B6;129S4-Gt(ROSA)26Sor^{tm1(rtTA*M2)Jae} Col1a1^{tm8(tetO-H3f3a*K5M)Hoch}/HochJ

Stock number: 041075
Product name: TetO-H3K4M/rtTA
Strain synonyms: TetO-H3K5M/rtTA
Research areas: Cell Biology Research, Research Tools

Description

These Col1a1-tetO-H3.3K4M::R26-M2rtTA mice may be used to regulate expression of mutant H3K4 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-H3.3K4M::R26-M2rtTA double heterozygous and homozyogus mice are viable and fertile. Expression of the mutant H3.3K4M 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 H3K4M can be regulated with the tetracycline analog doxycycline (dox) in these double mutant offspring. H3.3K4M has a lysine-to-methionine substitution at amino acid 4, leading to a severe reduction in H3K4 methylation.

These mice die from pancytopenia within 3-4 weeks after dox treatment, which is earlier than Col1a1-tetO-H3.3K36M::R26-M2rtTA mice (Stock No. 034365) with a lysine-to-methionine point mutation at amino acid 36 which die at 1-2 months. The donor states that the underlying mechanisms are different in the two lines. While H3K36M mice develop pancytopenia due to a gradual loss of hematopoietic stem cells, these H3K4M mice retain hematopoietic stem cells but instead exhibit a differentiation arrest of downstream progenitors, which is fully reversible once the H3K4M transgene is turned off and wildtype H3K4 methylation is restored. Also, H3K4M mice develop benign skin lesions in the face and on the paws, which has never been reported in H3K36M mice. H3K36M mice also exhibit defects in spermiogenesis and a secretory defect in the intestine, which is not present in this line of H3K4M mice.

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-H3.3K27M::R26-M2rtTA mice with a lysine-to-methionine point mutation at amino acid 27 are available as Stock No. 041076

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-H3.3K4M plasmid and Flpe recombinase. The H3.3K4M plasmid contained a splice acceptor-double polyA sequence and the tetracycline responsive element (tetO) upstream of the H3.3K4M sequence.

The resulting ES cells, now targeted with rtTA-M2 in the Gt(ROSA)26Sor locus (Rosa26-rtTA) and tetO-H3.3K4M in the Col1a1 locus (Col1a1-tetO-H3.3K4M), 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*K4M 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 5 (H3f3a*K5M) with respect to amino acid numbering the initiator methionine as position 1 of the Ensembl canonical transcript ENSMUST00000161308.8 H3f3a-205.

Allele

Symbol: Col1a1^{tm8(tetO-H3f3a*K5M)Hoch}
Name: targeted mutation 8, Konrad Hochedlinger
MGI accession ID: MGI:8244303
Generation method: Targeted
Attributes: Inducible; Inserted expressed sequence
Synonyms: tetO-H3.3 K4M
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 5 (K5M).
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.