Transgene expression in tick cells using Agrobacterium tumefaciens.

Area of Science:

• Vector biology
• Molecular entomology
• Genetic engineering

Background:

• Ticks transmit numerous infectious agents to humans and animals.
• Genetic modification of ticks can lead to novel disease control strategies.
• Agrobacterium tumefaciens is a tool for genetic modification in non-plant cells.

Purpose of the Study:

• To develop T-DNA constructs for Agrobacterium tumefaciens-mediated transgene expression in tick cells and larvae.
• To investigate the efficacy of different promoters (CaMV 35S, PBm, SV40) for transgene expression.
• To demonstrate the feasibility of expressing enhanced green fluorescent protein (eGfp) and Salp15 genes in ticks.

Main Methods:

• Construction of T-DNA vectors with translational fusions (eGfp:eGfp, Salp15:eGfp).
• Transformation of HeLa and Rhipicephalus microplus tick cells.
• In vivo transformation of Rhipicephalus microplus and Ixodes scapularis larvae using a soaking method with A. tumefaciens.
• Confirmation of transgene expression using confocal microscopy, RT-PCR, Western-blot, and nested-RT-PCR.

Main Results:

• Successful transgene expression (eGfp, Salp15) was demonstrated in both Rhipicephalus microplus tick cell lines and HeLa cells.
• Transgene expression was confirmed in vivo in Rhipicephalus microplus and Ixodes scapularis larvae.
• Nested-RT-PCR confirmed the presence of poly-A mRNA for eGfp and Salp15 in larvae.

Conclusions:

• Agrobacterium tumefaciens can be effectively used for transgene expression in tick cells and larvae.
• This method provides a novel approach for genetic manipulation of ticks.
• The strategy holds significant potential for advancing research in tick-host interactions and developing new disease control methods.