The Snap25-2A-GCaMP6s-D targeted mutation (also called Snap25-2A-GCaMP6s-Δhygro) has a viral 2A oligopeptide sequence (T2A) that mediates ribosomal skipping and a GCaMP6 slow variant calcium indicator (GCaMP6s), all inserted downstream of the synaptosomal-associated protein 25 (Snap25) coding region. The specific details are below.
(129S6/SvEvTac x C57BL/6)F1-derived G4 embryonic stem (ES) cells, already targeted with a LSL-2A-EGFP vector downstream of the Snap25 coding region (see Stock No. 021879), were re-targeted with a "T2A-GCaMP6s" vector and a FLP-expressing plasmid to facilitate recombination.
Correctly re-targeted ES cells had (from 5' to 3')
a partial Snap25 sequence spanning intron 6 through intron 7,
an frt3 site within Snap25 intron 7,
a partial Snap25 exon 8 sequence up (but not including) the endogenous translational stop codon,
a T2A peptide cleavage signal that is in-frame with the Snap25 coding sequence,
a GCaMP6s gene (described below) that is in-frame with the Snap25 coding sequence,
a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance the mRNA transcript stability),
a bovine growth hormone polyA sequence,
an AttB site,
a PGK promoter-hygromycin resistance gene-SV40polyA cassette (with an mRNA splice donor-frt5 site-mRNA splice acceptor in the hygromycin gene),
and an AttP site.
Correctly re-targeted ES cells were injected into recipient blastocysts. The resulting chimeric animals were bred to PhiC31-expressing mice (C57BL/6J congenic background; see Stock No. 007743) to delete the AttB/AttP-flanked sequences (PGK-5'hygro::mRNA splice donor::frt5::mRNA splice acceptor::3'hygro::SV40 polyA) and replace it with the recombined AttB/AttP site (AttL).
The resulting Snap25-2A-GCaMP6s-D mice were bred with C57BL/6J wildtype mice for several generations (and the PhiC31 gene was removed) prior to sending generation N6F1 males to The Jackson Laboratory Repository in 2015. Upon arrival, males were used to cryopreserve sperm. To establish the living Snap25-2A-GCaMP6s-D mouse colony, an aliquot of the frozen sperm was used to fertilize oocytes from C57BL/6J inbred females (Stock No. 000664).
The GCaMP6 slow variant calcium indicator (GCaMP6s) is an ultrasensitive detector of single neuronal action potentials with slow response kinetics (Chen et al. 2013 Nature 499:295), and is an improved version of GCaMP5G. The GCaMP6s fusion gene was designed by Drs. Douglas Kim and Loren Looger (Janelia Farm, HHMI). GCaMP6s has a chicken smooth muscle M13 fragment of myosin light chain kinase, a circularly permutated EGFP (cpEGFP; aa 149-238 followed by aa 1-144 [with M153K, V163A, S175G, D180Y, T203V, A206K and V93I mutations to increase dynamic range/baseline fluorescence, as well as K166H mutation for increased sensitivity/slower kinetics]), and a rat calmodulin DNA fragment (CaM; aa 2-148 with N60D, D78Y, T79R, S81T and R90G mutations [also called N362D, D380Y, T381R, S383T and R392G, respectively] to increase the fluorescence change for small calcium transients. The T302L and R303P amino acid substitutions in the cpEGFP-to-CaM linker improve sensor function.
