B6;129S4-Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae} Col1a1^{tm9(tetO-H3f3a*K28M)Hoch}/HochJ

Stock number: 041076
Product name: TetO-H3K27M/rtTA
Strain synonyms: TetO-H3K27M/rtTA
Research areas: Cell Biology Research, Research Tools

Description

These Col1a1-tetO-H3.3K27M::R26-M2rtTA mice may be used to regulate expression of mutant H3K27 via a tetracycline-controlled transactivator protein system and may be useful in studying the regulation of histone methylation in development, tissue regeneration, aging, cancer.

Detailed description

Col1a1-tetO-H3.3K27M::R26-M2rtTA double heterozygous mice are viable and fertile. The donating investigator did not test the fertility of homozygous mice. Expression of the mutant H3.3K27M histone A gene (H3f3a) is controlled by the tet-responsive element (tetO). Histones act to package DNA, which wraps around eight histones, into chromosomes. Histone modifications, such as methylation, act in diverse biological processes such as transcriptional activation/inactivation, chromosome packaging, and DNA damage/repair. This strain also contains the optimized form of reverse tetracycline controlled transactivator (rtTA-M2). Expression of H3K27M can be regulated with the tetracycline analog doxycycline (dox) in these double mutant offspring. H3.3K27M has a lysine-to-methionine substitution at amino acid 27, leading to a severe reduction in H3K27 methylation.

These mice develop a proB to preB cell arrest comparable a similar strain, Col1a1-tetO-H3.3K9M::R26-M2rtTA mice (Stock No. 034366) with a lysine-to-methionine point mutation at amino acid 9. Additionally, these H3K27M mice experience an exhaustion of hematopoietic stem cells, which kills 50% of animals after 3 months. The remaining 50% of H3K27M animals develop an aggressive form of B cell malignancy. In contrast, H3K9M mice experience an expansion of short-term hematopoietic stem cells, with a subset of mice developing an aggressive form of CD4⁺/CD8⁺ T cell malignancy.

Of note, a control strain containing wildtype H3.3, Col1a1-tetO-H3.3::R26-M2rtTA, is available as Stock No. 034364.
Similar strains with different lysine-to-methionine point mutations include:
Col1a1-tetO-H3.3K36M::R26-M2rtTA mice with a lysine-to-methionine point mutation at amino acid 36 are available as Stock No. 034365
Col1a1-tetO-H3.3K9M::R26-M2rtTA, with a lysine-to-methionine point mutation at amino acid 9 is available as Stock No. 034366
Col1a1-tetO-H3.3K4M::R26-M2rtTA mice with a lysine-to-methionine point mutation at amino acid 4 are available as Stock No. 041075

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 was re-targeted to insert an optimized form of reverse tetracycline controlled transactivator (rtTA-M2) followed by a rabbit beta-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-H3.3K27M plasmid and Flpe recombinase. The H3.3K27M plasmid contained a splice acceptor-double polyA sequence and the tetracycline responsive element (tetO) upstream of the H3.3K27M sequence.

The resulting ES cells, now targeted with rtTA-M2 in the Gt(ROSA)26Sor locus (Rosa26-rtTA) and tetO-H3.3K27M in the Col1a1 locus (Col1a1-tetO-H3.3K27M), were injected into recipient blastocysts. The resulting male mice were bred with C57BL/6 females and were maintained on a mixed background. Upon arrival at The Jackson Laboratory, sperm was cryopreserved. To establish our live colony, an aliquot of frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664).

The donating laboratory refers to this mutation as H3f3a*K27M with respect to amino acid numbering the initiator methionine as position 0. Sequence analysis performed by The Jackson Laboratory confirmed the K-to-M mutation is present at position 28 (H3f3a*K28M) with respect to amino acid numbering the initiator methionine as position 1 of the Ensembl canonical transcript ENSMUST00000161308.8 H3f3a-205

Allele

Symbol: Col1a1^{tm9(tetO-H3f3a*K28M)Hoch}
Name: targeted mutation 9, Konrad Hochedlinger
MGI accession ID: MGI:8244304
Generation method: Targeted
Attributes: Inducible; Inserted expressed sequence
Synonyms: tetO-H3.3 K27M
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
Molecular note: The targeting vector is designed to insert an FRT-flanked PGK-Neo-pA 500 bp downstream of the 3' UTR of the Col1a1 locus. The FRT-flanked PGK-Neo-pA in the 3' UTR of the Col1a1 locus is then replaced by co-injection of a mutant tetO-H3.3f3a plasmid and flpe recombinase. The plasmid contains a splice acceptor-double polyA sequence and the tetracycline responsive element (tetO) upstream of the H3.3 histone A gene carrying a lysine-to-methionine substitution at amino acid 28 (K28M).
General note: The allele was made in KH2 cells that carry Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae} and 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.