B6.Cg-Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae} Col1a1^{tm9(tetO-Dnmt3b_i1)Jae}/J

Stock number: 017983
Product name: Col1a1-tetO-Dnmt3b1::R26-M2rtTA
Research areas: Cancer Research, Research Tools

Description

These Col1a1-tetO-Dnmt3b1::R26-M2rtTA mice may be used to regulate expression of Dnmt3b1 via a tetracycline-controlled transactivator protein system, and may be useful in studying the regulation of tumorigenesis.

Detailed description

Mice homozygous for the Col1a1-tetO-Dnmt3b1::R26-M2rtTA targeted mutation are viable and fertile. Expression of the DNA methyltransferase 3B gene (Dnmt3b1) is controlled by the tet-responsive element (tetO). DNMT3B1 is a de novo methyltransferase which is able to methylate CpG sequences, a required step during development. Hypermethylation of DNA has been implicated in many neoplasms, altering gene expression patterns that contribute to neoplastic transformation, tumorigenesis, and tumor behavior. This strain also contains the optimized form of reverse tetracycline controlled transactivator (rtTA-M2). Expression of Dnmt3b1 can be regulated with the tetracycline analog doxycycline (dox) in these double mutant offspring.

When these mice are bred to C57BL/6J-Apc^Min/J mice (see Stock No. 002020), DNMT3B1 expression in dox induced mice results an increase in intestinal tumor number and size as compared to Apc^Min control mice.

 

Development

A targeting vector was designed to insert frt-flanked PGK-Neo-pA 500 bp downstream of the 3' UTR of the Col1a1 locus. This construct was electroporated into (C57BL/6 x 129S4Sv/Jae)F1-derived V6.5 embryonic stem (ES) cells. Correctly targeted ES cells were identified. One of these ES cells clones, C10, was re-targeted to insert an optimized form of reverse tetracycline controlled transactivator (rtTA-M2) followed by a β-globin intron, polyA signal and PGK-puromycin cassette, all downstream of the Gt(ROSA)26Sor promoter. Correctly targeted ES cells were identified. One of these ES cell clones, KH2, was selected and the frt-flanked PGK-Neo-pA in the 3' UTR of the Col1a1 locus was replaced by co-injection of a tetO-Dnmt3b1 plasmid. The tetO-Dnmt3b1 plasmid contained a splice acceptor-double polyA sequence and the tetracycline responsive element (tetO) upstream of the DNA methyltransferase 3B gene.

The resulting ES cells, now targeted with rtTA-M2 in the Gt(ROSA)26Sor locus (Rosa26-rtTA) and tetO-Dnmt3b1 in the Col1a1 locus (Col1a1-tetO-Dnmt3b1), were injected into recipient blastocysts. The donating investigator stated that the resulting chimeric mice were bred together and were subsequently backcrossed to C57BL/6J mice (see SNP note below) for at least 7 generations to establish the double mutant colony.

Upon arrival at The Jackson Laboratory Repository, these mice were bred to C57BL/6J inbred mice (Stock No. 000664) for at least one generation.

A 32 SNP (single nucleotide polymorphism) panel analysis, with 27 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 rederived living colony at The Jackson Laboratory Repository. While the 27 markers throughout the genome suggested a C57BL/6 genetic background, 4 of 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating. These data suggest the mice sent to The Jackson Laboratory Repository were on a C57BL/6N genetic background.

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.

Allele

Symbol: Col1a1^{tm9(tetO-Dnmt3b_i1)Jae}
Name: targeted mutation 9, Rudolf Jaenisch
MGI accession ID: MGI:5313405
Generation method: Targeted
Attributes: Inducible; Inserted expressed 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: Dnmt3b,DNA methyltransferase 3B,mouse, laboratory
Molecular note: RMCE on KH2 cells inserted a cassette that contains a splice acceptor-double polyA sequence and the tetracycline responsive element (tetO) upstream of the DNA methyltransferase 3B gene isoform 1 (Dnmt3b) 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}.