Electroporation of Horn Fly Embryos

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Ting Li, Ph.D., Postdoctoral Research Associate, Entomology and Plant Pathology, Auburn University
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Qiang Xu, et al. 2015 describe their efforts to transform the horn fly, Haematobia irritans, by elctroporation.

Transgenic technology has been considered an effective technique for the construction of genetically-modified insects for the purposes of insect control. Microinjection with piggyBac transposable element-mediated genetic transformation has been reported in a wide range of insect species but for many species of insects this is difficult or impossible.  Alternative delivery methods are needed.

Haematobia irritans

Qiang Xu, et al. 2015 compared microinjection and electroporation with vector pBac [3XP3 EGFP] and helper plasmid pBhs ∆sst into the horn fly embryos. Following microinjection they obtained  no survival of horn flies developing to the adult stage of G0 flies, but, in contrast,  electroporation produced transfected flies expressing EGFP in the G0, G1, and G2 generations as tested by both fluorescence screening and PCR.

In addition, following electroporation of another fluorescent marker, DsRed, it too was successfully transfected into horn fly embryos, with 12 out of 42 G0 individuals producing G1 lines after independently mating each with wild-type horn flies.   Expression of the marker gene was also detected in the G2 generation as determined by both fluorescence screening and PCR.

Basic Concepts of Electroporation

Basic Concepts of Electroporation

Qiang Xu, et al. 2015 reported that the expression of both EGFP and DsRed  when driven by the 3XP3 promoter in transgenic flies was only slightly higher than in the control flies, indicating in horn flies the 3xP3 promoter may not be highly functional.  The PUb (Polyubiquitin) promoter was more effective at driving foreign genes in the horn fly.

Furthermore, Qiang Xu, et al. 2015 determined the optimum voltages and showed that 800 and 1250 V/cm were appropriate for transgenic electroporation.

The dechorionation with sodium hypochlorite was utilized to treat horn fly embryos in both the microinjection and electroporation experiments. However,  Qiang Xu, et al. 2015 found that dechorionation caused mortality rates during both the processes of microinjection and electroporation, whereas non-dechorinated eggs survived with high rates following electroporation treatment.

Thus, the convenient use of non-dechorinated eggs can be utilized during electroporation of horn fly embryos.

To confirm transgene integration in horn fly germ-line, Qiang Xu, et al. 2015 used Southern blot hybridization but the data did not show any positive evidence of integration into the horn fly genome, although DsRed expression was detected in the G generation. The authors propose that bacterial symbionts may have been transformed and transgenerationally transmitted.

Overall, this study shows the potential of alternative delivery methods for introducing DNA into insect embryos that are not amenable to microinjection approaches.
Qiang Xu, Felix D. Guerrero, Azhahianambi Palavesam and Adalberto A. Pérez de León (2015) Use of electroporation as an option to transform the horn fly, Haematobia irritans, a species recalcitrant to microinjection Insect Science  DOI: 10.1111/1744-7917.12207

 

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