• 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 P2A-human MSH3 CDS expression cassette followed by the human 3’UTR is in-frame inserted immediately after exon 2 of the mouse Msh3 gene, replacing the sequence from part of exon 2 to the 3’UTR of the mouse Msh3 gene. The human MSH3 protein expression will be driven by the mouse endogenous Msh3 promoter, while the transcription and translation of the mouse Msh3 gene will be disrupted.
• Validation: Human MSH3 mRNA was detectable only in heterozygous B-hMSH3 mice but not in wild-type mice. And human MSH3 sequences were confirmed via Sanger sequencing. Human MSH3 was only detectable in brain from heterozygous B-hMSH3 mice, but not in wild-type mice.
• Application: This model is useful in studying the role of MSH3 and the therapeutic efficacy targeting MSH3 in HD.

Gene targeting strategy for B-hMSH3 mice. The P2A-human MSH3 CDS expression cassette followed by the human 3’UTR is in-frame inserted immediately after exon 2 of the mouse Msh3 gene, replacing the sequence from part of exon 2 to the 3’UTR of the mouse Msh3 gene. The human MSH3 protein expression will be driven by the mouse endogenous Msh3 promoter, while the transcription and translation of the mouse Msh3 gene will be disrupted.

Strain specific analysis of MSH3 mRNA expression in wild-type C57BL/6JNifdc mice and heterozygous B-hMSH3 mice by RT-PCR. Brain RNA were isolated from wild-type C57BL/6JNifdc mice (+/+) and heterozygous B-hMSH3 mice (H/+), then cDNA libraries were synthesized by reverse transcription, followed by PCR with mouse or human MSH3 primers. Mouse Msh3 mRNA was detectable both in wild-type mice and heterozygous B-hMSH3 mice. Human MSH3 mRNA was detectable only in heterozygous B-hMSH3 mice but not in wild-type mice. And human MSH3 sequences were confirmed via Sanger Sequencing.

Western blot analysis of MSH3 protein expression in heterozygous B-hMSH3 mice. Brain 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 species-specific anti-MSH3 antibodies. 40 μg total protein was loaded for western blotting analysis. Mouse MSH3 was absent in B-Msh3 KO mice but present in wild-type and heterozygous B-hMSH3 mice, with heterozygote B-hMSH3 mice showing reduced band intensity relative to wild-type mice for mouse-specific antibody results. Human MSH3 protein was only detectable in heterozygous B-hMSH3 in brain.

Strain specific analysis of MSH3 mRNA expression in wild-type C57BL/6JNifdc mice and heterozygous B-hMSH3 mice by RT-qPCR. Brain, spleen and liver RNA were isolated from wild-type C57BL/6JNifdc mice (+/+) and heterozygous B-hMSH3 mice (H/+, male, n=3, 9-week-old), then cDNA libraries were synthesized by reverse transcription, followed by RT-qPCR with mouse or human MSH3 primers. Mouse Gapdh served as an internal reference gene. Human MSH3 mRNA was detectable only in heterozygous B-hMSH3 mice but not in wild-type mice, and MSH3 expression in other tissues was normalized to that in the brain.