STOCK Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae} Hprt1^{tm1(tetO-KMT2A/MLLT3)Sguo}/J

Stock number: 034693
Product name: iMF9, iMLL-AF9

Research areas: Research Tools

Description

Widespread doxycycline-mediated, reversible expression of the mixed lineage leukemia oncofusion protein MLL-AF9 in this X-linked Hprt1 (hypoxanthine guanine phosphoribosyl transferase 1) mutant strain is regulated by a tetracycline operator (tetO). This strain may be useful in studies of leukemias in adults and infants.

Detailed description

The human KMT2A (formerly MLL)/MLLT3 (MLL – mixed lineage leukemia) oncofusion gene encodes the MLL-AF9 protein, which is associated with aggressive leukemias of both the myeloid and lymphoid lineage in infants, and in adults with acute myeloid leukemia. MLL-AF9 is a T(9;11) (p22:q23) translocation that contains the 5' portion of the MLL gene inserted into chromosome 9 at band p22, leaving the 3' region of the MLL gene on chromosome 11. Specifically, the N-terminal AT hook and DNA methyltransferase (1444 amino acids) from KMT2A (also known as MLL) is fused to the 192 C-terminal amino acids from MLLT3 (breakpoints are variable).

This knock in allele carries KMT2A/MLLT3 under the control of tetO, a tetracycline-responsive regulatory element, inserted into the endogenous Hprt1 locus on the X chromosome. The strain also carries the Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae} allele, providing inducible widespread expression of the KMT2A/MLLT3 gene. MLL-AF9 expression is monitored by the co-expressed ΔNGFR on the cell surface. Females heterozygous and males hemizygous for the Hprt1^{tm1(tetO-KMT2A/MLLT3)Sguo} allele (which is X-linked), and homozygous for the Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae} allele are viable and fertile. Wild-type recipient mice transplanted with iMLL-AF9 GMP (granulocyte-macrophage progenitor) cells express MLL-AF9 oncogene and develop myeloid leukemia with a survival time of around two months, when induced with doxycycline.

Development

The targeting vector was designed with tetO (TRE) promoter, upstream of human KMT2A/MLLT3 (MLL-AF9) cDNA followed by an IRES-ΔNGFR cassette. The KMT2A/MLLT3 translocation T(9:11) contains the KMT2A (1421 amino acids) at the N terminal fused to the 91 C-term amino acids from MLLT3. MLL-AF9 expression is monitored by the co-expressed ΔNGFR on the cell surface. 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 MLL-AF9 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/6 females. The donating lab reported the resulting offspring were bred to C57BL/6 mice for 6 generations and were bred to B6.Cg-Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae}/J (Stock No. 006965). See SNP note below regarding genetic background contributions and Ptprc locus.

In 2020, a 48 SNP (single nucleotide polymorphism) panel analysis, with 43 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the males sent to The Jackson Laboratory (and used to freeze sperm). The Chromosome 1 SNP marker within the Ptprc locus indicates the mice were homozygous for the Ptprc^a allele rather than the Ptprc^b allele normally present for C57BL/6 or 129. In addition, four markers throughout the genome, one each on Chromosomes 6, 15, 16 and X, were segregating with 129 allele-type. Two of those markers (Chromosomes 6 and 15) score the same for 129 or C57BL/6N. Taken together, these data suggest the mice were incompletely backcrossed and were on a mixed C57BL/6;129 background, and there may be C57BL/6N substrain contributions.

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
Synonyms: Gt(ROSA)26Sor^{tm1(M2rtTA)Jae}; M2; M2-rtTA; R26-M2rtTA; R26-rtTA; Rosa26-M2rtTA
Marker symbol: Gt(ROSA)26Sor
Marker name: gene trap ROSA 26, Philippe Soriano
Marker synonyms: beta geo; Gtrgeo26; Gtrosa26; R26; ROSA26; SETD5-AS1; Thumpd3as1
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: Hprt1^{tm1(tetO-KMT2A/MLLT3)Sguo}
Name: targeted mutation 1, Shangqin Guo
MGI accession ID: MGI:6385058
Generation method: Targeted
Attributes: Inserted expressed sequence; Humanized sequence
Synonyms: MLL-AF9
Marker symbol: Hprt1
Marker name: hypoxanthine phosphoribosyltransferase 1
Marker synonyms: HGPRT; Hgprtase; Hprt
Chromosome: X
Strain of origin: 129S5/SvEvBrd
Site of expression: tetO-inducible
Molecular note: The targeting vector was designed with tetO (TRE) promoter, upstream of human KMT2A/MLLT3 (MLL-AF9) cDNA followed by an IRES-deltaNGFR cassette. The KMT2A/MLLT3 translocation T(9:11) contains the AT hook and DNA methyltransferase from KMT2A (1444 amino acids) at the N terminal fused to the 192 C-term amino acids from MLLT3 (breakpoints are variable). MLL-AF9 expression is monitored by the co-expressed deltaNGFR on the cell surface. This construct was inserted into a cassette exchange locus upstream of the hypoxanthine guanine phosphoribosyl transferase (Hprt) gene on the X chromosome using Inducible Cassette Exchange (ICE) recombination.
General note: 129P2/OlaHsd-derived mutant A17 embryonic stem (ES) cell line (also called 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.