C57BL/6J-Timeless^em1Ljp/J

Stock number: 034832
Product name: Tim-R1078X

Research areas: Neurobiology Research

Description

Tim R1078X mice carry a CRISPR/Cas9 generated premature stop codon in exon 27 of the timeless circadian clock 1 (Timeless) gene, making this strain useful when studying Familial advanced sleep phase (FASP).

Detailed description

Tim R1078X mice carry a CRISPR/Cas9 generated premature stop codon in exon 27 of the timeless circadian clock 1 (Timeless) gene, resulting in a 128 aa loss in the C-terminal end of the conserved TIMELESS_C domain, which contains the fourth and last putative nuclear localization signal (NLS4). The causative change of an arginine codon at position 1078 to a stop codon (R1078X) mutation was identified in a family with Familial advanced sleep phase (FASP) in two independent screenings. FASP is an inherited condition where affected subjects wake and sleep early. Tim-RX mice exhibit FASP with altered photic entrainment but normal circadian period. The donating investigator reports that heterozygous mice are viable and fertile, while homozygous mice are lethal at the preimplantation stage.

Development

A guide RNA (G12-TGTACCTGTAGGAAACCTTC) and 200-nt single-stranded oligodeoxynucleotides (ssODN) were designed to change the arginine codon at position 1078 to a stop codon (AGA-TGA) in exon 27 of the timeless circadian clock 1 (Timeless) gene, resulting in a 128 aa loss in the C-terminal end of the conserved TIMELESS_C domain, which contains the fourth and last putative nuclear localization signal (NLS4). One nucleotide substitution in sgRNA was also introduced in order to prevent re-targeting. Cas9 mRNA, sgRNAs, and ssODN repair template were introduced into C57BL/6-derived zygotes with well recognized pronuclei. Correctly targeted embryos were transferred to pseudopregnant females. Tim R1078X pups were identified by sequencing and PCR and mice from line L1 were further bred to C57BL/6J mice for at least 10 generations to develop the colony. After backcrossing to C57BL/6 several times, most off-target sites should be replaced to backcrossed genomic sequences. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J (Stock No. 000664) oocytes.

Allele

Symbol: Timeless^em1Ljp
Name: endonuclease-mediated mutation 1, Louis J Ptacek
MGI accession ID: MGI:6369043
Generation method: Endonuclease-mediated
Attributes: Humanized sequence
Synonyms: Tim-R1078X
Marker symbol: Timeless
Marker name: timeless circadian clock 1
Marker synonyms: FASPS4; hTIM; rTIM; rTLP; tim; TIM1
Chromosome: 10
Strain of origin: C57BL/6
Molecular note: Using CRISPR/Cas9 technology with sgRNAs and a 200-nt single-stranded oligodeoxynucleotides (ssODN), a mutation was introduced to change arginine codon 1078 to a stop codon (p.R1078*). This mutation mimics the p.R1081* found in familial advanced sleep phase (FASP) patients.