The research reported is supported by the National Science Foundation I/UCRC, the Center for Arthropod Management Technologies, under Grant No IIP-1821936 and by industry partners, Agriculture and Food Research Initiative Competitive Grant No. 2019-67013-29351 and the National Institute of Food and Agriculture, US Department of Agriculture (2019-67013-29351 and 2353057000).

1. In vitro synthesis of hyPBase mRNA
NOTE: The complete coding sequence of the hyPBase sequence was synthesized and inserted into a pTD1-Cas9 vector (see the Table of Materials) to produce the pTD1-hyPBase construct, which contains a hyPBase-expressing cassette, T7 promoter: polyhedrin-5&#39; UTR: hyPBase: polyhedrin-3&#39; UTR: poly (A). The full sequence of the pTD1-hyPBase construct is provided in the Supplementary Material.
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The authors declare that they have no competing financial interests.

The low rate of transposition and difficulty of delivering transgenic components into fresh embryos limit the success of germline transformation in many non-model insects, especially those from order, Lepidoptera. To increase the germline transformation rate, a hyperactive version of the most widely used piggyBac transposase (hyPBase) was developed7,10. To date, successful germline transformation in lepidopteran insects is mainly reported in the model in...

The fall armyworm (FAW), Spodoptera frugiperda, is native to tropical and subtropical regions of America. Currently, this is a devastating insect herbivore in more than 100 countries worldwide1. FAW larvae feed on more than 350 host plants, including some important staple food crops2. The strong migration ability of FAW adults contributes to its recent rapid spread from the Americas to other places1,2. As a result, this insect is now threatening food security internationally. Applying new technologies may facilitate advanced studies in FAW and provide novel strategies to manage this pest.
Insect germline transformation has been used to study gene function and generate transgenic insects for use in genetic control3,4. Among the various methods used to achieve genetic transformation in insects, the piggyBac element-based method is the most used method5. However, due to the low rate of transposition, it is still challenging to conduct transgenesis in non-model insects. Recently, a hyperactive version of piggyBac transposase (hyPBase) was developed6,7. Germline transformation was achieved in FAW recently8, which is the first report that used the hyPBase in lepidopteran insects. In this report, detailed information on employing hyPBase mRNA in generating transgenic FAW is described. The method described here could be applied to achieve the transformation of other lepidopteran insects.

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			<td>Dissecting Scope</td>
			<td>Nikon</td>
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			<td>SCREENING</td>
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			<td>Microinjector</td>
			<td>Narishige</td>
			<td>IM-300</td>
			<td>INJECTION</td>
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			<td>Micropipette Puller</td>
			<td>Sutter Instruments</td>
			<td>P-1000</td>
			<td>INJECTION</td>
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			<td>SFA-RB-DIM</td>
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			<td>NI 225</td>
			<td>INJECTION</td>
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			<td>Benzon Research, Inc</td>
			<td>N/A [Request Species/Quantity]</td>
			<td>REARING</td>
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			<td>Taq MasterMix</td>
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hyperactive piggybac transposase-mediated germline transformation in the fall armyworm, spodoptera frugiperda

Stable insertion of genetic cargo into insect genomes using transposable elements is a powerful tool for functional genomic studies and developing genetic pest management strategies. The most used transposable element in insect transformation is piggyBac, and piggyBac-based germline transformation has been successfully conducted in model insects. However, it is still challenging to employ this technology in non-model insects that include agricultural pests. This paper reports on germline transformation of a global agricultural pest, the fall armyworm (FAW), Spodoptera frugiperda, using the hyperactive piggyBac transposase (hyPBase).
In this work, the hyPBase mRNA was produced and used in place of helper plasmid in embryo microinjections. This change led to the successful generation of transgenic FAW. Furthermore, the methods of screening transgenic animals, PCR-based rapid detection of transgene insertion, and thermal asymmetric interlaced PCR (TAIL-PCR)-based determination of the integration site, are also described. Thus, this paper presents a protocol to produce transgenic FAW, which will facilitate piggyBac-based transgenesis in FAW and other lepidopteran insects.

A construct for the expression of hyPBase-containing T7 promoter: polyhedrin-5&#39;UTR: hyPBase: polyhedrin-3&#39;UTR: poly (A) signal was generated (Figure 1A) and amplified as a ~2.2 kb PCR fragment to synthesize hyPBase mRNA in vitro (Figure 1B). Then, the hyPBase mRNA was produced and subjected to agarose gel electrophoresis. The mRNA of the expected size (~1.1 kb band) was detected on a 1% agarose gel (Figure 1C).
...

Watch this Scientific Journal Video about Hyperactive piggyBac Transposase-mediated Germline Transformation in the Fall Armyworm, Spodoptera frugiperda at JoVE.com

Hyperactive piggyBac Transposase-mediated Germline Transformation in the Fall Armyworm, Spodoptera frugiperda

Successful germline transformation in the fall armyworm, Spodoptera frugiperda, was achieved using mRNA of hyperactive piggyBac transposase.

Hyperactive piggyBac Transposase-mediated Germline Transformation in the Fall Armyworm, Spodoptera frugiperda

Stable insertion of genetic cargo into insect genomes using transposable elements is a powerful tool for functional genomic studies and developing genetic ...

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.

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4.0K 次观看.  University of Kentucky. 该协议显示了使用基于piggyBac的转化系统生产转基因秋粘虫。我们希望这些方法将有利于改善转化，不仅在秋粘虫中，而且在鳞翅目昆虫中。这里描述的方法使鳞翅目害虫昆虫的种系转化效率很高。演示该程序的将是我实验室的博士后研究员Xien Chen博士。首先将12只雄性蛹和12只雌性蛹放在一个盒子里。将10%蔗糖溶液放入盒子中喂养成人。用纸巾盖住盒子。在闭合后...