Gt(ROSA)26Sor^{tm1(TARDBP*M337V/Ypet)Tlbt}

The human TARDBP from BAC RP11-829B14 was modified by site-directed mutagenesis to create the M337V missense mutation in exon 6 (an A-to-G base change (A1009G) encoding a methionine-to-valine substitution at amino acid position 337). The final construct includes the TDP-43-M337V genomic DNA (44,886 bp) with a Ypet ORF cDNA sequence (717 bp; described below) fused to the 5' end of TARDBP exon 6, as well as 9.2 kbp of the original BAC backbone sequences. The TDP-43-M337V-Ypet BAC sequences are flanked with loxP sites. Embryonic stem (ES) cells with a pre-integrated Gt(ROSA)26Sor locus (splice acceptor::FRT::PGK promoter-attP-hygromycin resistance gene-pA::FRT::attP between exons 1 and 2) were re-transfected with the TDP-43-M337V-Ypet BAC sequence and a PhiC31 vector for integrase-mediated cassette exchange. The Ypet cDNA sequence is a codon-improved YFP variant YFP3 (with F46L, I47L, S208F, V224L, H231E, D234N mutations) further modified to include several Venus mutations (F64L, R79K, M153T, V163A, S175G), resulting in the YFP for energy transfer (YPet) variant. (J:259586, J:265544)