Abstract
Human and porcine cysticercosis is caused by the larval stage of the flatworm Taenia solium (Cestoda). Infestation of the human brain, also known as neurocysticercosis, is the most common parasite disease of the central nervous system worldwide. Significant advances in the understanding of the disease have been achieved using the Taenia crassiceps murine model. We describe here a successful transfection protocol of T. crassiceps cysticerci as the first step to approach a number of currently inaccessible biological questions on cysticercosis. T. crassiceps cysticerci (ORF strain) were microinjected with the plasmid pcDNA3.1/NT-GFP-TOPO, encoding the green fluorescent protein (GFP) driven by a cytomegalovirus promoter (CMV). Twelve hours after the microinjection, GFP fluorescence gradually developed in patches associated to bud structures in the bladder wall of cysts. Fluorescence reached a peak at 24–48 h and lasted up to 72 h after the microinjection. Immunohistochemical studies on tissue sections of transfected cysts using an anti-GFP antibody, demonstrated co-localization of the antibody and the GFP fluorescence in the tegumentary cytoplasm and subtegumentary cytons. To validate at the mRNA level the expression of GFP, we carried out RT-PCR using two pairs of nested primers. Results showed expression of GFP-mRNA at 24 h post-transfection. Moreover, western blot assays of crude extracts of transfected cysts, carried out using the anti-GFP specific antibody, showed the expected protein band of 27 kDa, demonstrating that the GFP expression started at 24 after plasmid microinjection and was maintained up to 72 h. These findings will facilitate the development of functional genomics approaches applied to this model of cysticercosis.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-015-1278-y) contains supplementary material, which is available to authorized users.
Highlights
Human and porcine cysticercosis is caused by the larval stage of the tapeworm Taenia solium (Cestoda)
green fluorescent protein (GFP) Fluorescence was evident in GFP‐TOPO microinjected cysticerci Around 40 % of the T. crassiceps cysticerci microinjected in the vesicular fluid with GFP-TOPO plasmid showed green fluorescence in patches on the bladder wall (Fig. 1a)
The fluorescent patches appeared in the buds of the plasmid microinjected cysts, Fig. 1 Time course of the GFP fluorescence after microinjection of intact Taenia crassiceps cysticerci. a GFP-TOPO plasmid (1–3) and a GFP-negative plasmid: pCMV-VSV-G (4) microinjected cysts, after 24 (1 and 4); 48 (2) and 72 h (3)
Summary
Human and porcine cysticercosis is caused by the larval stage of the tapeworm Taenia solium (Cestoda). An advantage to develop gene transfer protocols for platyhelminths is the availability of the whole genome sequence for several species, including the free-living planarian Schmidtea mediterranea (Robb et al 2008), the trematode parasites Schistosoma mansoni, S. japonicum, S. haematobium, (Berriman et al 2009; Young et al 2012; Zhou et al 2009), and the cestodes Spirometra erinaceieuropa (Bennett et al 2014), Echinococcus granulosus (Tsai et al 2013; Zheng et al 2013), E. multilocularis, T. solium and Hymenolepis microstoma (Tsai et al 2013). These studies have provided a reasonable understanding on the genome and gene structure in these organisms. Functional genomics tools are needed to assign the physiological roles of protein-coding genes, and identifying novel drug and/or vaccine targets
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