• Background: MSH3 is a key component of the DNA mismatch repair (MMR) system, forming the MutSβ heterodimer with MSH2. In Huntington’s disease, MutSβ aberrantly recognizes and process CAG repeat sequences in the HTT gene, leading to their progressive somatic expansion rather than correction, which accelerates disease onset and progression.
• Targeting strategy: The genome sequences between 2-24 of mouse Msh3 gene were depleted in B-Msh3 KO mice. The mouse Msh3 gene transcription and translation will be disrupted. As a result, mouse MSH3 protein will not be expressed anymore.
• Validation: Mouse Msh3 mRNA was only detectable in wild-type mice, but not in homozygous B-Msh3 KO mice. Mouse MSH3 protein was detectable in wild-type mice and heterozygous B-hMSH3 mice, but not in homozygous B-Msh3 KO mice.

Gene targeting strategy for B-Msh3 KO mice. The genome sequences between 2-24 of mouse Msh3 gene were depleted in B-Msh3 KO mice. The mouse Msh3 gene transcription and translation will be disrupted. As a result, mouse MSH3 protein will not be expressed anymore.

Strain specific analysis of Msh3 mRNA expression in wild-type C57BL/6JNifdc mice and homozygous B-Msh3 KO mice by RT-PCR. Brain RNA were isolated from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-Msh3 KO mice (-/-), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse Msh3 primers. Mouse Msh3 mRNA was only detectable in wild-type mice, but not in homozygous B-Msh3 KO mice.

Western blot analysis of MSH3 protein expression in homozygous B-Msh3 KO mice. Various tissue lysates were collected from wild-type C57BL/6JNifdc mice (+/+) , heterozygous B-hMSH3 mice (H/+) and homozygous B-Msh3 KO mice (-/-), and then analyzed by western blot with anti-mouse MSH3 antibody (Millipore, MABE324). 40 μg total proteins were loaded for western blotting analysis. Mouse MSH3 protein was detectable in wild-type mice and heterozygous B-hMSH3 mice, but not in homozygous B-Msh3 KO mice.