B6.129S1-Htt^em2(HTT)Chdi/J

Stock number: 036156
Product name: Q7-iExon49 (Line 7) B6J
Strain synonyms: Q7-iExon49 (Line 7)
Research areas: Mouse/Human Gene Homologs, Neurobiology Research, Research Tools

Description

The Q7-iExon49 (Line 7) knock-in allele has an entire human HTT intron 49, which contains a putative splicing site to produce an alternative exon (iExon 49), replacing the mouse Htt intron 49 sequence. These mice are wildtype for the polyglutamine expansion in exon 1 (~7 CAG repeats). Q7-iExon49 (Line 7) knock-in mice may be useful for studying Huntington's disease pathology as well as potential DNA and RNA-targeting therapies.

Note that the donating laboratory has also made available a Q107-iExon49 mouse line (Stock No. 036155) and a Q175-iExon49 mouse line (Stock No. 036157).

Detailed description

Huntington's disease (HD) is an autosomal dominant, fatal neurodegenerative disorder characterized by uncontrolled movements, psychiatric disturbances and cognitive impairment. HD is caused by an unstable trinucleotide (polyglutamine) repeat expansion in the human huntingtin gene (HTT).

The Q7-iExon49 (Line 7) knock-in line was generated by replacing mouse Htt intron 49 with an entire human HTT intron 49 sequence, which contains a putative splicing site to produce an alternative exon (iExon 49). These mice also carry the mouse wildtype Htt polyglutamine expansion in exon 1 (~7 CAG repeats). The genetic modification was accomplished by CRISPR-Cas9 mediated gene editing in the same project to generate a related mouse line Q175-iExpn49 (Stock No. 036157). Heterozygous Q7-iExon49 (Line 7) knock-in mice are viable and fertile. No additional characterization has been completed on this mouse line to date (September 2023). These mice may be useful controls when studying HD pathology as well as potential DNA and RNA-targeting therapies.

This Huntington's disease mouse model is available by way of a collaborative effort between CHDI Foundation and The Jackson Laboratory.

Note that the donating laboratory has also made available a Q107-iExon49 mouse line (Stock No. 036155) and a Q175-iExon49 mouse line (Stock No. 036157).

 

Development

The Q7-iExon49 (Line 7) knock-in allele was generated using CRISPR/Cas9 endonuclease-mediated genome editing. The targeting vector was designed to replace the mouse huntingtin (Htt) intron 49, on chromosome 5, with a human HTT intron 49 sequence, which contained a putative splicing site to produce an alternative exon (iExon 49). Two guide RNAs (gRNA) were used to target mouse Htt intron 49 sequences either close to the juncture with exon 49 (gRNA-1: 5'-CTCACATATCCTGTCCTCAC-3') or to exon 50 (gRNA-2: 5'-CTGTGTTAGAATGCTCACCC-3'). The donor template consisted of the human HTT intron 49 sequence flanked by mouse homology arms which resulted in replacement of the mouse Htt intron 49 region with human HTT intron 49 in its entirety without changing amino acid sequences in either flanking exon. The gRNAs, ssDNA donor template, and the Cas9 nuclease mRNA were introduced into the cytoplasm of zygotes, with well recognized pronuclei, derived from C57BL/6NTac females mated with heterozygous Htt-Q175 males (same embryos used in the project to generate a related line Q175-iExpn49 Stock No. 036157). Correctly targeted pups were identified by PCR and sequencing. PCR was also used to determine cis-configuration of iExon 49 knock-in with the wild-type mouse (Q7) allele. The resulting Q7-iExon49 (Line 7) mice were crossed to C57BL/6NTac mice for one generation, to reduce off-target events. Subsequently, the Q7-iExon49 (Line 7) mice were backcrossed to C57BL/6J (Stock No. 000664) using a speed congenic protocol (89-95% congenic on C57BL/6J as of August 2021). The colony was further maintained by backcrossing heterozygous males to C57BL/6J inbred females (January 2024). To establish a live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes.

Allele

Symbol: Htt^em2(HTT)Chdi
Name: endonuclease-mediated mutation 2, David Howland
MGI accession ID: MGI:7576151
Generation method: Endonuclease-mediated
Attributes: Humanized sequence
Marker symbol: Htt
Marker name: huntingtin
Marker synonyms: C430023I11Rik; Hd; Hdh; htt; IT15; LOMARS
Chromosome: 5
Strain of origin: C57BL/6NTac
Molecular note: The allele was generated using CRISPR/Cas9 endonuclease-mediated genome editing. The targeting vector was designed to replace the mouse huntingtin (Htt) intron 49, on chromosome 5, with a human HTT intron 49 sequence, which contained a putative splicing site to produce an alternative exon (iExon 49). This is Line7.