Fig. 1

Generation of a Tg vector based on conserved Prnp genomic elements. (a) Schematic of the rat Prnp genomic locus compared with mouse and Syrian hamster (SHa) Prnp using the Vista alignment tool. We define the rat Prnp locus in three regions: Region I spans a 6 kb upstream sequence, Exon 1 (E1), Intron 1, and Exon 2 (E2). Region II represents Intron 2. Region III defines Exon 3 (E3), the 3’UTR, and 2.3 kb of downstream sequence. Using a sliding window of contiguous regions of 100 bp with an identity greater than 50%, graphical outputs were generated comparing genomic position (x-axis) to percent identity (y-axis). Prnp genetic elements with high similarity are color coded: dark blue = exons, light blue = UTR, and red = non-coding sequence/intergenic region. (b) Schematic of the cloning steps to generate the RaPrnp vector. The RaPrnp vector includes Region I and Region III with E3 removed and replaced with an XhoI site but maintains the 3’UTR and 2.3 kb downstream sequence. Gray vertical bars refer to the 15 bp In-Fusion homology arms. NotI sites were incorporated at the ends of the RaPrnp transgene to remove the pUC19 backbone. The AmpR resistance cassette is designated by the orange curved arrow