title: Transgene expression in tick cells using Agrobacterium tumefaciens.
abstract: Ticks transmit infectious agents to humans and other animals. Genetic manipulation of vectors like ticks could enhance the development of alternative disease control strategies. Transgene expression using the phytopathogen Agrobacterium tumefaciens has been shown to promote the genetic modification of non-plant cells. In the present work we developed T-DNA constructs for A. tumefaciens to mediate transgene expression in HeLa cells as well as Rhipicephalus microplus tick cells. Translational fusions eGfp:eGfp or Salp15:eGfp, including the enhanced-green fluorescent protein and the Ixodes scapularis salivary factor SALP15 genes, were constructed using the CaMV 35S (cauliflower mosaic virus) promoter, "PBm" tick promoter (R. microplus pyrethroid metabolizing esterase gene) or the Simian Virus SV40 promoter. Confocal microscopy, RT-PCR and Western-blot assays demonstrated transgene(s) expression in both cell lines. Transgene expression was also achieved in vivo, in both R. microplus and I. scapularis larvae utilizing a soaking method including the A. tumefaciens donor cells and confirmed by nested-RT-PCR showing eGfp or Salp15 poly-A-mRNA(s). This strategy opens up a new avenue to express exogenous genes in ticks and represents a potential breakthrough for the study of tick-host pathophysiology.
authors.affiliation: Lab. Genética Molecular de Eucariontes e Simbiotes, Departamento de Genética, Instituto de Biologia, Universidade Federal do Rio de Janeiro, Ilha do Fundão, CCS, Bloco A, Sala A2-120, Rio de Janeiro, RJ, CEP 21941-617, Brazil.
authors.forename: Erik
authors.initials: E
authors.lastname: Machado-Ferreira
authors.forename: Emilia
authors.initials: E
authors.lastname: Balsemão-Pires
authors.forename: Gabrielle
authors.initials: G
authors.lastname: Dietrich
authors.forename: Andrias
authors.initials: A
authors.lastname: Hojgaard
authors.forename: Vinicius F
authors.initials: VF
authors.lastname: Vizzoni
authors.forename: Glen
authors.initials: G
authors.lastname: Scoles
authors.forename: Lesley
authors.initials: L
authors.lastname: Bell-Sakyi
authors.forename: Joseph
authors.initials: J
authors.lastname: Piesman
authors.forename: Nordin S
authors.initials: NS
authors.lastname: Zeidner
authors.forename: Carlos A G
authors.initials: CA
authors.lastname: Soares
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics; D000818:Animals; D060829:Arthropod Proteins / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics*; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D018884:Ixodes / Q000235:genetics* / Q000254:growth & development; D007814:Larva / Q000235:genetics; D048494:Rhipicephalus / Q000235:genetics* / Q000254:growth & development; D019076:Transgenes
chemical_list: D060829:Arthropod Proteins; D004269:DNA, Bacterial; C036483:T-DNA