Pcombo3

The vector pUASTattB MoPrP was generated previously (Murali et al. 2014 (link)). To avoid the introduction of mutations into the pUASTattB (Bischof et al. 2007 (link)) targeting vector, the MoPrP insert was transferred as a BglII/XhoI restriction fragment into the cloning vector pCombo3 (Mike Scott, UCSF) and site-directed mutagenesis performed using the GeneTailor system (Invitrogen, Grand Island, NY) to create pCombo3 P101L, Q171R, and D177N MoPrP constructs. The P101L, Q171R, and D177N MoPrP inserts were then cloned into pUASTattB as BglII/XhoI fragments and the plasmid sequence confirmed by sequencing prior to phiC31-integrase-mediated insertion at attP2 and attP40 performed by Genetic Services, Inc. (Cambridge, MA). Isogenic lines carrying UAS-P101L, Q171R, and D177N MoPrP were isolated, balanced, and maintained using standard Drosophila genetic techniques.