B6.129S6(C)-Smarca4^tm2.1Grc/J

Stock number: 031598
Product name: BRG1(N1507A)-Dendra2
Research areas: Neurobiology Research, Research Tools

Description

These knock-in mice express a Dendra2 photoswitchable reporter from the mouse Smarca4 (Brg1) promoter. An N1507A mutation in the Smarca4 bromodomain is useful in studies of histone/DNA binding and chromatin remodeling. Neither the N1507A or Dendra2 tag disrupt normal expression of the targeted gene. The N1507A mutation is believed to be the cause of homozygous embryonic lethality.

Detailed description

BRG1 (Smarca4; SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 4) and BRM (Smarca2) are central components of the Brg/Brahma-associated factors (BAF) chromatin remodeling complex that is critical to chromatin structure regulation. A bromodomain (BRD) in the ATPase subunit encoded by exon 11 of the mouse Smarca4 gene bears both DNA and histone binding activity. The mouse BRD sequence is identical to that of the human and has been implicated in mediating the response to histone acetylation.

These targeted mutant mice express a C-terminal Dendra2 photoswitchable tag from the mouse Smarca4/Brg1 promoter that is useful for fluorescence recovery after photobleaching (FRAP) experiments and for standard localization via microscopy. The bromodomain in exon 11 of the Smarca4 gene has also been modified to carry an N1507A mutation. Expression of the Smarca4 gene is unaffected by the tag insertion/bromodomain mutation.

Most bromodomains share a conserved asparagine (N) residue that mediates a hydrogen bond with acetylated histone peptides. Previous in vitro studies have shown that mutating the asparagine to an alanine (A) in multiple bromodomains eliminates acetyl-binding activity. These mice were created to study the in vivo function of the Smarca4/Brg1 bromodomain in the context of the entire BAF chromatin remodeling complex.

Heterozygotes are viable and fertile, but homozygotes do not progress past the blastocyst stage of development. Evidence suggests that the reduced viability is likely due to the N1507A mutation rather than the Dendra2 tag.

Development

A 2 base pair mutation (AAC to GCC; amino acid N1507A) was introduced to the exon 11 bromodomain of the Smarca4 (Brg1) gene, a linker/Dendra2 photoswitchable tag protein was placed directly 3' of the gene, and a loxP-flanked neomycin resistance cassette was inserted in the 3'UTR via homologous recombination in 129S6/SvEvTac-derived TC1 embryonic stem (ES) cells. The neomycin cassette was excised from the resultant mice via crosses with a C57BL/6 background CMV-cre strain (see Stock No. 006054). This strain was backcrossed to C57BL/6J for more than 5 generations by the donating laboratory.