This protocol shows production of transgenic Fall Armyworm using piggyBac-based transformation system. We hope that these methods will benefit improving transformation, not only in the Fall Armyworm, but also lepidopteran insects. The method described here makes the germline transformation of lepidopteran pest insects efficient.
Demonstrating the procedure will be Dr.Xien Chen, a postdoctoral fellow in my laboratory. Begin by placing 12 male pupae and 12 female pupae in a box. Place a 10%sucrose solution in the box for feeding adults.
Cover the box with a paper towel. On day two post-eclosion, expose the adults to intense light overnight. On day three post-eclosion, transfer the adults to a dark place.
Collect the embryos within two hours after over position. Add drops of water to the piece of the paper towel with fresh eggs kept in a Petri dish on ice. Transfer the eggs gently to a glass slide with a fine brush and align them one by one on a glass slide.
Keep the glass slides with the eggs aligned on ice before microinjection. Inject a mixture of the 200 nanograms per microliter piggyBac-based EGFP expressing plasmid, 200 nanograms per microliter hyperactive piggyBac transposase mRNA, and sterile distilled water into the eggs using the compensation pressure of the automated microinjector. Keep the injected eggs at 27 plus or minus one degree Celsius, 60 plus or minus 10%relative humidity until hatching.
Feed the hatched larvae on an artificial diet, and transfer each larva to one small cup at the early curd and star stage. Collect the pupae and place approximately 150 female and 150 male pupae in a cage. Hang several pieces of paper towels 30 centimeters by 15 centimeters in size in the cage to collect the eggs.
Collect the paper towels with eggs daily, and keep the eggs in appropriate conditions until hatching. Keep the neonate larvae at four degrees Celsius for five minutes to immobilize them, and then transfer them onto an ice cold plate. Screen the immobilized neonate larvae under a fluorescence stereo microscope.
Select the EGFP-positive neonates, raise them to the pupal stage, in around two weeks at 27 plus or minus one degree Celsius. The hyperactive piggyBac transposase mRNA synthesized in vitro was detected using a 1%Agarose gel. The PDS-injected embryos did not show EGFP signal, whereas the EGFP-expressing plasmid and hyperactive piggyBac transposase mRNA-injected embryos showed EGP fluorescence, indicating that the piggyBac construction was successful.
The G1 larvae obtained from eggs resulting from a self-cross of G0 adults showed a strong EGFP signal. A fragment containing the promoter and EGFP fragment was successfully amplified using the genomic DNA isolated from the EGFP-positive larvae as a template. However, the same fragment was not detected when genomic DNA was isolated from the wild-type larvae as the template.
The eggs for microinjection should be as fresh as possible and must always be kept on ice before microinjection to slow down their development. This technique paves the way for researchers to answer questions in pest insect functional genomics and develop genetic control methods for this global pest.
