B6.129-Hprt1^{tm1(tetO-EGFP/MIR125B1)Jlu}/J

Stock number: 031715
Product name: i125b
Research areas: Research Tools

Description

i125b mice possess a tetracycline operator (tetO) upstream of an EGFP sequence fused to a human microRNA 125b-1 (EGFP/MIR125B1) inserted on the X chromosome. These mice are useful when studying myeloid leukemia development.

Detailed description

i125b mice possess a tetracycline operator (tetO) upstream of an EGFP sequence fused to a human microRNA 125b-1 (EGFP/MIR125B1). This construct was inserted upstream of the hypoxanthine guanine phosphoribosyl transferase 1 (Hprt1) gene on the X chromosome. This strain has no known issues with viability or fertility. MIR125B1 is a regulator of hematopoiesis and is often overexpressed in human acute myeloid leukemia (AML).

When crossed to ROSA26-rtTAm2* mice (Stock No. 006965) and induced with doxycycline (dox), 98% of bone marrow cells became GFP positive, and bone marrow Mac-1+/GFP+ cells show ~1000-fold MIR-125B overexpression as compared with Mac-1+ bone marrow cells from wild-type controls. Continued dox induction leads to the development of myeloproliferative neoplasm-like symptoms without the development of AML, and increased mortality, with a median survival of 370 days after dox induction.

Granulocyte-monocyte progenitors from i125b mice were infected with a mixed lineage leukemia and lysine (K)-specific methyltransferase 2A (MLL AF9) fusion gene retrovirus, a translocation associated with aggressive leukemias of both the myeloid and lymphoid lineage in infants, whereas in adults this is mainly associated with acute myeloid leukemia. Infected cells were transplanted into WT mice. Mice treated with dox had accelerated onset of myeloid leukemia, which is partially dependent on continued overexpression of MIR125B1.

Development

The i125b targeting vector was designed with a human MIR125B1 cDNA inserted between 2 splicing sites in the 3’ untranslated region of an enhanced green fluorescent protein (GFP) sequence. This construct was inserted into a cassette exchange locus upstream of the hypoxanthine guanine phosphoribosyl transferase  1 (Hprt1) gene on the X chromosome using Inducible Cassette Exchange (ICE) recombination in 129P2/OlaHsd-derived mutant A17 embryonic stem (ES) cell line termed A2Lox.cre. These cells carry a doxycycline-inducible Cre Recombinase ORF, flanked by loxP and lox2272 sites, upstream of a non-functional neomycin resistance (neo) gene. Cre was thus replaced by EFGP/MIR125B1 cDNA, followed by a poly A signal, upon recombination in ES cells. Correctly targeted ES cells were injected into blastocysts and resulting chimeric males were bred with C57BL/6NCr (National Cancer Institute) females. Resulting offspring were bred to ROSA26-rtTAm2* mice (Stock No. 006965) and were subsequently bred to C57BL/6Ncr mice for at least 6 generations. Mice were further bred to C57BL/6J mice for two generations. Upon arrival at The Jackson Laboratory Repository, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664). The ROSA26-rtTAm2* allele was bred out of the colony.

Allele

Symbol: Hprt1^{tm1(tetO-EGFP/MIR125B1)Jlu}
Name: targeted mutation 1, Jun Lu
MGI accession ID: MGI:6161212
Generation method: Targeted
Attributes: Reporter; Inducible; Inserted expressed sequence; Humanized sequence
Synonyms: i125b
Marker symbol: Hprt1
Marker name: hypoxanthine phosphoribosyltransferase 1
Marker synonyms: HGPRT; Hgprtase; Hprt
Chromosome: X
Strain of origin: 129P2/OlaHsd
Expressed genes: MIR125B1,microRNA 125b-1,human; GFP,Green Fluorescent Protein,
Site of expression: Hematopoietic stem cells
Molecular note: The targeting vector was designed with a human MIR125B1 cDNA inserted between 2 splicing sites in the 3' untranslated region of an EGFP sequence and followed by a polyA signal. The targeting vector was then used to replace the Cre ORF in a mutant embryonic stem cell line (see note) and placed under the control of the tet response promoter (TRE). The construct was inserted into a cassette exchange locus upstream of the Hprt gene using Inducible Cassette Exchange (ICE) recombination.
General note: ES cell line = a mutant A17 line (A2Lox.cre) that carries a doxycycline-inducible Cre recombinase ORF flanked by loxP and lox2272 sites, upstream of a non-functional neomycin resistance (neo) gene.