B6;129-Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae} Col1a1^{tm2(tetO-Pou5f1)Jae}/J

Stock number: 006911
Product name: Oct-4/rtTA (R26-M2rtTA;Col1a1-tetO-Oct4)
Research areas: Cancer Research, Cell Biology Research, Research Tools

Description

These "Oct-4/rtTA" mutant mice (R26-M2rtTA;Col1a1-tetO-Oct4) may be useful for studies of tumorigenesis and pluripotent stem cells.

Detailed description

Mice heterozygous for both targeted mutations (R26-rtTA and Cola1a::tetO-Oct4) are viable and fertile. These "Oct-4/rtTA" mice express rtTA-M2, an optimized form of reverse tetracycline-controlled transactivator (rtTA) protein, in multiple tissues. In the absence of the tetracycline analog doxycycline (dox), Oct4 (Pou5f1) expression from the Col1a1 locus is not detected. Following dox administration, high Oct4 expression is induced in liver, bone marrow, stomach, intestine, and skin, with lower levels in the heart, lungs, kidney, spleen, and thymus; no expression was detected in the brain and testes. Dox-inducd activation of Oct4 results in dysplasia in epithelial tissues. These mutant mice may be useful for studies of tumorigenesis and pluripotent cells.

Development

The R26-M2rtTA targeted mutation has an optimized form of reverse tetracycline controlled transactivator (rtTA-M2) followed by a β-globin intron, polyA signal and PGK-puromycin cassette, all inserted downstream of the Gt(ROSA)26Sor promoter. The targeting vector was electroporated into (C57BL/6 x 129S4Sv/Jae)F1-derived V6.5 embryonic stem (ES) cells. In addition, these ES cells were retargeted to insert an frt-flanked PGK-Neo into the 3' UTR of the Col1a1 locus. Correctly targeted ES cells were selected and the frt-flanked PGK-Neo in the 3' UTR of the Col1a1 locus was replaced by injecting both a "flip-in plasmid" containing a splice acceptor-double polyA sequence and the tetracycline responsive element (TRE or tetO) upstream of the mouse Oct4 (Pou5f1) cDNA sequence, as well as a Flpe-expressing plasmid to facilitate tetO-Oct4 recombination into the 3' UTR of the Col1a1 locus. The resulting ES cells (now targeted with rtTA-M2 in the Gt(ROSA)26Sor locus and tetO-Oct4 in the Col1a1 locus) were injected into B6D2F1 tetraploid blastocysts. Chimeric mice were bred together for many generations prior to arrival at The Jackson Laboratory Repository.

Allele

Symbol: Col1a1^{tm2(tetO-Pou5f1)Jae}
Name: targeted mutation 2, Rudolf Jaenisch
MGI accession ID: MGI:3702325
Generation method: Targeted
Attributes: Inducible; Inserted expressed sequence; 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: Pou5f1,POU domain, class 5, transcription factor 1,mouse, laboratory
Site of expression: With doxycycline treatment, Pou5f1 expression from the Col1a1 locus is widespread with high levels in skin, intestine and stomach, while lower levels are seen in the heart, spleen, thymus and several other organs.
Molecular note: ES cells containing the Gt(ROSA)26Sor^{tm1(rtTA2S-M2)Jae} mutation were retargeted. Cells were injected with a "flip-in plasmid" containing a tetracycline responsive element (TRE or tetO) and mouse Oct4 (Pou5f1) cDNA sequence (and a Flpe-recombinase plasmid to facilitate tetO-Oct4 integration into the 3' UTR region of Col1a1). With doxycycline treatment, Pou5f1 expression from the Col1a1 locus is widespread with high levels in skin, intestine and stomach, while lower levels are seen in the heart, spleen, thymus and several other organs. No expression is seen in the brain or testes.

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.