B6;129S4-Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae} Col1a1^{tm7(tetO-HIST1H2BJ/GFP)Jae}/J

Stock number: 016836
Product name: R26-M2rtTA; TetOP-H2B-GFP
Research areas: Cell Biology Research, Hematological Research, Research Tools

Description

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.

Detailed description

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.

Development

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.

Allele

Symbol: Col1a1^{tm7(tetO-HIST1H2BJ/GFP)Jae}
Name: targeted mutation 7, Rudolf Jaenisch
MGI accession ID: MGI:5007779
Generation method: Targeted
Attributes: Inducible; Inserted expressed sequence; Humanized 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: GFP,Green Fluorescent Protein,; H2BC11,H2B clustered histone 11,human
Site of expression: Following doxycycline administration, H2B-GFP fusion protein is expressed in hematopoietic stem cells (HSCs).
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.
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.