These R26-M2rtTA;Col1a1-tetO-H2B-GFP compound mutant mice allow doxycycline-inducible, fluorescent labeling of cells (including hematopoietic stem cells), and may be used for monitoring cell proliferation.
Hanno R Hock, Massachusetts General Hospital
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 |
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Targeted (Inducible, Inserted expressed sequence, Humanized sequence, RMCE-ready) | Col1a1 | collagen, type I, alpha 1 |
This compound mutant strain is useful for monitoring cell proliferation. In the first targeted mutation, Human HIST1H2BJ (histone cluster 1, H2bj) produces a GFP fusion protein under the direction of the tetO minmal CMV promoter downstream of the Col1a1 (collagen, type I, alpha 1) gene. In the second mutation, Gt(ROSA)26Sor drives expression of an optimized rtTA. In conjunction, the two targeted mutations express GFP in a widespread pattern upon doxycycline induction. This cell labeling has several advantages over BrdU, including rapid induction of H2B-GFP in virtually all hematopoietic stem cells (HSCs) and higher labeling intensity. Fluorescence levels exceeding the background by several orders of magnitude can be observed in HSCs immediately after doxycycline pulse. Highly proliferative progenitors completely lose H2B-GFP expression after approximately 4-8 weeks, but a proportion of HSCs retain significant levels of H2B-GFP for more then 1.5 years.
A cDNA encoding human H2B (HIST1H2BJ) tagged at its carboxyl terminus with DNA encoding codon-optimized enhanced GFP was inserted downstream of the mouse Col1a1 locus under the transcriptional control of a tetracycline-dependent minimal CMV promoter. The mutation was created in V6.5 (C57BL/6 x 129S4/SvJae)F1-derived embryonic stem (ES) cells.
ES cells carrying the GFP mutation were selected and then injected with the R26-M2rtTA targeting vector. In this vector, Gt(ROSA)26Sor promoter drives expression of reverse tetracycline controlled transactivator optimized with mutations S12G, E19G, A56P, D148E, and H179R (see Stock No. 006965).
This compound mutant strain was backcrossed two times to C57BL/6J by the donating laboratory.
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 | H2BC11, H2B clustered histone 11, human |
Expressed Gene | GFP, Green Fluorescent Protein, |
Site of Expression | Following doxycycline administration, H2B-GFP fusion protein is expressed in hematopoietic stem cells (HSCs). |
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 7, Rudolf Jaenisch |
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Allele Type | Targeted (Inducible, Inserted expressed sequence, Humanized sequence, RMCE-ready) |
Allele Synonym(s) | TetOP-H2B-GFP |
Gene Symbol and Name | Col1a1, collagen, type I, alpha 1 |
Gene Synonym(s) | |
Promoter | tetO, tet operator, |
Expressed Gene | H2BC11, H2B clustered histone 11, human |
Expressed Gene | GFP, Green Fluorescent Protein, |
Site of Expression | Following doxycycline administration, H2B-GFP fusion protein is expressed in hematopoietic stem cells (HSCs). |
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 cells inserted a cassette that contains a cDNA encoding human H2B (HIST1H2BJ) tagged at its carboxyl terminus with DNA encoding codon-optimized enhanced GFP under the transcriptional control of a tetracycline-dependent minimal CMV promoter into the 3'UTR. |
When a live colony is maintained, compound homozygotes or heterozygotes may be bred.
When using the R26-M2rtTA; TetOP-H2B-GFP mouse strain in a publication, please cite the originating article(s) and include JAX stock #016836 in your Materials and Methods section.
Facility Barrier Level Descriptions
Service/Product | Description | Price |
---|---|---|
Heterozygous for Gt(ROSA)26Sor<tm1(rtTA*M2)Jae> , Heterozygous for Col1a1<tm7(tetO-HIST1H2BJ/GFP)Jae> |
Frozen Mouse Embryo | B6;129S4-Gt(ROSA)26Sor<tm1(rtTA*M2)Jae> Col1a1<tm7(tetO-HIST | $2595.00 |
Frozen Mouse Embryo | B6;129S4-Gt(ROSA)26Sor<tm1(rtTA*M2)Jae> Col1a1<tm7(tetO-HIST | $2595.00 |
Frozen Mouse Embryo | B6;129S4-Gt(ROSA)26Sor<tm1(rtTA*M2)Jae> Col1a1<tm7(tetO-HIST | $3373.50 |
Frozen Mouse Embryo | B6;129S4-Gt(ROSA)26Sor<tm1(rtTA*M2)Jae> Col1a1<tm7(tetO-HIST | $3373.50 |
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