C57BL/6J-Tg(C9orf72_i3)112Lutzy/J

Stock number: 023099
Product name: Tg(C9orf72_3) line 112
Research areas: Neurobiology Research

Description

Tg(C9orf72_3) line 112 mice have several tandem copies of the C9orf72_3 transgene, with each copy encoding the human C9orf72 with a hexanucleotide repeat expansion in the intron between the alternatively-spliced non-coding first exons 1a and 1b. Each transgene copy has between 100-1000 repeats ([GGGGCC]_100-1000). Hemizygous mice exhibit RNA foci and poly(GP) dipeptides in most neuronal populations of the brain by three months of age. These mice may be useful in applications related to amyotrophic lateral sclerosis and frontotemporal dementia.

Detailed description

The most common genetic mutation associated with human amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a hexanucleotide repeat expansion in a non-coding region of the chromosome 9 open reading frame 72 gene (C9orf72). The C9orf72 transgenes C9orf72_3 and C9orf72_2 encode the human C9orf72 gene with a hexanucleotide repeat expansion in the intron between the alternatively-spliced non-coding first exons 1a and 1b.

The Tg(C9orf72_3) line 112 mice (Stock No. 023099) have several tandem copies of the C9orf72_3 transgene, with each copy having between 100-1000 repeats ([GGGGCC]_100-1000).

The Tg(C9orf72_2) line 8 mice (Stock No. 023088) have the C9orf72_2 transgene with 15 hexanucleotide repeats ([GGGGCC]₁₅). Line 8 may be used as a control strain for Line 112.

Hemizygous mice from Tg(C9orf72_3) line 112 and Tg(C9orf72_2) line 8 are viable, fertile, born in Mendelian ratios and do not develop any locomotor or cognitive phenotype by 16 months of age.

By three months of age, however, hemizygous Tg(C9orf72_3) line 112 animals exhibit RNA foci (formed by hexanucleotide-repeat expansion-containing human C9ORF72 RNAs) and poly(GP) dipeptides (resulting from a non-ATG initiated translation of the repeat expansion) in most neuronal populations of the brain. No RNA foci or poly(GP) dipeptide phenotype is observed for hemizygous Tg(C9orf72_2) line 8.

The donating investigator reports that homozygous mice from Tg(C9orf72_3) line 112 and Tg(C9orf72_2) line 8 are viable and fertile, with no phenotype until at least three months of age. The homozygous colony at The Jackson Laboratory shows no overt paralysis with age (16 months old).

The Jackson Laboratory Southern Blot Protocol for this transgene is available here [2019]. Using this protocol, the expected band size for Stock No. 023099 is ~5 kbp.

Development

The C9orf72_3 transgene was designed by Dr. Cat Lutz (The Jackson Laboratory) and Dr. Robert Baloh (Cedars-Sinai) by first obtaining a bacterial artificial chromosome (BAC) clone derived from fibroblasts of a patient with amyotrophic lateral sclerosis (ALS), carrying the human chromosome 9 open reading frame 72 gene (C9orf72) with a hexanucleotide repeat expansion (GGGGCC) in the intron between the alternatively-spliced non-coding first exons 1a and 1b. Sequence isolated from the BAC included a ~166 kbp sequence (~36 kbp human C9orf72 genomic sequence, with ~110 kbp upstream and ~20 kbp downstream sequences).

Upon amplification of different BAC subclones in bacteria, the repeat expansion underwent contraction in some clones. A BAC subclone with a limited contraction to 100-1000 GGGGCC repeats was used, non-linearized, for microinjection into C57BL/6J blastocysts. Founder animals were bred to C57BL/6J inbred mice (Stock No. 000664) to establish founder line 112. Several copies of the transgene integrated in tandem; each copy harboring 100-1000 GGGGCC repeats. The Tg(C9orf72_3) line 112 colony was backcrossed to C57BL/6J for at least three generations prior to freezing sperm at The Jackson Laboratory Repository. To establish our living colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J females.

Allele

Symbol: Tg(C9orf72_i3)112Lutzy
Name: transgene insertion 112, Cathy Lutz
MGI accession ID: MGI:5618284
Generation method: Transgenic
Attributes: Inserted expressed sequence; Humanized sequence
Marker symbol: Tg(C9orf72_i3)112Lutzy
Marker name: transgene insertion 112, Cathy Lutz
Chromosome: UN
Strain of origin: C57BL/6J
Expressed genes: C9orf72,chromosome 9 open reading frame 72,human
Molecular note: The transgenic construct was designed using a bacterial artificial chromosome (BAC) clone derived from fibroblasts of a patient with amyotrophic lateral sclerosis (ALS), carrying the human chromosome 9 open reading frame 72 gene (C9orf72) with a hexanucleotide repeat expansion (GGGGCC) in the intron between the alternatively-spliced non-coding first exons 1a and 1b (variant 3). The BAC isolated a ~166 kbp sequence (~36 kbp human C9orf72 genomic sequence, with ~110 kbp upstream and ~20 kbp downstream sequences). Upon amplification of different BAC subclones in bacteria, the repeat expansion underwent contraction in some clones. A BAC subclone with a limited contraction to 100-1000 GGGGCC repeats was used.