B6.Cg-Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae} Col1a1^{tm1(tetO-U2AF1*S34F)Mjwa}/J

Stock number: 029784
Product name: U2af1(S34F)
Research areas: Cancer Research, Research Tools

Description

These U2AF1(S34F)/rtTA mice express a doxycycline-inducible human S34F mutant U2AF1. These mice may be suitable for use in studies related to myelodysplastic syndromes, acute myeloid leukemia, lung adenocarcinomas, as well as other cancers harboring this missense mutation.

Detailed description

The U2AF1 gene encodes a component of the U2 small nuclear ribonucleoprotein auxiliary factor complex. Mutations in this gene have been associated with myelodysplastic syndromes, lung adenocarcinomas, and AML. The missense mutation coding for S34F substitution in U2AF1 is a common mutation found in myelodysplastic syndromes. These U2AF1(S34F)/rtTA mice express a doxycycline-inducible mutant human U2AF1(S34F). U2AF1(S34F)/rtTA mice carry 2 mutations: Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae} allows widespread expression of the optimized form of reverse tetracycline-controlled transactivator (rtTA-M2) protein under the control of the endogenous Gt(ROSA)26Sor promoter, and the doxycycline-inducible mutant human U2AF1 under the control of the tetO, tetracycline-responsive regulatory element, in Col1a1^{tm1(tetO-U2AF1*S34F)Mjwa}. Doxycycline induced U2AF1(S34F) expression is detected by quantitative RT-PCR in c-Kit+Lin-Sca-1+(KLS) hematopoietic stem cells, common myeloid progenitor cells, granulocyte-monocyte progenitor cells, megakaryocyte-erythroid progenitor cells, neutrophils, monocytes, B cells and T cells. In the absence of the tetracycline analog doxycycline (dox), expression of the dox-inducible U2AF1(S34F) from the Col1a1 locus is not detected in bone marrow cells by pyrosequencing assay. Expression of U2AF1(S34F) is doxycycline dose dependent, with higher doses inducing higher levels of expression. Following administration of 625 ppm doxycycline chow, mice that are heterozygous for both alleles express mutant U2AF1 in bone marrow cells at levels similar to endogenous mouse U2af1 expression levels, based on pyrosequencing assay for human and mouse single nucleotide polymorphisms found in U2af1 RNA. In lethally irradiated wildtype recipient mice engrafted with U2AF1(S34F)/rtTA bone marrow, doxycycline induction results in decreased number of peripheral blood total white blood cells, B cells and monocytes, as well as reduced numbers of B cells and monocytes in bone marrow, compared to controls. Mice homozygous for the single mutant Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae} allele are viable and fertile. Double heterozygotes are viable and fertile. Viability/fertility of mice homozygous for both alleles has not been tested (June 2017).

In an attempt to offer alleles on well-characterized or multiple genetic backgrounds, alleles are frequently moved to a genetic background different from that on which an allele was first characterized. This is the case for the strain above. It should be noted that the phenotype could vary from that originally described. We will modify the strain description if necessary as published results become available.

Development

RMCE (recombinase-mediated cassette exchange) was utilized to insert tetO-U2AF1(S34F) into the 3' UTR of the Col1a1 locus. KH2 embryonic stem cells (C57BL/6 x 129S4Sv/Jae), carrying the Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae} allele (see Stock No. 006965), were previously re-targeted to insert a FRT site-flanked PGK-Neo cassette and a promoterless hygromycin resistance cassette in the 3' UTR of the Col1a1 locus, thus adapting the ES cell line for RMCE. The U2AF1(S34F)-pBS31' "flip-in plasmid" vector was then co-electroporated with a FLPe recombinase expressing vector into the RMCE-ready KH2 ES cells. The U2AF1(S34F)-pBS31' vector contains a PGK promoter, ATG start codon (to initiate the expression of the promoterless/ATG-less hygromycin- resistance cassette in the KH2 ES cells), FRT site, a splice acceptor-double polyA sequence (SA-2xpA), tetO (tetracycline responsive element) with a CMV minimal promoter, cDNA of human U2AF1 (isoform a) with the S34F amino acid conversion mutation, and SV40 polyA signal. The resulting correctly targeted ES cells, carrying rtTA*M2 in the Gt(ROSA)26Sor locus and tetO-U2AF1(S34F) in the Col1a1 locus, were injected into blastocysts. The resulting chimeric animals were tested for germline transmission. The mice were maintained by breeding double heterozygotes with (C57BL/6 x 129S4Sv/Jae)F1 mice, and then backcrossed to the C57BL/6J background for 10 generations using a marker assisted protocol. Upon arrival, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

Allele

Symbol: Col1a1^{tm1(tetO-U2AF1*S34F)Mjwa}
Name: targeted mutation 1, Matthew J Walter
MGI accession ID: MGI:5752388
Generation method: Targeted
Attributes: Inducible; Inserted expressed sequence
Synonyms: U2AF1(S34F)
Marker symbol: Col1a1
Marker name: collagen, type I, alpha 1
Marker synonyms: CAFYD; Col1a-1; Cola1; Cola-1; COLIA1; EDSARTH1; EDSC; Mov-13; OI1; OI2; OI3; OI4
Chromosome: 11
Strain of origin: (C57BL/6 x 129S4/SvJae)F1
Expressed genes: U2AF1,U2 small nuclear RNA auxiliary factor 1,human
Site of expression: Upon doxycycline induction, expression is detected in hematopoietic stem cells, common myeloid progenitor cells, granulocyte-monocyte progenitor cells, megakaryocyte-erythroid progenitor cells, neutrophils, monocytes, and B and T cells.
Molecular note: RMCE on KH2 ES cells introduced into the 3' UTR a tetracycline responsive element driving inducible expression of a human U2AF1 cDNA with an S34F mutation.
General note: 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
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.