Transgene expression in tick cells using Agrobacterium tumefaciens
- 309 Downloads
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.
KeywordsTransgene expression Tick cells Agrobacterium tumefaciens Rhipicephalus microplus Ixodes scapularis
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.
- Gaudin V, Vrain T, Jouanin L (1994) Bacterial genes modifying hormonal balances in plants. Plant Physiol Biochem 32:11–29Google Scholar
- Haas JH, Moore LW, Ream W, Manulis S (1995) Universal primers for detection of pathogenic Agrobacterium strains. Appl Environ Microbiol 6:2879–2884Google Scholar
- Hojgaard A, Biketov SF, Shtannikov AV, Zeidner NS, Piesman J (2009) Molecular identification of Salp15, a key salivary gland protein in the transmission of lyme disease spirochetes, from Ixodes persulcatus and Ixodes pacificus (Acari: Ixodidae). J Med Entomol 46:1458–1463Google Scholar
- Kurtti TJ, Mattila JT, Herron MJ, Felsheim RF, Baldridge GD, Burkhardt NY, Blazar BR, Hackett PB, Meyer JM, Munderloh UG (2008) Transgene expression and silencing in tick cell line: a model system for functional tick genomics. Insect Biochem Mol Biol 38:963–968PubMedCentralCrossRefPubMedGoogle Scholar
- Lo N, Beninati T, Sassera D, Bouman EA, Santagati S, Gern L, Sambri V, Masuzawa T, Gray JS, Jaenson TG, Bouattour A, Kenny MJ, Guner ES, Kharitonenkov IG, Bitam I, Bandi C (2006) Widespread distribution and high prevalence of an alpha-proteobacterial symbiont in the tick Ixodes ricinus. Environ Microbiol 7:1280–1287CrossRefGoogle Scholar
- Mougel C, Cournoyer B, Nesme X (2001) Novel tellurite-amended media and specific chromosomal and TI plasmid probes for direct analysis of soil popuations of Agrobacterium biovars 1 and 2. Appl Environ Microbiol 67:65–74Google Scholar
- Scherer WF, Syverton JT, Gey GO (1953) HeLa: studies on the propagation in vitro of poliomyelitis viruses—IV: viral multiplication in a stable strain of human malignant epithelial cells (strain HeLa) derived from an epidermoid carcinoma of the cervix. J Exp Med 97:695–710PubMedCentralCrossRefPubMedGoogle Scholar
- Sonenshine DE (1993) Biology of ticks, vol 2. Oxford University Press, New York, NYGoogle Scholar