Following doxycycline (dox) administration, these single-gene transgenic R26rtTA;Col1a13F2A mice express the dox-inducible polycistronic 3F2A cassette (three mouse reprogramming genes Oct4 [Pou5f1], Klf4, and c-Myc [Myc]) from the Col1a1 locus. While somatic expression of these three reprogramming factors alone does not allow the generation of induced pluripotent stem cells (iPSCs), addition of other reprogramming factors (such as Sox2) allows multiple somatic cell types to be directly reprogrammed to generate iPSCs by culture in dox. As such, somatic cells derived from R26rtTA;Col1a13F2A mice are useful to screen for small molecules that can replace Sox2 for reprogramming.
Rudolf Jaenisch, Whitehead Institute, Massachusetts Institute of Technology
Genetic Background | Generation |
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Allele Type | Gene Symbol | Gene Name |
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Targeted (Transactivator) | Gt(ROSA)26Sor | gene trap ROSA 26, Philippe Soriano |
Allele Type | Gene Symbol | Gene Name |
---|---|---|
Targeted (Inducible, Inserted expressed sequence, RMCE-ready) | Col1a1 | collagen, type I, alpha 1 |
Mice heterozygous for both targeted mutations (R26-rtTA and Col1a1::tetO-3F2A) 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 R26rtTA;Col1a13F2A 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 3F2A cassette from the Col1a1 locus is not detected. Following dox administration, this single-gene transgenic mouse strain expresses the polycistronic 3F2A cassette (three mouse reprogramming genes Oct4 [Pou5f1], Klf4, and c-Myc [Myc]). While somatic expression of these three reprogramming factors alone does not allow the generation of induced pluripotent stem cells (iPSCs), addition of other reprogramming factors (such as Sox2) allows multiple somatic cell types to be directly reprogrammed to generate iPSCs by culture in doxycycline. As such, somatic cells derived from R26rtTA;Col1a13F2A mice are useful to screen for small molecules that can replace Sox2 for reprogramming. Because the reprogramming factors are carried on a single polycistronic construct, these double mutant R26rtTA;Col1a13F2A mice can be easily maintained, and the transgene can be easily transferred into other genetic backgrounds if needed.
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. Next, these targeted ES cells (termed V19 ES cells) were retargeted at the 3' UTR of the Col1a1 locus for insertion of (from 5' to 3') a loxP-flanked pgkNEOpolyA cassette, the tetracycline responsive element (TRE or tetO), the single polycistronic expression cassette 3F2A, and SV40 intron plus polyA signal. The 3F2A cassette contains the three mouse reprogramming genes Oct4 (Pou5f1; POU domain, class 5, transcription factor 1), Klf4 (Kruppel-like factor 4 (gut)), and c-Myc (Myc; myelocytomatosis oncogene) separated by two different viral 2A oligopeptides that mediate ribosomal skipping (T2A [from insect Thosea asigna virus] and E2A [equine rhinitis A virus], respectively). The resulting ES cells, now targeted with rtTA-M2 in the Gt(ROSA)26Sor locus and tetO-3F2A in the Col1a1 locus, were injected into B6D2F1 tetraploid blastocysts. The double mutant chimeric males were bred with B6D2F1 females to generate the colony. Double mutant pups on the resulting mixed genetic background were sent to The Jackson Laboratory Repository. Upon arrival, double mutant R26rtTA;Col1a13F2A mice were bred with B6129SF1/J mice (Stock No. 101043) for at least one generation to establish the colony. Double mutant mice were then bred together to maintain the colony.
Expressed Gene | rtTA, reverse tetracycline-controlled transactivator, E. coli |
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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. |
Expressed Gene | Myc, myelocytomatosis oncogene, mouse, laboratory |
Expressed Gene | Pou5f1, POU domain, class 5, transcription factor 1, mouse, laboratory |
Expressed Gene | Klf4, Kruppel-like factor 4 (gut), mouse, laboratory |
Site of Expression | widely expressed. |
Allele Name | targeted mutation 1, Rudolf Jaenisch |
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Allele Type | Targeted (Transactivator) |
Allele Synonym(s) | Gt(ROSA)26Sortm1(M2rtTA)Jae; M2; M2-rtTA; R26-M2rtTA; R26-rtTA; Rosa26-M2rtTA |
Gene Symbol and Name | Gt(ROSA)26Sor, gene trap ROSA 26, Philippe Soriano |
Gene Synonym(s) | |
Expressed Gene | 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. |
Strain of Origin | (C57BL/6 x 129S4/SvJae)F1 |
Chromosome | 6 |
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 |
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. |
Mutations Made By | Rudolf Jaenisch, Whitehead Institute, Massachusetts Institute of Technology |
Allele Name | targeted mutation 5, Rudolf Jaenisch |
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Allele Type | Targeted (Inducible, Inserted expressed sequence, RMCE-ready) |
Allele Synonym(s) | Col1a1 3F2A; Col1a1::tetO-3F2A; Col1a13F2A; Col1a1tetO-3F2A; Col1a1-3F2A |
Gene Symbol and Name | Col1a1, collagen, type I, alpha 1 |
Gene Synonym(s) | |
Promoter | tetO, tet operator, |
Expressed Gene | Myc, myelocytomatosis oncogene, mouse, laboratory |
Expressed Gene | Pou5f1, POU domain, class 5, transcription factor 1, mouse, laboratory |
Expressed Gene | Klf4, Kruppel-like factor 4 (gut), mouse, laboratory |
Site of Expression | widely expressed. |
Strain of Origin | (C57BL/6 x 129S4/SvJae)F1 |
Chromosome | 11 |
General Note | The allele was made in KH2 cells that carry Gt(ROSA)26Sortm1(rtTA*M2)Jae and Col1a1tm2(tetO-Pou5f1)Jae (J:159351). |
Molecular Note | RMCE on KH2 ES cells inserted a cassette containing (from 5' to 3') a loxP-flanked pgkNEOpolyA cassette, the tetracycline responsive element (TRE or tetO), the single polycistronic expression cassette 3F2A, and SV40 intron plus polyA signal into the 3' UTR. The 3F2A cassette contains the three mouse reprogramming genes Oct4 (Pou5f1; POU domain, class 5, transcription factor 1), Klf4 (Kruppel-like factor 4 (gut)), and c-Myc (Myc; myelocytomatosis oncogene) separated by two different viral 2A oligopeptides that mediate ribosomal skipping (T2A [from insect Thosea asigna virus] and E2A [equine rhinitis A virus], respectively). |
When maintaining a live colony, mice that are homozygous for both mutations may be bred together.
When using the R26rtTA;Col1a13F2A mouse strain in a publication, please cite the originating article(s) and include JAX stock #011013 in your Materials and Methods section.
Facility Barrier Level Descriptions
Service/Product | Description | Price |
---|---|---|
Heterozygous or wildtype for Gt(ROSA)26Sor<tm1(rtTA*M2)Jae>, Heterozygous or wildtype for Col1a1<tm5(tetO-Pou5f1,-Klf4,-Myc)Jae> |
Frozen Mouse Embryo | STOCK Gt(ROSA)26Sor<tm1(rtTA*M2)Jae> Col1a1<tm5(tetO-Pou5f1- | $2595.00 |
Frozen Mouse Embryo | STOCK Gt(ROSA)26Sor<tm1(rtTA*M2)Jae> Col1a1<tm5(tetO-Pou5f1- | $2595.00 |
Frozen Mouse Embryo | STOCK Gt(ROSA)26Sor<tm1(rtTA*M2)Jae> Col1a1<tm5(tetO-Pou5f1- | $3373.50 |
Frozen Mouse Embryo | STOCK Gt(ROSA)26Sor<tm1(rtTA*M2)Jae> Col1a1<tm5(tetO-Pou5f1- | $3373.50 |
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