B6.Cg-Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae} Col1a1^{tm6(tetO-MSI2)Jae}/J

Stock number: 014588
Product name: Coll-TetO-MSI2
Research areas: Cancer Research, Research Tools

Description

These Coll1a1-TetO-MSI2 (Coll-TetO-MSI2) double targeted mutation mice have doxycycline (tetracycline) inducible overexpression of human MSI2 (musashi homolog 2) and may be useful in conjunction with other oncogenic mutations for studies of the role of MSI2 in proliferation and differentiation of hematopoietic stem and myeloid progenitor cells in diseases such as human leukemia.

Detailed description

These Coll1a1-TetO-MSI2 (Coll-TetO-MSI2) double mutant mice carry two targeted mutations: (1) an optimized form of reverse tetracycline controlled transactivator (rtTA*M2) in the Gt(ROSA)26Sor locus and (2) a tetracycline responsive element (tetO or TRE)-controlled human MSI2 (musashi homolog 2) gene in the Col1a1 locus. MSI2 plays a role in regulation of hematopoiesis. Aberrant MSI2 expression is associated with aggressive myeloid leukemia and poor prognosis in blast crisis chronic myelogenous leukemia. Doxycycline administration induces widespread overexpression of human MSI2 (musashi homolog 2), resulting in expansion of hematopoietic stem and progenitor cells. While mutant mice treated continuously with doxycycline for 1 year exhibit increased spleen and liver weights, and a reduction in MSI2 induction over time, they do not develop leukemia. Mice that are homozygous for the targeted mutations and untreated with doxycycline are viable, fertile, normal in size and do not display any gross physical or behavioral abnormalities. Recipient control mice transplanted with bone marrow from these Coll1a1-TetO-MSI2 double mutant mice exhibit (with doxycycline treatment) increased hematopoietic stem and progenitor cells, increased hematocrit and mean corpuscular volume; and decreased myeloid progenitor, myeloid erythroid progenitor cells, neutrophils, lymphocytes and platelets.

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. Correctly targeted ES cells (called KH2) carrying the Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae} allele, were retargeted to insert an FRT site-flanked PGK-Neo cassette and a promoterless hygromycin resistance cassette downstream of the Col1a1 locus. Correctly retargeted ES cells were selected and the FRT site-flanked PGK-Neo downstream of the Col1a1 locus was replaced by electroporating both a "flip-in plasmid" containing a PGK promoter, ATG start codon, FRT site, a splice acceptor-double polyA sequence, tetO (tetracycline responsive element) with a CMV minimal promoter, and SV40 polyA signal, upstream of the human MSI2 cDNA sequence, and a FLPe recombinase expressing vector to facilitate tetO-MSI2 recombination into the 3' UTR of the Col1a1 locus. The resulting correctly targeted ES cells, carrying rtTA*M2 in the Gt(ROSA)26Sor locus and tetO-MSI2 in the Col1a1 locus, were injected into recipient blastocysts. The mice were backcrossed to C57BL/6 for 5 generations. Upon arrival at The Jackson Laboratory, the mice were crossed to C57BL/6J (Stock No. 000664) at least once to establish the colony.

Allele

Symbol: Col1a1^{tm6(tetO-MSI2)Jae}
Name: targeted mutation 6, Rudolf Jaenisch
MGI accession ID: MGI:4822360
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: MSI2,musashi RNA binding protein 2,human
Site of expression: MSI2 is normally expressed in hematopoietic stem cells.
Molecular note: RMCE on KH2 cells inserted a cassette that contains a tetracycline operator and minimal CMV promoter drives inducible expression of the human cDNA preceded by a splice acceptor and polyadenylation sequence and followed by an FRT flanked PGK-neo cassette and promotorless hygro cassette 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.