B6.Cg-Htt^{tm1(HTT*)Chdi} Htt^em1(HTT)Chdi/J

Stock number: 036155
Product name: Q107-iExon49 (Line 1) B6J
Strain synonyms: Q107-iExon49 (Line 1)
Research areas: Mouse/Human Gene Homologs, Neurobiology Research, Research Tools

Description

The Q107-iExon49 (Line 1) knock-in allele has the human HTT exon 1 sequence with a polyglutamine expansion comprised of a pure CAG expansion [(CAG)₁₀₅CAACAG] 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. 036155 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-Q107; Htt^{tm1(HTT*)Chdi}) and mouse intron 49 replaced with the human HTT intron 49 (iExon 49; Htt^em1(HTT)Chdi). Because the iExon 49 targeting/modification was unlikely to affect the original Htt-Q107 mutation, the strain nomenclature lists both mutations.

Note that the donating laboratory has also made available a Q7-iExon49 control mouse line (Stock No. 036156) 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 Q107-iExon49 (Line 1) 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)₁₀₅CAACAG] and 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). The genetic modification was accomplished by CRISPR-Cas9 mediated gene editing to insert the human intron 49 sequence in embryos derived from a Q107 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 Q107-iExon49 (Line 1) 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 for 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 Q7-iExon49 control mouse line (Stock No. 036156) and a Q175-iExon49 mouse line (Stock No. 036157).

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 Q107-iExon49 (Line 1) 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-Q107 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-Q107 allele. The resulting Q107-iExon49 (Line 1) mice were crossed to C57BL/6NTac mice for one generation, to reduce off-target events. Subsequently, the Q107-iExon49 (Line 1) mice were backcrossed to C57BL/6J (Stock No. 000664) for at least 4 generations using a speed congenic protocol (90-97% congenic on C57BL/6J as of March 2022). 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.

The Htt-Q107 knock-in allele was designed to replace mouse huntingtin (Htt) exon 1 with a human HTT exon 1 sequence containing a polyglutamine expansion comprised of a pure CAG expansion [~(CAG)₁₀₉CAACAG]. The knock-in (KI) was accomplished by homologous recombination in C57BL/6NTac-derived ES cells. The resulting Htt-Q107 knock-in mice were maintained on a C57BL/6NTac background by the donating laboratory and utilized to generate the Q107-iExon49 (Line 1) knock-in mouse line as described above.

Allele

Symbol: Htt^em1(HTT)Chdi
Name: endonuclease-mediated mutation 1, David Howland
MGI accession ID: MGI:7576089
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 Line1.

Allele

Symbol: Htt^{tm1(HTT*)Chdi}
Name: targeted mutation 1, David Howland
MGI accession ID: MGI:7576912
Generation method: Targeted
Attributes: Humanized sequence
Synonyms: Htt Q107
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 designed to replace mouse huntingtin (Htt) exon 1 with a human HTT exon 1 sequence containing a polyglutamine expansion comprised of a pure CAG expansion ~(CAG)109CAACAG. The knock-in (KI) was accomplished by homologous recombination in C57BL/6NTac-derived ES cells.