qRT-PCR quantitation of the mCherry transcript in schistosomes transfected with vectors expressing a reporter gene at day 2 and day 7.

<p>The viral promoter CMV and the endogenous schistosome promoter SmActin1 were assayed for their ability to express an mCherry reporter at different time frames, without maintaining the schistosomula under transfection conditions.</p

<i>In vitro</i> cultured schistosomula in the presence or absence of SmCaspase7 expression plasmids.

<p>Parasites were incubated in a total volume of 2°C and 5% CO₂ for 60 h. Larvae showing dark and opaque in the images may still be alive and are motile when observed under a light microscope. Only parasites displaying a complete loss of motility or a distinctive loss of morphological integrity were considered dead. (A) Schistosomula were cultured in complete Basch medium with PEI agent alone. After 60 h, most parasites were still alive. Similar results were observed with samples treated with DNA constructs alone (data not shown) or samples treated without any transfection agent (wild-type). (B) Schistosomula were incubated with both PEI and plasmid constructs expressing SmCaspase7 under control of the SmActin1 promoter. Overexpression of the SmCaspase 7 gene in schistosomula caused parasite death and lysis as indicated by the large amount of body debris (arrows). Images were obtained under 40X magnification.</p

qRT-PCR quantitation of the mCherry transcript in schistosomes transfected with vectors expressing this reporter gene regulated by different promoters.

<p>Two viral promoters and four endogenous schistosome promoters were assayed for their ability to express an mCherry reporter. Schistosome samples treated with PEI only or DNA only were used as negative controls. All samples were normalized against the CMV samples.</p

Assessment of 7-day schistosomula viability with propidium iodide.

<p>At 7-days, schistosomula were stained with 2 µg/mL propidium iodide and observed under a microscope with either polarized light or a rhodamine filter (536 nm). Dead parasites fluoresce under the rhodamine filter. Schistosomula were cultured in the Basch medium with PEI agent alone (A–D), transfected with plasmid constructs expressing mCherry under control of SmActin1 promoter (E–H), or transfected with plasmid constructs expressing SmCaspase7 under control of SmActin1 promoter (I–L). (A), (C), (E), (G), (I), (K) were detected by a rhodamine filter. (B, D, F, H, I, L) were the corresponding samples visualized by polarized light. Images were obtained under 40X magnification.</p

Evaluation of Schistosome Promoter Expression for Transgenesis and Genetic Analysis

<div><p>Schistosome worms of the genus <i>Schistosoma</i> are the causative agents of schistosomiasis, a devastating parasitic disease affecting more than 240 million people worldwide. Schistosomes have complex life cycles, and have been challenging to manipulate genetically due to the dearth of molecular tools. Although the use of gene overexpression, gene knockouts or knockdowns are straight-forward genetic tools applied in many model systems, gene misexpression and genetic manipulation of schistosome genes <i>in vivo</i> has been exceptionally challenging, and plasmid based transfection inducing gene expression is limited. We recently reported the use of polyethyleneimine (PEI) as a simple and effective method for schistosome transfection and gene expression. Here, we use PEI-mediated schistosome plasmid transgenesis to define and compare gene expression profiles from endogenous and nonendogenous promoters in the schistosomula stage of schistosomes that are potentially useful to misexpress (underexpress or overexpress) gene product levels. In addition, we overexpress schistosome genes <i>in vivo</i> using a strong promoter and show plasmid-based misregulation of genes in schistosomes, producing a clear and distinct phenotype- death. These data focus on the schistosomula stage, but they foreshadow strong potential for genetic characterization of schistosome molecular pathways, and potential for use in overexpression screens and drug resistance studies in schistosomes using plasmid-based gene expression.</p></div

The <i>Sm</i>Hsf1 antibody recognizes the <i>Sm</i>Hsf1 protein.

<p>Purified IgG from <i>Sm</i>Hsf1-immunized rabbit bleeds (lanes 1–4) or pre-immune serum (lane 5) were used in a Western blot to test for reactivity against bacterially expressed recombinant proteins and cercarial extract (lane 1), 1 µg MBP negative control (lane 2), 1 µg MBP- <i>Sm</i>Hsf1 fusion protein (lane 3), 7 µg MBP- <i>Sm</i>Hsf1 fusion protein (lanes 4 & 5), 7 µg cercarial extract.</p

Gene specific overexpression affects schistosome viability.

<p>The mCherry gene, SmActin 1 gene, SmCyclin B gene, SmCaspase 3 and 7 genes were cloned under regulation of the SmActin promoter, and expressed in schistosomula for up to 7 days. Viability of schistosomula was assessed and quantified. Samples incubated with neither PEI or plasmid DNA (wildtype) or in the presence of PEI agent alone, were used as experimental controls. Data are shown as the mean percentage of surviving larvae from three biological replicates.</p

mCherry protein was produced in schistosomula transfected with plasmids expressing mCherry regulated by the SV40 promoter.

<p>Total protein was extracted from schistosomula expressing mCherry regulated by the SV40 promoter (Lane 1) and assayed by Western blot analysis using a primary antibody targeting the mCherry protein. The mCherry expression regulated by the CMV promoter was used as a positive control (Lane 2) and total protein from untransfected schistosomula was used as a negative control (Lane 3).</p

Schematic of expression plasmids used for transfection experiments.

<p>(A) Six promoters, two viral (CMV and SV40) and four endogenous (SmHsp70, SmActin1, Sm23, and SmCalcineurinA), were used to regulate expression of an mCherry reporter gene. These promoters were separately subcloned into vector pCI-Neo to make plasmids pEJ1175, pEJ1500, pEJ1501, pEJ1502, pEJ1503, pEJ1504, respectively. Forward arrow (a) and reverse arrow (b) represent forward oligonucleotide (a) and reverse oligonucleotide (b), used to quantify mCherry transcript levels by qRT-PCR. (B) The schistosome Actin1, CyclinB, Caspase3, and Caspase7 genes are separately regulated by the plasmid-based SmActin1 promoter. Forward and reverse arrows represent DNA oligonucleotides used for qRT-PCR analysis of each transcript (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098302#pone.0098302.s002" target="_blank">Table S2</a>). (C) Transcript levels of the schistosome Caspase 7 gene were regulated by plasmid-based SmActin1, SmHsp70, or CMV promoters. DNA oligos (c) and (d) were used for qRT-PCR analysis to measure SmCaspase7 transcript levels directed by each promoter.</p

Caspase 3/7 activity in schistosomula is increased by overexpression of SmCaspase7 under different promoters.

<p>Caspase 3/7 activity was measured from 8000 schistosomula transfected with plasmids containing either SmCaspase3 or SmCyclinB genes regulated by the SmActin1 promoter, or the SmCaspase7 gene is regulated by three different promoters (SmActin1, SmHsp70, or CMV) and measured after 48 hours. Caspase activity was assayed from cell extracts using the Caspase-Glo 3/7 assay kit (Promega, Madison, WI) following the manufacturer's protocol. Experiments were done in biological triplicates. Untransfected schistosomes are used as a negative control.</p