B6.Cg-Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae} Tg(tetO-Pou5f1,-Sox2,-Klf4,-Myc)1Srn/J

Stock number: 023749
Product name: i4F-A
Strain synonyms: R26:rtTA;TetO-OSKM, R26:rtTA;TetO-4F2A
Research areas: Cancer Research, Cell Biology Research, Research Tools

Description

i4F-A mice have expression of the 4F2A lentiviral vector (four mouse reprogramming genes Oct4 [Pou5f1], Klf4, Sox2, and c-Myc [Myc]) under the control of the tet-operator with CMV minimal enhancer-less promoter. They also express rtTA allowing expression of Tet-O-FUW-OSKM to be regulated with the tetracycline analog doxycycline (dox). Somatic expression of the reprogramming factors allows multiple somatic cell types to be directly reprogrammed to generate induced pluripotent stem cells (iPSCs).

Detailed description

i4F-A mutant mice are viable and fertile. These mice contain the Tet-O-FUW-OSKM lentiviral vector, expressing four mouse reprogramming genes (Oct4 [Pou5f1], Sox2, Klf4, and c-Myc [Myc]) controlled by the tet-responsive element (tetO; also called tetracycline operator, tet-operator, or tetracycline-responsive element [TRE]) with CMV minimal enhancer-less promoter. These i4F-A mice also express reverse tetracycline-controlled transactivator protein (rtTA), allowing the expression of the Tet-O-FUW-OSKM cassette to be regulated with the tetracycline analog doxycycline (dox) in the double mutant mice. Expression of the reprogramming factors allows multiple somatic cell types to be directly reprogrammed to generate induced pluripotent stem cells (iPSCs) in vivo. Dox treated mice develop and succumb to teratomas which are present in a variety of tissues, including the stomach, intestine, pancreas and kidney. Circulating iPSCs are also detected in the bloodstream and can produce embryo-like structures when injected intraperitoneally.

Development

The Tet-O-FUW-OSKM lentiviral vector was designed with a tetracycline responsive element (TRE, tetOP, or tetO) and a minimal CMV promoter driving expression of the OSKM cassette (4F2A cassette).

The OSKM cassette (4F2A cassette) contains the four mouse reprogramming genes Oct4 (Pou5f1; POU domain, class 5, transcription factor 1), Sox2 (SRY-box containing gene 2), Klf4 (Kruppel-like factor 4 (gut)) and c-Myc (Myc; myelocytomatosis oncogene) separated by three different viral 2A oligopeptides that mediate ribosomal skipping (P2A [from porcine teschovirus-1], T2A [from insect Thosea asigna virus], and E2A [equine rhinitis A virus], respectively).

C57BL/6-derived mouse embryonic fibroblasts (MEFs), carrying a doxycycline-inducible transcriptional activator (rtTA) within the Rosa26 locus (see Stock No. 006965) were transduced with the lentivirus Tet-O-FUW-OSKM. Treatment of MEFs with doxycycline reprogrammed the MEFs into induced pluripotent stem cells (iPSCs). iPSCs were injected into B6(Cg)-Tyr^c-2J/J blastocysts. Resulting offspring were bred with C57BL6/J mice until the lentiviral transgenes were transmitted at Mendelian proportions, indicative of single integration site. Upon arrival at The Jackson Laboratory Repository, sperm was cryopreserved. To generate the living i4F-A colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J female mice (Stock No. 000664). The double mutant mice were then bred together to maintain the colony.

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.

Allele

Symbol: Tg(tetO-Pou5f1,-Sox2,-Klf4,-Myc)1Srn
Name: transgene insertion 1, Manuel Serrano
MGI accession ID: MGI:5512672
Generation method: Transgenic
Attributes: Inducible; Inserted expressed sequence
Marker symbol: Tg(tetO-Pou5f1,-Sox2,-Klf4,-Myc)1Srn
Marker name: transgene insertion 1, Manuel Serrano
Chromosome: 8
Strain of origin: C57BL/6
Expressed genes: Pou5f1,POU domain, class 5, transcription factor 1,mouse, laboratory; Klf4,Kruppel-like factor 4 (gut),mouse, laboratory; Sox2,SRY (sex determining region Y)-box 2,mouse, laboratory; Myc,myelocytomatosis oncogene,mouse, laboratory
Site of expression: Expression of Pou5f1, Sox2, Klf4, and Myc can be induced by doxycycline to generate pluripotent stem cells.
Molecular note: The Tet-O-FUW-OSKM lentiviral vector was designed with a tetracycline responsive element (TRE, tetOP, or tetO) and a minimal CMV promoter driving expression of four mouse genes: Oct4 (Pou5f1; POU domain, class 5, transcription factor 1), Klf4 (Kruppel-like factor 4 (gut)), Sox2 (SRY-box containing gene 2), and c-Myc (Myc; myelocytomatosis oncogene). Expression of Pou5f1, Klf4, Sox2, and Myc were separated by three different viral 2A oligopeptides that mediate ribosomal skipping (P2A [from porcine teschovirus-1], T2A [from insect Thosea asigna virus], and E2A [equine rhinitis A virus], respectively). C57BL/6-derived mouse embryonic fibroblasts (MEFs), carrying a doxycycline-inducible transcriptional activator (rtTA) within the Rosa26 locus (see Stock No. 006965) were transduced with the lentivirus Tet-O-FUW-OSKM. Treatment of MEFs with doxycycline reprogrammed the MEFs into induced pluripotent stem cells (iPSCs). The lentiviral transgenes were transmitted at Mendelian proportions, indicative of single integration site. Inducible four factors lines i4F-A (also known as line 1) and i4F-B were generated. In this line, the transgene integrated within the fourth intron of the Neto2 gene, which resides on Chr 8.