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Experimental and Applied Acarology

, Volume 67, Issue 2, pp 269–287 | Cite as

Transgene expression in tick cells using Agrobacterium tumefaciens

  • Erik Machado-Ferreira
  • Emilia Balsemão-Pires
  • Gabrielle Dietrich
  • Andrias Hojgaard
  • Vinicius F. Vizzoni
  • Glen Scoles
  • Lesley Bell-Sakyi
  • Joseph Piesman
  • Nordin S. Zeidner
  • Carlos A. G. SoaresEmail author
Article

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.

Keywords

Transgene expression Tick cells Agrobacterium tumefaciens Rhipicephalus microplus Ixodes scapularis 

Notes

Acknowledgments

Erik Machado-Ferreira’s fellowship was supported by CDC and the Brazilian agency CAPES in the Departamento de Genética-Instituto de Biologia (Universidade Federal do Rio de Janeiro). E. Balsemão-Pires was supported by a PhD fellowship from CAPES/Brazil and a SWE fellowship from CNPq/Brazil. The BME/CTVM2 cell line was provided by the Tick Cell Biobank. The anti-Salp15 antibody was supplied by Dr. Amy Ullmann-Moore of the CDC.

Supplementary material

10493_2015_9949_MOESM1_ESM.pdf (309 kb)
Supplemental fig. 1 Confocal microscopy of N. benthamiana leaves infiltrated with A. tumefaciens pP35S-eGFPeGFP. Free eGFP (green) and chlorophyll-a (red) emissions captured at 505-530 nm and 685nm, respectively, are presented in the left-side and central panels. Merged images are displayed in the right-side panels. Emission for the infiltrations with T-DNA construct with P35S:eGfp:eGfp (eGFP T-DNA) and with bacteria-free buffer (Mock transformation) are shown in each figure row. Bar = 50 µM (PDF 309 kb)

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Copyright information

© Springer International Publishing Switzerland 2015

Authors and Affiliations

  • Erik Machado-Ferreira
    • 1
    • 2
  • Emilia Balsemão-Pires
    • 1
  • Gabrielle Dietrich
    • 2
  • Andrias Hojgaard
    • 2
  • Vinicius F. Vizzoni
    • 1
  • Glen Scoles
    • 3
  • Lesley Bell-Sakyi
    • 4
  • Joseph Piesman
    • 2
  • Nordin S. Zeidner
    • 2
  • Carlos A. G. Soares
    • 1
    Email author
  1. 1.Lab. Genética Molecular de Eucariontes e Simbiotes, Departamento de Genética, Instituto de BiologiaUniversidade Federal do Rio de JaneiroRio de JaneiroBrazil
  2. 2.Bacterial Zoonoses Branch, Division of Vector-Borne Infectious DiseasesCenters for Disease Control and PreventionFort CollinsUSA
  3. 3.Agricultural Research ServicesUSDAPullmanUSA
  4. 4.The Pirbright InstitutePirbrightUK

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