Col1a1^{tm5(tetO-Cas9/Dntt*)Fcam}
Targeted Allele Detail

Summary

• Symbol: Col1a1^{tm5(tetO-Cas9/Dntt*)Fcam}
• Name: collagen, type I, alpha 1; targeted mutation 5, Fernando Camargo
• MGI ID: MGI:7526271
• Synonyms: Cas9-TdT-gRNAs
• Gene: Col1a1 Location: Chr11:94827050-94843868 bp, + strand Genetic Position: Chr11, 59.01 cM, cytoband D
• Alliance: Col1a1^{tm5(tetO-Cas9/Dntt*)Fcam} page

Mutation origin

• Germline Transmission: Earliest citation of germline transmission: J:341616
• Parent Cell Line: KH2 (ES Cell)
• Strain of Origin: (C57BL/6 x 129S4/SvJae)F1

Mutation description

• Allele Type: Targeted (Inducible, Inserted expressed sequence)
• Mutation: Insertion
• Mutation details: RMCE (recombinase-mediated cassette exchange) was utilized to insert the Cas9-Tdt (Dntt)-gRNA) construct into the Col1a1 locus. KH2 embryonic stem cells, carrying the Gt(ROSA)26Sor(tm1(rtTA*M2)Jae) allele, were previously re-targeted to insert an FRT-flanked PGK-Neo cassette and a promoterless hygromycin resistance cassette in the 3' UTR of the Col1a1 locus, thus adapting the ES cell line for RMCE. The Cas9-Dntt fusion is composed of the SpCas9 (derived from S. pyogenes) coding sequence (that includes an N-terminal 3xFLAG peptide, the SV40-NLS [nulcear localization sequence], and a C-terminal NLS), and the coding sequence of the mutant d138 Tdt (formal designation Dntt) containing an N-terminal 6xHis tag. The fusion was created by replacing the Cas9 stop codon with a linker sequence followed by the entire d138 Tdt coding sequence. The 3' UTR includes a WPRE and polyA sequence. Ten tandem gRNAs driven by separate U6 promoters were inserted downstream (in reverse orientation). The Cas9-Tdt-gRNA construct was cloned into the pBS31 targeting vector containing an FRT site and a TetO promoter. The construct was then co-electroporated with a FLPe recombinase-expressing vector (pCAGGS-FLPe-puro) into the RMCE-ready KH2 ES cells. (J:341616)

Find Mice (IMSR)

• Mouse strains and cell lines available from the International Mouse Strain Resource (IMSR)
• Carrying this Mutation: Mouse Strains: 1 strain available Cell Lines: 0 lines available
• Carrying any Col1a1 Mutation: 161 strains or lines available

Notes

KH2 embryonic stem cells carrying the Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae} allele, were previously re-targeted to insert an FRT-flanked PGK-Neo cassette and a promoterless hygromycin resistance cassette in the 3' UTR of the Col1a1 locus.

References

• Original: J:341616 Li L, et al., A mouse model with high clonal barcode diversity for joint lineage, transcriptomic, and epigenomic profiling in single cells. Cell. 2023 Oct 12
• All: 1 reference(s)