B6;129S4-Gt(ROSA)26Sor^{tm1(UBC-EGFP)Fcam} Igs7^{em1(CAG-mCherry)Fcam} Col1a1^{tm4(CAG-EGFP)Fcam}/J

Stock number: 038750
Product name: CA/TA/RA
Strain synonyms: CA/TA/RA
Research areas: Neurobiology Research, Research Tools

Description

CA/TA/RA triple target-array knock-in mice carry three targeted alleles in Col1a1, Igs7, and Gt(ROSA)26Sor, respectively. CA has 10 guide RNAs (gRNA) with separate U6 promoters inserted upstream of a CAG-EGFP followed by the sequence targeted by the gRNAs. TA has a CAG-mCherry reporter followed the same target sequences in a different order. RA has a UBC-EGFP reporter followed the same target sequences in a different order.

The DARLIN model is created when CA/TA/RA triple target-array mice are combined with Cas9-TdT-gRNAs-M2 mice (Stock No. 038749). DARLIN is a lineage tracing mouse line with a 10¹⁸ lineage barcoding capacity and single cell lineage coverage with a 60% efficiency that can be induced at any time and across a wide range of tissues.

Detailed description

CA/TA/RA triple target-array knock-in mice carry three targeted mutations: (1) CA is an expression cassette containing 10 guide RNAs (gRNA) with separate U6 promoters, a CAG-driven GFP reporter in which the sequence targeted by the gRNAs is inserted upstream of its polyadenylation site and placed into the Col1a1 locus (2) TA has the same target sequences arranged into a different order and placed into the 3'UTR of CAG-mCherry reporter, the construct is inserted into the Igs7 locus, (3) RA has the same target sequences arranged into a different order and placed into the 3'UTR of a UBC-EGFP reporter and inserted into the Gt(ROSA)26Sor locus. The target sequences can be edited by the existing 10 tandem gRNAs inserted into the CA allele.

To create the DARLIN model, CA/TA/RA triple target-array mice are combined with Cas9-TdT-gRNAs-M2 mice (B6;129S4-Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae} Col1a1^{tm5(tetO-Cas9/Dntt*)Fcam}/J, Stock No. 038749). Administration of doxycycline to DARLIN mice activates expression of Cas9/Dntt allowing cut and repair of the target sequence creating a unique and inheritable "barcode". DARLIN can generate an estimated 10¹⁸ unique lineage barcodes and has 60% of captured cells that have edited and rare lineage barcodes in single-cell assays.

Development

The triple array (CA/TA/RA) strain contains three alleles carrying the same 10 target sequences each arranged in a different order.

 RMCE (recombinase-mediated cassette exchange) was utilized to insert CARLIN (CRISPR array repair lineage tracing or CA) into the Col1a1 locus. KH2 embryonic stem cells (C57BL/6 x 129S4/SvJae), 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 CARLIN targeting construct is designed as a multiplex containing 10 single guide RNAs (sgRNA) driven by separate U6 promoters inserted into the 3’ UTR of an EGFP reporter driven by constitutive CAG promoter.  A "barcode" 276 bp array containing target sites matching each of the expressed sgRNAs is inserted upstream of the EGFP polyadenylation site. The vector was then co-electroporated with a FLPe recombinase-expressing vector into the RMCE-ready KH2 ES cells. The resulting correctly targeted ES cells, carrying rtTA*M2 in the Gt(ROSA)26Sor locus and CARLIN in the Col1a1 locus, were injected into blastocysts. The rtTA has been bred out of the strain by crossing to C57BL/6 mice. The colony is maintained on a mixed B6 and 129S4 background. The CA strain is maintained separately as B6;129S4-Col1a1^tm4Fcam/Mmjax (MMRRC Stock No. 067061).

The TA targeting vector contains the same 10 target sequences inserted into the Col1a1 locus but arranged into a different order. The construct contains the mCherry reporter open reading frame with a WPRE and the 10 target sequences inserted into the 3’ UTR driven by a CAG promoter and inserted into the Igs7 locus on chromosome 9 using CRISPR/cas9 genome editing. The target sequences can be edited by the existing 10 tandem gRNAs inserted into the CA allele. The targeting vector, cas9 endonuclease and an Igs7 (Tigre) specific single guide RNA were introduced into C57BL/6N-A^tm1Brd-derived JM8A3 embryonic stem (ES) cells by nucleofection. Correctly targeted ES cells were injected into blastocysts. The resulting chimeric animals were crossed to C57BL/6J and maintained on the C57BL/6J background.

The RA targeting vector contains the same 10 target sequences inserted into the Col1a1 locus but arranged into a different order. The construct contains a EGFP reporter with a WPRE, the 10 target sequences, an SV40-polyA signal, and 2xHS4 insulator elements inserted into the GFP 3’ UTR driven by a UBC (ubiquitin C) promoter and inserted in reverse orientation into the Gt(ROSA)26Sor locus on chromosome 6. The target sequences can be edited by the existing 10 tandem gRNAs inserted into the CA allele. A CAG-puromycin selection cassette is present in the construct. The construct was introduced into C57BL/6N-A^tm1Brd-derived JM8A3 embryonic stem (ES) cells by nucleofection. Correctly targeted ES cells were injected into blastocysts. The resulting chimeric animals were crossed to C57BL/6J and maintained on the C57BL/6J background.

Mice carrying the CA, TA, and RA alleles are combined to create the triple array strain.

Allele

Symbol: Col1a1^{tm4(CAG-EGFP)Fcam}
Name: targeted mutation 4, Fernando Camargo
MGI accession ID: MGI:6442576
Generation method: Targeted
Attributes: Reporter; Inserted expressed sequence; Lineage barcode
Synonyms: CARLIN; Col1a1^tm1(cCARLIN)
Marker symbol: Col1a1
Marker name: collagen, type I, alpha 1
Marker synonyms: CAFYD; Col1a-1; Cola1; Cola-1; COLIA1; EDSARTH1; EDSC; Mov-13; OI1; OI2; OI3; OI4
Chromosome: 11
Strain of origin: (C57BL/6 x 129S4/SvJae)F1
Site of expression: widespread pattern of expression
Molecular note: The targeting construct is designed as a multiplex containing 10 single guide RNAs (sgRNA) driven by separate U6 promoters inserted into the 3' UTR of an EGFP reporter driven by constitutive CAG promoter. A "barcode" 276 bp array containing target sites matching each of the expressed sgRNAs is inserted upstream of the EGFP polyadenylation site.
General note: 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.

Allele

Symbol: Gt(ROSA)26Sor^{tm1(UBC-EGFP)Fcam}
Name: targeted mutation 1, Fernando Camargo
MGI accession ID: MGI:7526265
Generation method: Targeted
Attributes: Reporter; Lineage barcode
Synonyms: RA
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Marker synonyms: beta geo; Gtrgeo26; Gtrosa26; R26; ROSA26; SETD5-AS1; Thumpd3as1
Chromosome: 6
Strain of origin: C57BL/6N-A^tm1Brd
Molecular note: The targeting construct construct contains a EGFP reporter with a WPRE, 10 target sites, an SV40-polyA signal, and 2xHS4 insulator elements inserted into the GFP 3’ UTR driven by a UBC (ubiquitin C) promoter and inserted in reverse orientation into the Gt(ROSA)26Sor locus. A "barcode" 276 bp array containing 10 target sites in a unique arrangement is inserted upstream of the EGFP polyadenylation site.

Allele

Symbol: Igs7^{em1(CAG-mCherry)Fcam}
Name: endonuclease-mediated mutation 1, Fernando Camargo
MGI accession ID: MGI:7526267
Generation method: Endonuclease-mediated
Attributes: Reporter; Lineage barcode
Synonyms: TA
Marker symbol: Igs7
Marker name: intergenic site 7
Marker synonyms: Iis7; TIGRE
Chromosome: 9
Strain of origin: C57BL/6N-A^tm1Brd
Molecular note: The targeting construct contains the mCherry reporter open reading frame with a WPRE and the 10 target sequences (see below) inserted into the 3’ UTR driven by a CAG promoter and inserted into the Igs7 locus using CRISPR/cas9 genome editing. A "barcode" 276 bp array containing 10 target sites in a unique arrangement is inserted upstream of the EGFP polyadenylation site.