G93A) mice. Various tissue lysates were collected from wild-type C57BL/6 mice (+/+)  and transgenic B-Tg(hSOD1*G93A) mice (TG/+), and then analyzed by western blot with anti-SOD1 antibody (abcam, ab52950) and anti-misfolded human SOD1 (medimabs, MM-0070-P). 40 μg total proteins were loaded for western blotting analysis. Misfold human SOD1 was exclusively detectable in hippocampus, cortex, cerebellum, spinal cord, heart and muscle.

The inhibitory efficiency of the nucleic acid drugs against human SOD1 in B-Tg(hSOD1*G93A) mice. B-Tg(hSOD1*G93A) mice (male, 8-week-old) were randomly divided into two groups (n=3). The human SOD1 targeted nucleic acid drugs, Tofersen analog (125 μg/mouse, Day 0, purchased from MCE, HY-132580) and vehicle (artificial cerebrospinal fluid) were administered by intracerebroventricular injection to the mice individually and brain samples were collected at the indicated time points (Days 28). (A) Schematic timeline of the in vivo experimental procedure. (B) Human SOD1 mRNA expression levels in cortex, hippocampus, cerebellum were decreased in Tofersen-analog treatment group (G2) compared to vehicle group (G1). B-Tg(hSOD1*G93A) mice can be used in small nucleic acid drug screening research. Significance was determined by t-test, *P<0.05, **P<0.01. Values are expressed as mean ± SEM.