STOCK Gt(ROSA)26Sor^{em4(CAG-BMPR1A*,-Venus)Akg}/J

Stock number: 040351
Product name: BMPR-QD
Research areas: Cancer Research, Developmental Biology Research, Internal/Organ Research, Research Tools

Description

BMPR-QD knock-in mice have a Cre-inducible human BMPR1A allele carrying the constitutively activating Q233D variant, together with Venus fluorescent protein. When bred with mice that express Cre recombinase, offspring will express the constitutively active form of BMPR1A in cells of interest. These mice may be useful for studying bone morphogenetic protein (BMP) signaling.

Detailed description

BMPR-QD knock-in mice are viable, fertile, normal in size and do not display any gross physical or behavioral abnormalities. These transgenic mice possess a Cre-inducible constitutively active mutant form of human bone morphogenetic protein (BMP) receptor type IA (BMPR1A Q233D), a P2A sequence, and the Venus coding sequence driven by a CAG promoter from the Rosa26 locus. When bred with mice that express Cre recombinase, BMPR1A Q233D and Venus are expressed in Cre-recombined cells; Venus fluorescent protein is visible without antibody amplification. These mice may be useful for studying BMP signaling in cartilage and muscle, osteoclastogenesis (via RANKL-OPG pathway and/or canonical Wnt signaling), age dependent bone loss, osteoporosis, chondrodysplasia, fibrodysplasia ossificans progressive, and mineralization in muscle/skeletal development.

Development

The human BMPR1A coding sequence was cloned into the pENTR22 vector, and a constitutively active Q233D mutant was generated by in vitro site-directed mutagenesis. The targeting vectors were constructed by modifying the Ai3 targeting construct (Addgene #22797; Madisen et al., 2010). The EGFP insert in Ai3 was replaced with the coding region of the human BMPR1A receptor with Q233D variant, followed by a GSG-P2A sequence and the coding region of the Venus fluorescent protein downstream of CAG promoter followed by loxP-STOP-loxP cassette. The targeting vector also contains a PGK-Neo cassette flanked by AttB-AttP downstream of BMPR1A-P2A-Venus. The targeting constructs were cloned into a p15a-based targeting vector containing homology arms for the Rosa26 locus (5': 1057 bp; 3': 1231 bp) and electroporated into 129S7/SvEvBrd-Hprt^b-m2 derived AB2.2 embryonic stem (ES) cells with a plasmid expressing Cas9 and a sgRNA sequence to target the Rosa26 locus just inside the 5' homology arm (ACTG GAGT TGCA GATC ACGA GGG). The ES clones were picked, verified for correct targeting of the Rosa26 locus, expanded, and injected into recipient blastocysts to create chimeras, which were then bred with C57BL/6 mice to establish germline founders. The strain was subsequently bred to ICR mice and maintained by the donating laboratory. 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 (Stock No. 000664) oocytes.

Allele

Symbol: Gt(ROSA)26Sor^{em4(CAG-BMPR1A*,-Venus)Akg}
Name: endonuclease-mediated mutation 3, Andrew K Groves
MGI accession ID: MGI:7869451
Generation method: Endonuclease-mediated
Attributes: Conditional ready (e.g. floxed); Reporter; Constitutively active; Inserted expressed sequence
Synonyms: BMPR-QD
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: 129S7/SvEvBrd-Hprt1^b-m2
Molecular note: Using CRSPR technology, the human BMPR1A coding sequence with a constitutively active Q233D mutant followed by a GSG-P2A and coding region of the Venus fluorescent protein were inserted into Gt(ROSA)26Sor downstream of CAG promoter followed by loxp-STOP.