Gt(ROSA)26Sor^{tm1(CAG-CHRM4*,-mCitrine)Ute}
Targeted Allele Detail

Summary

• Symbol: Gt(ROSA)26Sor^{tm1(CAG-CHRM4*,-mCitrine)Ute}
• Name: gene trap ROSA 26, Philippe Soriano; targeted mutation 1, Ute Hochgeschwender
• MGI ID: MGI:5614050
• Synonyms: hM4Di, LSL-hM4Di, R26^hM4D, R26-hM4Di/mCitrine, R26-LSL-Gi-DREADD, R26-LSL-hM4Di/mCitrine
• Gene: Gt(ROSA)26Sor Location: Chr6:113044389-113054205 bp, - strand Genetic Position: Chr6, 52.73 cM
• Alliance: Gt(ROSA)26Sor^{tm1(CAG-CHRM4*,-mCitrine)Ute} page

Mutation origin

• Germline Transmission: Earliest citation of germline transmission: J:236327
• Parent Cell Line: R1 (ES Cell)
• Strain of Origin: (129X1/SvJ x 129S1/Sv)F1-Kitl⁺

Mutation description

• Allele Type: Targeted (Conditional ready, No functional change)
• Mutation: Insertion
• Mutation details: The targeting vector was designed with (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG), an FRT site, a loxP-flanked STOP cassette (with stop codons in all 3 reading frames and a triple polyA signal), a hemagglutinin epitope tag (HA), the hM4Di sequence, a porcine teschovirus (PTA) cleavage site, the mCitrine fluorescent protein, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability), a BGH polyA signal, and an attB/attP-flanked PGK-FRT-Neo-polyA cassette. This entire construct was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus.

  The hM4Di sequence is a Gⁱ-coupled human M4 muscarinic DREADD (designer receptor exclusively activated by designer drug). To create the hM4Di sequence, the wildtype human muscarinic 4 receptor (CHRM4) sequence was modified via site-directed mutagenesis to harbor two amino acid substitutions (Y113C<3.33>/A203G<5.46>) that abolish receptor affinity for the native ligand, acetylcholine (ACh), but allow receptor binding and subsequent activation by the small drug-like molecule clozapine-N-oxide (CNO).

  mCitrine is a yellow-green variant of Citrine YFP additionally harboring the A206K mutation. mCitrine is a approx 5.7 pKa monomer with 516nm excitation and 529nm emmission spectra. Compared to wildtype GFP, Citrine harbors the S65G, V68L, Q69M, S72A and T203Y mutations, and mCitrine additionally has the A206K mutation (enhancing its ability to be a stable monomer). (J:236327)

Find Mice (IMSR)

• Mouse strains and cell lines available from the International Mouse Strain Resource (IMSR)
• Carrying this Mutation: Mouse Strains: 1 strain available Cell Lines: 0 lines available
• Carrying any Gt(ROSA)26Sor Mutation: 993 strains or lines available

References

• Original: J:236327 Zhu H, et al., Cre-dependent DREADD (Designer Receptors Exclusively Activated by Designer Drugs) mice. Genesis. 2016 Aug;54(8):439-46
• All: 58 reference(s)