Gt(ROSA)26Sor^{tm1.1(CAG-HL9-MG*V70A,-mBFP,-SNCA,-mTFP1,-SNCA*A30P,-mKO2,-SNCA*A53T)Rali}

A targeting vector contains (from 5' to 3') a strong CAG (CMV-IE enhancer/chicken beta-actin/rabbit beta-globin hybrid) promoter, three pairs of orthogonal lox sites (LoxN, Lox2272, and LoxP), and the alphasynuclein-Crainbow (SNCAbow) construct (described in greater detail below), followed by an FRT-flanked neomycin resistance (neo) cassette. This entire construct was inserted between exons 1 and 2 of the Gt(ROSA)26Sor locus. The alphasynuclein-Crainbow construct contains three pairs of orthogonal lox sites (LoxN, Lox2272, and LoxP) that separate four cassettes, three of which are associated with a corresponding alpha-synuclein gene sequence. The first cassette expresses a chemically inducible nearinfrared fluorogenactivating peptide (referred to as FAPMars1). Mars1 is a V70A mutation of the human single-chain antibody (scFv)-based fluorogen isolated from HL9-MG (human library 9 malachite green). The next three cassettes encode a unique nuclear localized fluorescent protein (TagBFP:blue, mTFP1:turquoise, or mKO2:orange) and a corresponding human alpha-synuclein protein sequence (SNCA(WT), SNCA(A30P), or SNCA(A53T), respectively) each separated by a P2A peptide to mediate ribosomal skipping. Each alpha-synuclein sequence contains a C-terminal epitope tag: SNCA(WT) tagged with V5, SNCA(A30P) tagged with 3XHA, or SNCA(A53T) tagged with Myc. To ensure that only one fluorescent protein is expressed at any one time, an SV40 polyadenylation signal was inserted downstream of each open reading frame. Upon cre activation, the lox sites recombine to create three mutually exclusive excision possibilities with the fluorescent protein that is placed first after the CAG promoter being expressed. FLP-mediated recombination removed the neomycin cassette. (J:101977)