B6.Cg-Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae} Col1a1^{tm1(tetO-mCherry)Eggn}/J

Stock number: 014602
Product name: Rosa26-rtTA::Col1a1-tetO-H2B-mCherry
Research areas: Cancer Research, Cell Biology Research, Research Tools

Description

These double mutant Rosa26-rtTA::Col1a1-tetO-H2B-mCherry mice harbor the Rosa26-rtTA targeted mutation (an optimized form of reverse tetracycline controlled transactivator (rtTA-M2) downstream of the Gt(ROSA)26Sor promoter) and the Col1a1-tetO-H2B-mcherry targeted mutation (a tet-responsive element (tetO) and a histone H2B-mCherry fusion protein, targeted to the collagen, type I, alpha 1 (Col1a1) locus). These Rosa26-rtTA::Col1a1-tetO-H2B-mCherry mice may be useful for doxycycline-inducible studies which utilize the rtTA/tet-O (tet-on/TRE) system for visualizing pluripotent cells.

Detailed description

The Rosa26-rtTA::Col1a1-tetO-H2B-mCherry double mutant mice were generated by breeding Rosa26-rtTA mice (from Stock No. 006965) with Col1a1-tetO-H2B-mCherry mice (from Stock No. 014592). The Rosa26-rtTA targeted mutation has an optimized form of reverse tetracycline controlled transactivator (rtTA-M2) downstream of the Gt(ROSA)26Sor promoter. The Col1a1-tetO-H2B-mcherry targeted mutation contains a tet-responsive element (tetO) and a histone H2B-mCherry fusion protein, targeted to the collagen, type I, alpha 1 (Col1a1) locus.

Rosa26-rtTA::Col1a1-tetO-H2B-mCherry double mutant mice that are heterozygous for both targeted mutations are viable and fertile (mice homozygous for both targeted mutations were not generated by the donating investigator but are expected to be viable and fertile as well). These double mutant Rosa26-rtTA::Col1a1-tetO-H2B-mCherry mice have widespread expression of the optimized form of reverse tetracycline-controlled transactivator (rtTA-M2) protein directed to multiple tissues by the Gt(ROSA)26Sor promoter. In the absence of the tetracycline analog doxycycline (dox), expression of the dox-inducible histone H2B-mCherry fusion protein cassette from the Col1a1 locus is not detected. Following dox administration, double mutant mice express the H2B-mCherry fusion protein in embryonic stem (ES) cells, and differentiated trophoblast stem (TS) cells. These double mutant Rosa26-rtTA::Col1a1-tetO-H2B-mCherry mice may be useful for doxycycline-inducible studies which utilize the rtTA/tet-O (tet-on/TRE) system for visualizing pluripotent cells. Mice homozygous for the Col1a1-tetO-H2B-mCherry mutation may exhibit a subtle kink in the tail.

Development

These mice harbor the Rosa26-rtTA targeted mutation and the Col1a1-tetO-H2B-mCherry targeted mutation. The Rosa26-rtTA targeted mutation was created by Dr. Rudolf Jaenisch (Whitehead Institute, MIT) and sent to The Jackson Laboratory Repository as Stock No. 006965. The Col1a1-tetO-H2B-mCherry targeted mutation was created by Dr. Kevin Eggan (Harvard University) and sent to The Jackson Laboratory Repository as Stock No. 014592. The Rosa26-rtTA::Col1a1-tetO-H2B-mCherry double mutant mice were generated at The Jackson Laboratory Repository by breeding Rosa26-rtTA mice (from Stock No. 006965) with Col1a1-tetO-H2B-mCherry mice (Stock No. 014592). Double mutant mice were then bred together to maintain the colony (Stock No. 014602).

A 32 SNP (single nucleotide polymorphism) panel analysis, with 27 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 F3-F5 generation living colony at The Jackson Laboratory Repository. This revealed 3 of 32 markers, one each on chromosomes 7, 11 and 13, that were not fixed for C57BL/6 allele-type (e.g., still segregating for 129S4 allele-type markers) in some animals. Collectively, these data suggest the mice are >90% C57BL/6J with some (but not all) animals still segregating for 129S4 regions from the Col1a1-tetO-H2B-mCherry donor strain.

Allele

Symbol: Col1a1^{tm1(tetO-mCherry)Eggn}
Name: targeted mutation 1, Kevin Eggan
MGI accession ID: MGI:4868220
Generation method: Targeted
Attributes: Reporter; Null/Knockout; RMCE-ready
Marker symbol: Col1a1
Marker name: collagen, type I, alpha 1
Chromosome: 11
Strain of origin: (C57BL/6 x 129S4/SvJae)F1
Expressed genes: RFP,Red Fluorescent Protein,coral
Site of expression: Following doxycycline administration, H2B-mCherry fusion protein is expressed in embryonic stem (ES) cells, and differentiated trophoblast stem (TS) cells.
Molecular note: Using KH2 ES cells inserted a tetO-mCherry "flip-in plasmid" into the 3'UTR. The tetO-mCherry "flip-in plasmid" contained a splice acceptor-double polyA sequence and the tetracycline responsive element (tetO) upstream of a histone H2B minimal promoter-mCherry fluorescent protein fusion protein.
General note: The allele was made in KH2 cells that carry Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae} and Col1a1^{tm13(neo/hygro*)Jae}. This allele was generated with Col1a1^{tm13(neo/hygro*)Jae}.

Allele

Symbol: Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae}
Name: targeted mutation 1, Rudolf Jaenisch
MGI accession ID: MGI:3702294
Generation method: Targeted
Attributes: Transactivator
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Chromosome: 6
Strain of origin: (C57BL/6 x 129S4/SvJae)F1
Expressed genes: rtTA,reverse tetracycline-controlled transactivator,E. coli
Site of expression: Expresses an optimized rtTA protein (rtTA-M2). Inducible target gene expression is detected in liver, bone marrow, stomach, intestine, and skin, with lower levels in the heart, lungs, kidney, spleen, and thymus; no expression is detected in the brain and testes.
Molecular note: An optimized form of reverse tetracycline controlled transactivator (rtTA-M2) was inserted downstream of the Gt(ROSA)26Sor promoter and was followed by a PGK-puro selection cassette. This mutant form of rtTA termed M2 has five amino acid substitutions in the tetR moiety of tTA: S12G, E19G, A56P, D148E and H179R. This mutated form of transactivator protein has increased doxycycline sensitivity. Mice have widespread expression of the rtTA-M2 protein.
General note: This mutation was created during the generation of mutant ES cell line KH2, which also contains a frt docking site downstream of the endogenous Col1a1 locus on Chr 11 (Col1a1). The two mutations have subsequently been bred apart.