B6.129-H3f3a^tm1Hjf/J

Stock number: 038594
Product name: H3.3A-K27M-FLAG
Research areas: Cancer Research, Cell Biology Research, Research Tools

Description

H3.3A-K28M conditional knock-in mice carry a K27M (see note) amino acid substitution, a 3XFLAG tag, and Venus reporter preceded by loxP-flanked wildtype H3f3a cDNA and an frt-flanked neo cassette in the H3f3a (H3.3 histone A) gene. This strain may be useful for research in cancers associated with the H3.3 variant.

Detailed description

H3f3a (H3.3 histone A) encodes the replication-independent core H3.3 histone protein, which forms a component of the nucleosome. Histone modifications, such as methylation, act in diverse biological processes such as transcriptional activation/inactivation, chromosome packaging, and DNA damage/repair. In humans, the pLys27Met amino acid substitution in the H3F3A is identified as an oncogenic driver in glioblastoma, leukemia and possibly other cancers.

H3.3A-K27M knock-in mice conditionally express a K27M (lysine to methionine, see note about numbering below) amino acid substitution with a 3xFLAG epitope tag and a Venus YFP reporter. The mutation is orthologous to the human K27M mutation. The K27M mutation is preceded by a loxP-flanked H3f3a cDNA (to avoid loss of H3f3a expression from the knock-in) and an frt-flanked neomycin cassette. Prior to Cre recombinase exposure, mice homozygous for the mutation are viable, fertile, and have no overt phenotype.

Upon exposure to Cre recombinase, the wildtype H3f3a cDNA and neo cassette are removed and the human K27M mutation (with FLAG tag and Venus reporter) is expressed. Expression of the mutant allele results in global undermethylation of position K27 in all histone H3 proteins (H3.1, H3.2. H3.3A, H3.3B), not just in the mutated version of H3.3, where methylation at position K27 is no longer possible. Expression of the mutant allele may be monitored by flow cytometry at the single cell level using Venus fluorescent protein. The FLAG tag may be used for detection of mutant protein by Western blot or flow cytometry.

Development

The H3f3a-K27M-FLAG targeting vector was designed to insert a splice acceptor (SA), a loxP site, H3f3a cDNA, an frt-flanked neomycin cassette, a second loxP site, H3f3a cDNA carrying the K27M (lysine to methionine, AAG to ATG, see note) mutation and a FLAG epitope followed by an IRES site and a Venus YFP reporter inserted into exon 2 of the H3.3 histone A (Hf3fa) locus on chromosome 1. The construct was electroporated into (129X1/SvJ x 129S1/Sv)F1-Kitl⁺ derived R1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into blastocysts. The resulting male chimeras were crossed to C57BL/6J females. The strain has been backcrossed to C57BL/6J for at least 10 generations by the donating laboratory. Upon arrival, mice were bred to C57BL/6J for at least 1 generation to establish the colony.

Note: If codons are counted alone based on the open reading frame of the message, the correct codon would be 28. However, the first amino acid (methionine encoded by the start codon ATG) is not part of the mature H3F3A histone protein, as like for many proteins, the amino-terminal Met is removed co-translationally by methionine amino peptidase in a well-known processing step. The lysine that is subject to methylation by Ezh2 of the polycomb complex 2 is numbered in the literature as position 27 (and not 28).

Allele

Symbol: H3f3a^tm1Hjf
Name: targeted mutation 1, Hans Joerg Fehling
MGI accession ID: MGI:7509717
Generation method: Targeted
Attributes: Conditional ready (e.g. floxed); Reporter; Epitope tag
Synonyms: H3f3a-K27M-FLAG
Marker symbol: H3f3a
Marker name: H3 histone, family 3A
Marker synonyms: BRYLIB1; H3.3A; H3.3B; H3.3C; H3.5; H3-3a; H3-3B; H3F3; H3F3A; H3F3C
Chromosome: 1
Strain of origin: (129X1/SvJ x 129S1/Sv)F1-Kitl⁺
Molecular note: The targeting vector is designed to insert a splice acceptor (SA), a loxP site, H3f3a cDNA, an frt-flanked neomycin cassette, a second loxP site, H3f3a cDNA carrying the K27M (lysine to methionine, AAG to ATG) mutation and a FLAG epitope followed by an IRES site and a Venus YFP reporter inserted into exon 2. Please note: if codons are counted alone based on the open reading frame of the message, the correct codon would be 28. However, the first amino acid (methionine encoded by the start codon ATG) is not part of the mature H3F3A histone protein, as like for many proteins, the amino-terminal Met is removed co-translationally by methionine amino peptidase in a well-known processing step. The lysine that is subject to methylation by Ezh2 of the polycomb complex 2 is numbered in the literature as position 27 (and not 28).