The R26-LSL-Gi-DREADD mice were created at Duke University by Drs. Ute Hochgeschwender (while at Duke University) and Bryan L. Roth (University of North Carolina Chapel Hill Medical School). The Rosa-CAG-LSL-HA-hM4Di-pta-mCitrine targeting vector was designed with (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (CAG), a 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 (described in detail below), a porcine teschovirus (PTA) cleavage site, the mCitrine fluorescent protein (described in detail below), 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 via electroporation of (129X1/SvJ x 129S1/Sv)F1-Kitl+-derived R1 embryonic stem (ES) cells. Correctly targeted ES cells were injected into recipient blastocysts. The resulting chimeric animals were bred with C57BL/6NCrl mice. Heterozygotes were further crossed to C57BL/6NCrl for several generations by the donating lab (see SNP notes below). In 2015, heterozygous males from generation N6 were sent to The Jackson Laboratory Repository. Upon arrival, sperm was cryopreserved. To generate the living colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6NJ female mice (Stock No. 005304). The R26-LSL-Gi-DREADD mice were then bred together, to wildtype mice from the colony or to C57BL/6NJ inbred animals. The donating investigator reports they bred a R26-LSL-Gi-DREADD female to a C57BL/6N male (thus the Y chromosome is from the C57BL/6N background).
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 (Y113C3.33/A203G5.46) 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).
Additional information on hM4Di:
More information on hM4Di or other DREADDs may be available at Designer Receptors Exclusively Activated by Designer Drugs DREADD wiki webpage.
mCitrine is a yellow-green variant of Citrine YFP additionally harboring the A206K mutation. mCitrine is a ~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).
In 2020, a 48 SNP (single nucleotide polymorphism) panel analysis, with 43 markers covering all 19 chromosomes and the X chromosome, as well as 5 markers that distinguish between the C57BL/6J and C57BL/6N substrains, was performed on the rederived living colony at The Jackson Laboratory Repository. One of the 43 markers, on Chromosome 7, was segregating with 129 suggesting an incomplete backcross. Also, one of the 5 markers that determine C57BL/6J from C57BL/6N was found to be segregating. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6N;C57BL/6J genetic background.
