The Rosa-CAG-LSL-GCaMP3-WPRE targeting vector was designed by Dr. Hongkui Zeng (Allen Institute for Brain Science) to have (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 GCaMP3 fusion gene, 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.
The GCaMP3 fusion gene contains a cDNA sequence encoding the chicken smooth muscle M13 fragment of myosin light chain kinase, a circularly permutated EGFP (amino acid residues 149-238 followed by amino acid residues 1-144 (including M153K, V163A, S175G, D180Y, T203V, A206K, and V93I mutations to increase dynamic range and baseline fluorescence), and a DNA fragment encoding amino acid residues 2-148 of the rat calmodulin (modified to harbor the N60D mutation to increase the fluorescence change for small calcium transients).
The entire Rosa-CAG-LSL-GCaMP3-WPRE targeting vector was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus via electroporation of (129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem (ES) cells. Correctly targeted ES cells (clone Ai38) were selected.
Chimeric mice were bred with C57BL/6 wildtype mice and/or PhiC31-expressing mice (C57BL/6J congenic background; see Stock No. 007743); but some mice retaining the attB/attP-flanked PGK-frt-Neo-polyA cassette were propagated (see more details below).
The donating investigator reported that these mutant mice were then bred with C57BL/6J wildtype mice (but see SNP note below) for approximately four additional generations prior to sending to The Jackson Laboratory Repository. Upon arrival, sperm from Ai38 males was used to fertilize oocytes from C57BL/6J inbred mice (Stock No. 000664) in order to rederive the living colony at The Jackson Laboratory Repository.
All of the mice originally sent to The Jackson Laboratory Repository, as well as all the first generation of rederived offspring genotyped as completely co-segregating for the neo cassette and GCaMP3 portion of the Ai38 construct. As such, these mice harbor the Ai38 mutation (retaining the attB/attP-flanked PGK-frt-Neo-polyA cassette) rather than the Ai38Δneo mutation.
A 32 SNP (single nucleotide polymorphism) panel analysis, with 27 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. While the 27 markers throughout the genome suggested a C57BL/6 genetic background, 3 of 5 markers that determine C57BL/6J from C57BL/6N were found to be segregating. These data suggest the mice sent to The Jackson Laboratory Repository were on a mixed C57BL/6J ; C57BL/6N genetic background.
