B6.129S1-Htt^tm8(HTT*)Szi Htt^em3(HTT)Chdi/J

Stock number: 036157
Product name: Q175-iExon49 (Line 4) B6J
Strain synonyms: Q175-iExon49 (Line 4)
Research areas: Mouse/Human Gene Homologs, Neurobiology Research, Research Tools

Description

The Q175-iExon49 (Line 4) knock-in allele has the human HTT exon 1 sequence with a polyglutamine expansion comprised of a pure CAG expansion [(CAG)₁₉₀CAACAG] replacing the mouse Htt exon 1 as well as the 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 may be useful for studying Huntington's disease pathology as well as potential DNA and RNA-targeting therapies.

Stock No. 036157 harbors an Htt mutant allele with two modifications on the same homologous chromosome (in cis): mouse exon 1 replaced with human HTT exon 1 having polyglutamine expansion (Htt-Q175; Htt^tm8(HTT*)Szi) and mouse intron 49 replaced with the human HTT intron 49 (iExon 49; Htt^em3(HTT)Chdi). Because the iExon 49 targeting/modification was unlikely to affect the original Htt-Q175 mutation, the strain nomenclature lists both mutations.

Note that the donating laboratory has also made available a Q107-iExon49 mouse line (Stock No. 036155) and a control Q7-iExon49 mouse line (Stock No. 036156).

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 Q175-iExon49 (Line 4) knock-in line was generated by replacing mouse Htt exon 1 with a human HTT exon 1 sequence containing a polyglutamine expansion comprised of a pure CAG expansion [~(CAG)₁₉₀CAACAG] and replacing mouse Htt intron 49 with an entire human HTT intron 49, which contains a putative splicing site to produce an alternative exon (iExon 49). The genetic modification was accomplished by CRISPR-Cas9 mediated gene editing to insert the human intron 49 sequence in embryos derived from the Q175 HD mouse. Of note, the CAG repeat number is subject to germline and somatic instability, and may expand or contract (see note on CAG instability below). Heterozygous Q175-iExon49 (Line 4) 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 control Q7-iExon49 mouse line (Stock No. 036156).

Note on CAG instability: Expanded CAG sequences in huntingtin exon 1 may be subject to germline or somatic instability and may expand or contract. When using lines with unstable CAG repeat length, it is strongly recommended the CAG repeat number be quantified in all the experimental animals; all animals in all experimental groups should carry comparable CAG repeat sizes. CAG repeat sizing of HD mice should be done using high-resolution methods as assays based on agarose gel electrophoresis typically do not provide sufficient resolution to accurately measure CAG repeat numbers. If labs do not have access to the appropriate equipment for determining CAG repeat length, CAG repeats can be evaluated on a fee-for-service basis by TransnetYX, Inc.

Development

The Q175-iExon49 (Line 4) 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. Correctly targeted pups were identified by PCR and sequencing. PCR was also used to determine cis-configuration of iExon 49 knock-in with the Htt-Q175 allele. The resulting Q175-iExon49 (Line 4) mice were crossed to C57BL/6NTac mice for one generation, to reduce off-target events. Subsequently, the Q175-iExon49 (Line 4) mice were backcrossed to C57BL/6J (Stock No. 000664) for 4 generations using a speed congenic protocol (89-96% congenic on C57BL/6J as of December 2021). The colony was further maintained by backcrossing heterozygous males to C57BL/6J inbred females (January 2024). To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes.

The Htt-Q175 knock-in (KI) mouse line was generated as a substrain from the Htt^LacQ140 strain (Stock No. 037918). The Htt^LacQ140 targeting vector was designed, by Dr. Scott Zeitlin, to insert a cassette containing synthetic LacO sequences flanking the Htt transcriptional start site followed by a chimeric mouse/human Htt/HTT sequence with human exon 1 into the mouse Htt locus on chromosome 5. The human sequence encodes the N terminal sequence including an expanded CAG repeat containing 140Q and the adjacent sequence encoding the human proline-rich region. A loxP-flanked PGK-neomycin cassette was inserted in reverse orientation 1.3 kb upstream of transcription initiation site. The construct was electroporated into 129S1/Sv-derived W9.5 embryonic stem (ES) cells and correctly targeted ES cells were injected into blastocysts. The resulting chimeras were bred with C57BL/6J mice and maintained on a congenic C57BL/6J background as Stock No. 037918. A subset of Htt^LacQ140 mice with expanded CAG repeats (~Q175) were utilized to generate the Htt-Q175 KI substrain and utilized to generate the Q175-iExon49 (Line 4) knock-in mouse line as described above.

Allele

Symbol: Htt^em3(HTT)Chdi
Name: endonuclease-mediated mutation 3, David Howland
MGI accession ID: MGI:7576152
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 Line4.

Allele

Symbol: Htt^tm8(HTT*)Szi
Name: targeted mutation 8, Scott Zeitlin
MGI accession ID: MGI:7442513
Generation method: Targeted
Attributes: Inserted expressed sequence; Humanized sequence
Synonyms: Htt^LacQ140; Htt Q175
Marker symbol: Htt
Marker name: huntingtin
Marker synonyms: C430023I11Rik; Hd; Hdh; htt; IT15; LOMARS
Chromosome: 5
Strain of origin: 129S1/Sv-Oca2⁺ Tyr⁺ Kitl⁺
Molecular note: The targeting vector was designed to insert a cassette containing synthetic LacO sequences flanking the Htt transcriptional start site followed by a chimeric mouse/human Htt sequence with human exon 1 into the mouse Htt locus on chromosome 5. The human sequence encodes the N terminal sequence including an expanded CAG repeat containing 140Q and the adjacent sequence encoding the human proline-rich region. A loxP-flanked PGK-neomycin cassette was inserted in reverse orientation 1.3 kb upstream of transcription initiation site