B6J.Cg-Tg(C9orf72*)30Rhbr/J

Stock number: 030222
Product name: C9BAC
Research areas: Neurobiology Research, Research Tools

Description

C9BAC is a C9orf72 BAC transgenic line expressing a familial ALS/FTD human patient-derived C9orf72 genomic DNA sequence (140.5 kbp upstream region, exon 1 through part of exon 6) with a hexanucleotide expansion (~300-500 GGGGCC repeats) in intron 1. Hemizygous mice recapitulate distinctive histopathological features observed in C9ORF72 ALS/FTD patients, including sense/antisense intranuclear RNA foci and RAN-translated dipeptide repeat proteins (poly[GC]) - but exhibit no overt behavioral, motor or neurodegeneration phenotype. This is a model for studying C9orf72 repeat length-dependent gain-of-toxicity in familial amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease) and frontotemporal dementia (FTD).

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 mouse 3110043O21Rik locus is the orthologue of human C9orf72.

C9BAC is a C9orf72 BAC transgenic line expressing a familial ALS/FTD human patient-derived C9orf72 genomic DNA sequence that contains exon 1 through part of exon 6, with a hexanucleotide expansion (~300-500 GGGGCC repeats) in intron 1. The hexanucleotide repeat sizes are reported to be stable, but routine monitoring is recommended (see further discussion below). The transgene insertion site, copy number and expression levels are also described in greater detail below. The BAC includes 140.5 kbp of sequence transcriptionally upstream of C9orf72 exon 1 that is likely to contain regulatory regions needed to control sense/antisense transcript expression in a temporal and spatial pattern that mimics expression in the human disease.

The truncated human C9orf72 sequences in the transgene express three human C9ORF72 splice variant isoforms (V1, V2 and V3), of which transcripts V1 and V3 contain the hexanucleotide expansion. Human C9ORF72 mRNA transcripts were detected in all tissues examined. Human-specific probes targeting each variant mRNA detected all three transcripts at expression levels relative to their abundances in human frontal cortex. Moreover, the total level of the transgenic human transcripts was roughly comparable to that of the endogenous mouse C9ORF72 ortholog transcripts and to expression levels of human control and ALS/FTD cases.

Hemizygous C9BAC mice exhibit no overt behavioral, motor or neurodegeneration phenotype - but recapitulate distinctive histopathological features observed in C9ORF72 ALS/FTD patients, including sense/antisense intranuclear RNA foci and repeat-associated non-ATG (RAN)-translated dipeptide repeat proteins (poly[glycine-proline]). These features first appear at ~3 months old and increase with age.
Specifically, 24 month old C9BAC are devoid of other ALS characteristics (including altered activation of microgliosis/astrogliosis or cytoplasmic TDP-43 mislocalization and TDP-43 aggregation in motor cortex). Furthermore, 24 month old C9BAC lack other FTD characteristics (including altered activation of gliosis in prefrontal cortex or hippocampus).
The donating investigator reports that hemizygous mice are viable and fertile, with normal breeding characteristics/litter sizes and exhibit the phenotype described above. They report that C9BAC homozygous mice are viable and fertile, but the homozygous phenotype has not been fully characterized to date (July 2017). Compared to hemizygotes, however, the homozygotes would be expected to have greater transgenic C9ORF72 expression levels and could exhibit a more severe phenotype.

Hexanucleotide repeat size/stability, transgene copy number and transgene insertion: The donating investigator reports that Southern blot analysis identified hemizygous C9BAC mice to have two transgene insertions - one with ~500 hexanucleotide repeats (GGGGCC_[500]) and the other with ~300 hexanucleotide repeats (GGGGCC_[300]). While the genomic location of the two transgene insertion sites was not initially identified (see additional details below), they report the two sites segregate together. Through the first eight breeding generations, they report repeat sizes were intergenerationally stable and tissue-type stable. Additional characterization of repeat stability has not been performed to date (July 2017).
In December 2017, SNP analysis of hemizygous and wildtype (noncarrier) mice suggest transgene insertion on chromosome 4 [~20 Mbp].
In July 2019, The Jackson Laboratory performed additional transgene insertion analyses on hemizygous mice. This suggested ~3-5 copies of the transgene have integrated on chromosome 4 (23,004,513-23,004,540 [mouse mm10]) and resulted in a 27 bp deletion of genomic region in between the two identified breakpoints. There are currently no genes annotated in this region. In addition, a total of 224 kb of E. coli sequence appears to be co-integrated after the transgene sequence.
When using mouse models where repeat number is subject to germline and somatic instability, and may expand or contract, it is strongly recommended the repeat number be quantified in all the experimental animals; all animals in all experimental groups should carry comparable hexanucleotide repeat sizes.

Southern Blot Protocol: The Jackson Laboratory Southern Blot Protocol for this transgene is available here [2019]. Using this protocol, Stock No. 030222 has two expected band sizes - one at ~6 kbp and the other at ~5 kbp.

In an attempt to offer alleles on well-characterized or multiple genetic backgrounds, alleles are frequently moved to a genetic background different from that on which an allele was first characterized. The phenotype described above is for C9BAC mice on a C57BL/6;SJL genetic background. It should be noted that Stock No. 030222 is backcrossed onto C57BL/6J for 13 generations or more. The donating investigator reports they observed no differences in phenotype between C57BL/6;SJL C9BAC mice and C57BL/6J-congenic C9BAC mice. We may modify the strain description if necessary as published results become available.

Development

The C9orf72 BAC transgenic line C9BAC was created by Dr. Robert H. Brown, Jr. (University of Massachusetts Medical School). First, the 153.2 kbp human bacterial artificial chromosome (BAC) CH523-111K12 (Chr9:27,561,112-27,714,301 : Hg19) was obtained containing the familial FTD/ALS human patient-derived C9orf72 gene. Specifically, this genomic C9orf72 DNA fragment has 140.5 kbp of upstream sequences, the promoter region and exon 1 through part of exon 6 - including ~500 hexanucleotide repeat motifs (GGGGCC_[500]) in the non-coding intron 1 and part of the 3' UTR of the short isoform V1. The BAC has 140.5 kbp of transcriptionally-upstream (telomeric) sequences reported to contain no other complete loci or confirmed genes (July 2017). The 153.2 kbp unmodified BAC was microinjected into the pronuclei of B6SJLF2/J embryos (B6SJLF1/J oocytes fertilized by B6SJLF1/J sperm). Founder animals were bred to B6SJLF1/J mice for germline transmission, and a single founder mouse (F30) was successful - establishing the C9BAC colony. The donating investigator reports that Southern blot analysis of hemizygous C9BAC identified it to have two transgene insertions - one with ~500 GGGGCC repeats and the other with ~300 GGGGCC repeats (see additional details below). The C9BAC transgenic colony was then maintained by breeding transgenic males to C57BL/6J females each generation for 13 generations, and then hemizygous males with black coat color were sent to The Jackson Laboratory Repository in 2017. Upon arrival, sperm was cryopreserved. To establish our living colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J inbred females (Stock No. 000664).
Of note, the donating investigator reports that the Y chromosome may not have been fixed to the C57BL/6J background during backcrossing. While the genomic location of the two transgene insertion sites was not specifically identified (see more details below), they report the two sites segregate together. Through the first eight breeding generations, they report repeat sizes were intergenerationally stable and tissue-type stable.

In 2017, a 48 SNP (single nucleotide polymorphism) panel analysis, with 43 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the males sent to The Jackson Laboratory Repository and our first generation rederived living colony. All markers were homozygous for C57BL/6J allele-type with one exception - one marker on chromosome 4 [~20 Mbp] was C57BL/6J;SJL/J in all the hemizygous animals tested. These data suggest transgene insertion on chromosome 4 [~20 Mbp].
In July 2019, The Jackson Laboratory performed additional transgene insertion analyses on hemizygous mice. This suggested ~3-5 copies of the transgene have integrated on chromosome 4 (23,004,513-23,004,540 [mouse mm10]) and resulted in a 27 bp deletion of genomic region in between the two identified breakpoints. There are currently no genes annotated in this region. In addition, a total of 224 kb of E. coli sequence appears to be co-integrated after the transgene sequence.

Allele

Symbol: Tg(C9orf72*)30Rhbr
Name: transgene insertion 30, Robert Brown Jr
MGI accession ID: MGI:6101162
Generation method: Transgenic
Attributes: Inserted expressed sequence; Humanized sequence
Synonyms: C9BAC; C9-BAC
Marker symbol: Tg(C9orf72*)30Rhbr
Marker name: transgene insertion 30, Robert Brown Jr
Chromosome: 4
Strain of origin: (C57BL/6J x SJL/J)F1
Expressed genes: C9orf72,chromosome 9 open reading frame 72,human
Site of expression: C9BAC is a C9orf72 BAC transgenic line expressing a familial ALS/FTD human patient-derived C9orf72 genomic DNA sequence that contains exon 1 through part of exon 6, with a hexanucleotide expansion (~300-500 GGGGCC repeats) in intron 1 and part of the 3' UTR of the short isoform V1. The BAC includes 140.5 kbp of sequence transcriptionally upstream of C9orf72 exon 1 that is likely to contain regulatory regions needed to control sense/antisense transcript expression in a temporal and spatial pattern that mimics expression in the human disease. The truncated human C9orf72 sequences in the transgene express three human C9ORF72 splice variant isoforms (V1, V2 and V3), of which transcripts V1 and V3 contain the hexanucleotide expansion. Human C9ORF72 mRNA transcripts were detected in all tissues examined. Human-specific probes targeting each variant mRNA detected all three transcripts at expression levels relative to their abundances in human frontal cortex. Moreover, the total level of the transgenic human transcripts was roughly comparable to that of the endogenous mouse C9ORF72 ortholog transcripts and to expression levels of human control and ALS/FTD cases. See Detailed Description section for greater detail of transgene insertion site, copy number and expression levels.
Molecular note: A 153.2 kbp human bacterial artificial chromosome (BAC) CH523-111K12 (Chr9:27,561,112-27,714,301 : Hg19) was obtained containing the familial FTD/ALS human patient-derived C9orf72 gene. This genomic C9orf72 DNA fragment has 140.5 kbp of upstream sequences, the promoter region and exon 1 through part of exon 6 - including ~500 hexanucleotide repeat motifs (GGGGCC>[500]<) in the non-coding intron 1 and part of the 3' UTR of the short isoform V1. The BAC has 140.5 kbp of transcriptionally-upstream (telomeric) sequences reported to contain no other complete loci or confirmed genes. Southern blot analysis of hemizygous mice identified two transgene insertions - one with ~500 GGGGCC repeats and the other with ~300 GGGGCC repeats. The truncated human C9orf72 sequences in the transgene express three human C9ORF72 splice variant isoforms (V1, V2 and V3), of which transcripts V1 and V3 contain the hexanucleotide expansion. Human C9ORF72 mRNA transcripts were detected in all tissues examined. Human-specific probes targeting each variant mRNA detected all three transcripts at expression levels relative to their abundances in human frontal cortex. Moreover, the total level of the transgenic human transcripts was roughly comparable to that of the endogenous mouse C9ORF72 ortholog transcripts and to expression levels of human control and ALS/FTD cases. 3-5 copies of the transgene have integrated on chromosome 4 (23,004,513-23,004,540 [mouse mm10]) and resulted in a 27 bp deletion of genomic region in between the two identified breakpoints. No genes are annotated in this region. In addition, a total of 224 kb of E. coli sequence appears to be co-integrated after the transgene sequence.