The Ai96(RCL-GCaMP6s) allele (also called Ai96) was designed by Dr. Hongkui Zeng (Allen Institute for Brain Science) to have a floxed-STOP cassette upstream of the GCaMP6 slow variant calcium indicator (GCaMP6s), all inserted into the Gt(ROSA)26Sor locus. Specific details below.
The Rosa-pCAG-LSL-GCaMP6s-WPRE-bGHpA-AttB-PGK-neo-polyA-AttP targeting vector was designed with (from 5' to 3') a CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid promoter (from pCAGGS), a loxP-flanked STOP cassette (stop codons in all three reading frames linked to synthetic pA-hGHpA-PGKpA), the GCaMP6s coding sequence, a WPRE sequence (to enhance mRNA transcript stability), a bovine growth hormone polyA signal, the GCaMP6s coding sequence (described below), a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; to enhance mRNA transcript stability), a bovine growth hormone polyA signal, an AttB site, a PGK promoter-Neomycin resistance gene-PGK polyA cassette, and an AttP site. This entire 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 were selected. The resulting Ai96 mice were bred to C57BL/6J wildtype mice for at least one additional generation. In 2014, Ai96 heterozygous males on a mixed genetic background (estimated 75% C57BL/6 ; with black coat color) were sent to The Jackson Laboratory Repository. Upon arrival, sperm was cryopreserved. An aliquot of that frozen sperm was used to fertilize C57BL/6J oocytes (Stock No. 000664) and rederive our live colony, which was thereafter maintained by breeding heterozygous mice with wildtype mice from the colony or with C57BL/6J inbred mice.
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.
