title: Transient transgenesis of the tapeworm Taenia crassiceps.
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.
authors.affiliation: Institute for Biomedical Research, Universidad Nacional Autónoma de México, Av. Universidad 3000, Coyoacán, C.P. 04510 México DF, México.
authors.forename: Bárbara
authors.initials: B
authors.lastname: Moguel
authors.affiliation: Institute for Biomedical Research, Universidad Nacional Autónoma de México, Av. Universidad 3000, Coyoacán, C.P. 04510 México DF, México.
authors.forename: Norma
authors.initials: N
authors.lastname: Moreno-Mendoza
authors.affiliation: Institute for Biomedical Research, Universidad Nacional Autónoma de México, Av. Universidad 3000, Coyoacán, C.P. 04510 México DF, México.
authors.forename: Raúl J
authors.initials: RJ
authors.lastname: Bobes
authors.affiliation: Institute for Biomedical Research, Universidad Nacional Autónoma de México, Av. Universidad 3000, Coyoacán, C.P. 04510 México DF, México.
authors.forename: Julio C
authors.initials: JC
authors.lastname: Carrero
authors.affiliation: Institute for Biomedical Research, Universidad Nacional Autónoma de México, Av. Universidad 3000, Coyoacán, C.P. 04510 México DF, México.
authors.forename: Jesús
authors.initials: J
authors.lastname: Chimal-Monroy
authors.affiliation: Institute for Biomedical Research, Universidad Nacional Autónoma de México, Av. Universidad 3000, Coyoacán, C.P. 04510 México DF, México.
authors.forename: Martha E
authors.initials: ME
authors.lastname: Díaz-Hernández
authors.affiliation: National Laboratory of Genomics for Biodiversity-cinvestav, Km 9.6 Libramiento Norte Carretera Irapuato-León, C.P. 36821 Irapuato, Gto México.
authors.forename: Luis
authors.initials: L
authors.lastname: Herrera-Estrella
authors.affiliation: Institute for Biomedical Research, Universidad Nacional Autónoma de México, Av. Universidad 3000, Coyoacán, C.P. 04510 México DF, México.
authors.forename: Juan P
authors.initials: JP
authors.lastname: Laclette
keywords: Cysticerci; Cytomegalovirus promoter; Green fluorescent protein; Taenia crassiceps; Taenia solium; Transfection