STOCK Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae} Col1a1^{tm5(tetO-Pou5f1,-Klf4,-Myc)Jae}/J

Stock number: 011013
Product name: R26^rtTA;Col1a1^3F2A
Research areas: Cancer Research, Cell Biology Research, Research Tools

Description

Following doxycycline (dox) administration, these single-gene transgenic R26^rtTA;Col1a1^3F2A 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 R26^rtTA;Col1a1^3F2A mice are useful to screen for small molecules that can replace Sox2 for reprogramming.

Detailed description

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 R26^rtTA;Col1a1^3F2A 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 R26^rtTA;Col1a1^3F2A 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 R26^rtTA;Col1a1^3F2A mice can be easily maintained, and the transgene can be easily transferred into other genetic backgrounds if needed.

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. 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 R26^rtTA;Col1a1^3F2A 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.

Allele

Symbol: Col1a1^{tm5(tetO-Pou5f1,-Klf4,-Myc)Jae}
Name: targeted mutation 5, Rudolf Jaenisch
MGI accession ID: MGI:4430600
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; Myc,myelocytomatosis oncogene,mouse, laboratory; Klf4,Kruppel-like factor 4 (gut),mouse, laboratory
Site of expression: widely expressed.
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).
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
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.