C57BL/6J-Zcchc8^em1Arman/J

Stock number: 033335
Product name: Zcchc8 null
Research areas: Cell Biology Research, Developmental Biology Research, Neurobiology Research, Research Tools

Description

Zcchc8 null is a CRISPR/cas9-made knock-out allele with a 33 bp insertion (BamHI site, Flag tag, STOP) in Zcchc8 exon 2 that creates a premature stop codon at amino acid 93. Homozygous mice (Zcchc8-/-) develop a progressive and fatal neurodevelopmental pathology with features of a ciliopathy. These mice may be useful for studying telomerase RNA (TR) maturation and global RNA processing.

Detailed description

Telomerase is a specialized ribonucleoprotein that synthesizes telomere DNA. Telomerase has two essential components - the telomerase reverse transcriptase TERT and the specialized non-coding telomerase RNA (TR; also known as TERC) which provides the template for telomere repeat addition. The Zcchc8 gene encodes a protein important in TR maturation and global RNA processing.

Zcchc8 null is a CRISPR/cas9-made knock-out allele with a 33 bp insertion (BamHI site, Flag tag, STOP) in Zcchc8 exon 2 that creates a premature stop codon at amino acid 93. No ZCCHC8 protein is detectable from the Zcchc8 null allele.
Heterozygous mice (Zcchc8+/-) are viable and fertile, with no gross or histological abnormalities compared to wildtype littermates. The donating investigator reports that heterozygous mice have TR insufficiency and are expected to show short telomere syndrome phenotypes also similar to TR and TERT mutant mice. Whereas consecutive generations of breeding TR mutant mice together can lead to worsening of telomere issues, the donating investigator predicts that given the length of telomeres in wildtype mice of this genetic background (C57BL/6J) and the extent of TR insufficiency in Zcchc8+/- mice, this telomere shortening and the related phenotypes would not be expected until after very high numbers of consecutive generations of breeding Zcchc8+/- mice together (perhaps greater than 50-100 generations).
Homozygous mice (Zcchc8-/-) have severe, progressive and ultimately fatal neurodevelopmental pathology with features of ciliopathy. They exhibit small brain size during development and at birth, with obvious cranial deformities and domed-shaped heads (hydrocephalus) by 3-4 weeks of age. The Zcchc8-/- brain transcriptome at E12.5 was highly dysregulated, showing accumulation and 3' end misprocessing of other low-abundance RNAs, including those encoding cilia components as well as the intronless replication-dependent histones. ~90-100% of knock-out mice do not survive beyond ~70 days of age. [Gable et al. 2019 Genes Dev 33:1381 (PMID:31488579)]

Development

The Zcchc8 null allele was created by Dustin Gable and Mary Armanios (Johns Hopkins University School of Medicine) using CRISPR/cas9 endonuclease-mediated genome editing. First, a single guide RNA (5'-TGAACATTCTGACAAGACCC-3') was designed to target sequences in exon 2 of the zinc finger, CCHC domain containing 8 locus (Zcchc8) on chromosome 5. Next, a DNA oligo for homology directed repair (HDR) was designed to target a 33 bp insertion (BamHI site, Flag tag sequence and STOP sequence) into Zcchc8 exon 2 that introduced a premature stop codon at amino acid 93 (P83delinsGSDYKDDDDK*). The guide RNA, HDR DNA oligo and cas9 nuclease were all injected into the cytoplasm C57BL/6J-derived fertilized eggs with well-recognized pronuclei. Embryos were transferred to pseudopregnant females. Founder mice were screened by PCR and sequencing of the Zcchc8 exon 2 region. The correctly targeted founder was bred to C57BL/6J mice for germline transmission. The Zcchc8 null colony was then backcrossed to C57BL/6J inbred mice for five generations (to minimize the possibility of off-target mutations). In 2017, the mutant mice were sent to the Jackson Laboratory. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).
The donating investigator reports the mice have not been specifically screened for the absence/presence of off-target mutations to date (November 2019).

Allele

Symbol: Zcchc8^em1Arman
Name: endonuclease-mediated mutation 1, Mary Armanios
MGI accession ID: MGI:6378994
Generation method: Endonuclease-mediated
Attributes: Null/Knockout
Synonyms: Zcchc8^-
Marker symbol: Zcchc8
Marker name: zinc finger, CCHC domain containing 8
Marker synonyms: 5730565F05Rik; PFBMFT5
Chromosome: 5
Strain of origin: C57BL/6J
Molecular note: First, a single guide RNA (5'-TGAACATTCTGACAAGACCC-3') was designed to target sequences in exon 2. Next, a DNA oligo for homology directed repair (HDR) was designed to target a 33 bp insertion (BamHI site, Flag tag sequence and STOP sequence) into exon 2 that introduced a premature stop codon at amino acid 93 (P83delinsGSDYKDDDDK*).