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Title: Transgene expression in tick cells using agrobacterium tumefaciens

Author
item Machado-ferreira, E - Universidade Federal Do Rio De Janeiro
item Balsemao-pires, E - Universidade Federal Do Rio De Janeiro
item Dietrich, G - Centers For Disease Control And Prevention (CDCP) - United States
item Hojgaard, A - Centers For Disease Control And Prevention (CDCP) - United States
item Vizzoni, Vinicius - Universidade Federal Do Rio De Janeiro
item Scoles, Glen
item Bell-sakyi, Lesley - Pirbright Laboratory
item Piesman, Joseph - Centers For Disease Control And Prevention (CDCP) - United States
item Zeidner, Nordin - Centers For Disease Control And Prevention (CDCP) - United States
item Soares, Carlos - Universidade Federal Do Rio De Janeiro

Submitted to: Experimental and Applied Acarology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/11/2015
Publication Date: 7/19/2015
Citation: Machado-Ferreira, E., Balsemao-Pires, E., Dietrich, G., Hojgaard, A., Vizzoni, V.F., Scoles, G.A., Bell-Sakyi, L., Piesman, J., Zeidner, N.S., Soares, C.A. 2015. Transgene expression in tick cells using agrobacterium tumefaciens. Experimental and Applied Acarology. 67(2):269-287.

Interpretive Summary: Ticks transmit infectious diseases to humans and other animals. Genetic manipulation of these arthropods would allow the development of alternative disease control strategies. Interestingly, Agrobacterium tumefaciens (At) mediated T-DNA transfer has been recently shown to promote the genetic modification of non-plant cells. In the present work we developed T-DNA constructs for successful At-mediated transgene transfer into both human HeLa cells and Rhipicephalus microplus tick cell line BME26. Translational fusions eGfp:eGfp or Salp15:eGfp, with the enhanced-green fluorescent protein (eGFP) and the Ixodes scapularis salivary factor SALP15 genes were constructed, using either the CaMV 35S (cauliflower mosaic virus) promoter, the tick promoter “PBm”, for the R. microplus pyrethroid metabolizing esterase gene, or the Simian Virus SV40 promoter. Confocal and fluorescence microscopy, RT-PCR, and Western-blot assays demonstrated transgene(s) expression in both cell lines. Unprecedented genetic modification of ticks was also achieved with the insert of T-DNAs in both R. microplus and Ixodes scapularis larvae utilizing a soaking method for At infection. Transgenesis was confirmed by nested-RT-PCR showing eGfp or Salp15 poly-A-mRNAs specifically in ticks infected with At designed for T-DNAs delivery. This strategy opens up a new venue to manipulate the tick genome and represents a breakthrough for the study of tick biology.

Technical Abstract: Ticks transmit infectious diseases to humans and other animals. Genetic manipulation of these arthropods would allow the development of alternative disease control strategies. Interestingly, Agrobacterium tumefaciens (At) mediated T-DNA transfer has been recently shown to promote the genetic modification of non-plant cells. In the present work we developed T-DNA constructs for successful At-mediated transgene transfer into both human HeLa cells and Rhipicephalus microplus tick cell line BME26. Translational fusions eGfp:eGfp or Salp15:eGfp, with the enhanced-green fluorescent protein (eGFP) and the Ixodes scapularis salivary factor SALP15 genes were constructed, using either the CaMV 35S (cauliflower mosaic virus) promoter, the tick promoter “PBm”, for the R. microplus pyrethroid metabolizing esterase gene, or the Simian Virus SV40 promoter. Confocal and fluorescence microscopy, RT-PCR, and Western-blot assays demonstrated transgene(s) expression in both cell lines. Unprecedented genetic modification of ticks was also achieved with the insert of T-DNAs in both R. microplus and Ixodes scapularis larvae utilizing a soaking method for At infection. Transgenesis was confirmed by nested-RT-PCR showing eGfp or Salp15 poly-A-mRNAs specifically in ticks infected with At designed for T-DNAs delivery. This strategy opens up a new venue to manipulate the tick genome and represents a breakthrough for the study of tick biology.