CYP5122A1, a Novel Cytochrome P450 of Leishmania Figure 4. Generation of CYP5122A1 half knock-outs. A: Schematic representation of replacement construct inserted into the genome with positions of primers used for confirmation marked by arrows. 59: 59 homologous sequence; 39: 39 homologous sequence; Neomycin: ORF encoding for resistance to neomycin; F2: forward primer designed in the intergenic region upstream of CYP5122A1 gene; P2: internal primer in CYP5122A1 encoding sequence that has been replaced in the replacement construct; NI: primer designed in the internal sequence of ORF for neomycin resistance; Neo: primer pair amplifying neomycin resistance ORF, 59-Neo: Primers that amplify sequence spanning the 59 homologous region through to the end of neomycin resistance ORF. B: Insertion of allelic replacement construct was confirmed by PCR for neomycin and knockout-neomycin construct (59-Neo) using primers that span the 59 homologous sequence (Forward primer) and the neomycin gene (Reverse primer). Lane 1: positive control (Pos) for neomycin resistance gene (plasmid); Lane 2: fragment obtained after amplification of neomycin resistance gene from genomic DNA (G DNA); Lane 3: 1 kb DNA Ladder; Lane 4: Positive control for knock out–neomycin construct (plasmid); Lane 5, Fragment amplified for the knockout- neomycin construct. C: Confirmation of insertion of the replacement construct at the correct locus was assessed by PCR. Lane 1: amplicons generated from WT genomic DNA as template with primers F2/P2 indicating the presence of intact CYP5122A1 allele; Lane 2: Primers F2/NI did not generate any amplicons with WT genomic DNA as template, showing the absence of neomycin resistance ORF; Lane 3: amplicons generated from HKO genomic DNA as template with primers F2/P2 indicating the presence of an intact CYP5122A1 allele; Lane 4: amplicons generated from HKO genomic DNA as template with primers F2/NI indicating the presence of insertion of neomycin resistance ORF at the correct locus. D: Western blot analysis of cell lysates from WT and HKO parasites probed with anti-CYP5122A1 antibody showing decreased level of CYP5122A1 protein in the HKO parasites. Loading was normalized with Tubulin–a (50 kDa). E: Bar graph representing averaged densitometric analysis of immunoblots represented in D (mean6 SE, n = 3), * P#0.05. F: Photomicrographs of L. donovani WT (a, b) and HKO (c, d) parasites immunostained with anti-CYP5122A1. Scale, 10 mm. G: Bar graph representing mean fluorescence intensities of CYP5122A1 fluorescence in WT versus HKO parasites (mean6 SE, n = 3), * P#0.05. doi:10.1371/journal.pone.0025273.g004 PLoS ONE | www.plosone.org 6 September 2011 | Volume 6 | Issue 9 | e25273