B6;129S4-Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae} Col1a1^{tm7(tetO-Pou5f1,-Klf4,-Sox2,-mCherry)Hoch}/J

Stock number: 034917
Product name: tetOP-OKSmC
Research areas: Cell Biology Research, Research Tools

Description

Col1a1-tetO-OKSmC mice have expression of the OKSmC cassette consisting of three mouse reprogramming genes, Pou5f1, Klf4, and Sox2, followed by an mCherry sequence, under the control of the bi-directional tet-responsive element (tetO) with CMV minimal enhancer-less promoter. They also contain Rosa26-rtTA mutation consisting of a reverse tetracycline-controlled transactivator protein (rtTA) expressed from the Gt(ROSA)26Sor locus. Expression of the OKSmC cassette can be regulated with the tetracycline analog doxycycline (dox) in the resulting double mutant offspring. Somatic expression of the OKSmC reprogramming factors allows multiple somatic cell types to be directly reprogrammed to generate induced pluripotent stem cells that are easily tracked by direct fluorescence.

Detailed description

Mice homozygous for the Col1a1-tetO-OKSmC and Rosa26-rtTA targeted mutations are viable and fertile. Expression of the OKSmC cassette (three mouse reprogramming genes Oct4 [Pou5f1], Klf4, and Sox2, and an enhanced red fluorescent protein [mCherry]) is controlled by the tet-responsive element (tetO; also called tetracycline operator, tet-operator, or tetracycline-responsive element [TRE]) with CMV minimal enhancer-less promoter. Expression of the OKSmC cassette can be regulated with the tetracycline analog doxycycline (dox) in double mutant offspring. Somatic expression of the OKSmC reprogramming factors allows multiple somatic cell types to be directly reprogrammed to generate induced pluripotent stem cells (iPSCs) that are easily tracked by direct fluorescence.

Col1a1-tetO-OKSM mice are a similar strain, and is available as (Stock No. 011001). Col1a1-tetO-OKSM mice contain a myelocytomatosis oncogene (c-Myc) sequence instead of mCherry. In contrast to Col1a1-tetO-OKSmC mice, chimeras derived from reprogrammed cells of Col1a1-tetO-OKSM mice (heterozygous for Col1a1-tetO-OKSM and homozygous for Rosa26-rtTA) have ~80% incidence of an aggressively growing tumor by three months of age after dox induction. In contrast, incidence is reduced to ~30% by three months of age when heterozygous for both mutations.

Development

A targeting vector was designed to insert frt-flanked PGK-Neo-pA into the 3' UTR of the Col1a1 locus. This construct was electroporated into (C57BL/6 x 129S4Sv/Jae)F1-derived V6.5 embryonic stem (ES) cells. Correctly targeted ES cells were identified. One of these ES cells clones, C10, was re-targeted to insert 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. Correctly targeted ES cells were identified. One of these ES cell clones, KH2, was selected and the frt-flanked PGK-Neo-pA in the 3' UTR of the Col1a1 locus was replaced by co-injection of a tetOP-OKSmC "flip-in plasmid" and a CAG-Flpe plasmid. The tetOP-OKSmC "flip-in plasmid" contained a splice acceptor-double polyA sequence and the tetracycline responsive element (TRE, tetOP, or tetO) upstream of the three mouse reprogramming genes Oct4 (Pou5f1; POU domain, class 5, transcription factor 1), Klf4 (Kruppel-like factor 4 (gut)), and Sox2 (SRY-box containing gene 2), and an enhanced red fluorescence sequence, mCherry. Internal ribosome entry site (IRES) sequences were placed between the coding sequences for Klf4, Sox2, and mCherry. Expression of Pou5f1 and Klf4, and that of Sox2 and mCherry was linked by self-cleaving peptides that mediate ribosomal skipping (F2A [from foot-and-mouth disease virus] and E2A [from equine rhinitis A virus], respectively). Internal ribosome entry site (IRES) sequences were placed between the coding sequences for Klf4 and Sox2, yielding a transgene with the following order of elements: Pou5f1-F2A-Klf4-IRES-Sox2-E2A-mCherry.

The resulting ES cells, now targeted with rtTA-M2 in the Gt(ROSA)26Sor locus (Rosa26-rtTA) and tetOP-OKSmC in the Col1a1 locus (Col1a1-tetO-OKSmC), were injected into recipient blastocysts. Chimeric mice were bred together to establish the double mutant colony on a C57BL/6;129S4Sv/Jae mixed genetic background. Upon arrival The Jackson Laboratory Repository, 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: Col1a1^{tm7(tetO-Pou5f1,-Klf4,-Sox2,-mCherry)Hoch}
Name: targeted mutation 7, Konrad Hochedlinger
MGI accession ID: MGI:6392017
Generation method: Targeted
Attributes: Reporter; Inducible; Inserted expressed sequence
Synonyms: tetOP-OKSmC
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
Molecular note: A targeting vector was designed to insert a FRT-flanked PGK-Neo-pA into the 3' UTR of the Col1a1 locus. This construct was electroporated into (C57BL/6 x 129S4Sv/Jae)F1-derived V6.5 embryonic stem (ES) cells. Correctly targeted ES cells were identified. One of these ES cells clones, C10, was re-targeted to insert an optimized form of reverse tetracycline controlled transactivator (rtTA-M2) followed by a beta-globin intron, polyA signal and PGK-puromycin cassette, all inserted downstream of the Gt(ROSA)26Sor promoter. Correctly targeted ES cells were identified. One of these ES cell clones, KH2, was selected and the FRT-flanked PGK-Neo-pA in the 3' UTR of the Col1a1 locus was replaced by co-injection of a tetOP-OKSM "flip-in plasmid" and a CAG-Flpe plasmid. The tetOP-OKSmC "flip-in plasmid" contained a splice acceptor-double polyA sequence and the tetracycline responsive element (TRE, tetOP, or tetO) upstream of the three mouse reprogramming genes Pou5f1, Klf4, and Sox2, and an enhanced red fluorescence sequence, mCherry. Internal ribosome entry site (IRES) sequences were placed between the coding sequences for Klf4, Sox2, and mCherry. Expression of Pou5f1, Klf4, and Sox2 was linked by self-cleaving peptides that mediate ribosomal skipping (F2A [from foot-and-mouth disease virus] and E2A [from equine rhinitis A virus], respectively).
General note: The allele was made in KH2 cells that carry Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae} and Col1a1^{tm13(neo/hygro*)Jae} (J:159351). 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
Synonyms: Gt(ROSA)26Sor^{tm1(M2rtTA)Jae}; M2; M2-rtTA; R26-M2rtTA; R26-rtTA; Rosa26-M2rtTA
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/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.