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.
<ol>
	<li>Preparation of the template for in vitro synthesis
	<ol>
		<li>Perform a PCR reaction to amplify the hyPBase-expressing cassette using a forward primer, 5&#39;- atgcggtgtgaaataccgcacagatgcg-3&#39;, and a reverse primer 5&#39;- tagaggccccaaggggttatgctag-3&#39;, high-fidelity polymerase, and pTD1-hyPBase plasmid as a template. 
		NOTE: The PCR reaction (final volume of 50 &#181;L) contains 5.0 &#181;L of 10x Reaction buffer, 4.0 &#181;L of 10 mM deoxynucleoside triphosphate (dNTP), 1.0 &#181;L of plasmid (50 &#181;g/&#181;L), 1.0 &#181;L of high-fidelity polymerase, 1.0 &#181;L each of 10 &#181;M forward and reverse primers, and 33 &#181;L of nuclease-free water. The settings were as follows: an initial incubation of 95 &#176;C for 3 min, followed by 35 cycles of 98 &#176;C for 10 s, 60 &#176;C for 15 s, and 68 &#176;C for 2 min.</li>
		<li>Check the PCR product on a 1% agarose gel at 120 V in fresh Tris-acetate-ethylenediamine tetraacetic acid (TAE) buffer for 30 min and purify the product with the gel extraction kit. 
		NOTE: Do not use the PCR product purification kit. Even a trace amount of the pTD1-hyPBase plasmid significantly decreases the efficiency of in vitro synthesis.</li>
	</ol>
	</li>
	<li>In vitro synthesis of hyPBase mRNA using a commercial kit
	<ol>
		<li>Completely thaw the frozen reagents, keep the enzyme and 2x NTP/CAP solution on ice, and leave the thawed 10x Reaction buffer at room temperature.</li>
		<li>Assemble the reaction at room temperature by adding the following components: 2x NTP/CAP solution: 10 &#181;L; 10x Reaction buffer: 2 &#181;L; Template DNA: 0.1-0.2 &#181;g; Enzyme mix: 2 &#181;L; Nuclease-free water to 20 &#181;L.</li>
		<li>Mix the reagents thoroughly by gently pipetting the mixture up and down and incubate at 37 &#176;C for 2-4 h.</li>
	</ol>
	</li>
	<li>Recovery of synthesized mRNA by lithium chloride precipitation
	<ol>
		<li>Add 30 &#181;L of LiCl Precipitation Solution, mix thoroughly by flicking the tube, and then keep at -20 &#176;C for &#8805; 30 min.</li>
		<li>Centrifuge at 4 &#176;C for 15 min at the maximum speed, and carefully remove the supernatant.</li>
		<li>Wash the pellet once with 1 mL of 70% ethanol, re-centrifuge, and remove the supernatant carefully.</li>
		<li>Dry the pellet at room temperature for 1-2 min, and then resuspend the pellet with 20-40 &#181;L of nuclease-free water.</li>
		<li>Dilute 1 &#181;L of mRNA solution with 9 &#181;L of nuclease-free water. Take 2 &#181;L to determine the mRNA concentration and use 8 &#181;L to check the mRNA quality on a 1% agarose gel at 100 V in fresh TAE buffer for 40 min.</li>
		<li>Aliquot the mRNA solution and store frozen at -70 &#176;C for up to one year. 
		&#8203;NOTE: Do not completely dry the mRNA pellet; it may be harder to dissolve it in water.</li>
	</ol>
	</li>
</ol>
2. Microinjection
<ol>
	<li>Egg collection
	<ol>
		<li>Place 12 male pupae and 12 female pupae in a 15 cm x 15 cm x 10 cm box. Cover the box with a paper towel. Place a 10% sucrose solution in the box for feeding adults.</li>
		<li>On day 2 post eclosion, expose the adults to intense light overnight.</li>
		<li>On day 3 post eclosion, transfer the adults from step 2.1.2 to a dark place. Collect the embryos within 2 h after oviposition.</li>
		<li>Add drops of water to the piece of a 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 aligned eggs on ice before microinjection.</li>
	</ol>
	</li>
	<li>Microinjection setup
	<ol>
		<li>Inject a mixture of a transgenic enhanced green fluorescent protein (EGFP)-expressing plasmid (200 ng/&#181;L), pBac: hr5ie1-EGFP-SV40, hyPBase mRNA (200 ng/&#181;L), and sterile distilled water into the eggs, using the compensation pressure of the automated microinjector (see the Table of Materials).</li>
		<li>Keep the injected eggs at 27 &#177; 1 &#176;C, 60 &#177; 10% relative humidity until hatching.</li>
		<li>Feed the hatched larvae on an artificial diet, and transfer each larva to one small cup at the early 3rd instar stage (~6 mm in length, and body-color turns black).</li>
	</ol>
	</li>
</ol>
3. Fluorescence screening and genetic crossing
<ol>
	<li>Collect the pupae and place ~150 female pupae and ~150 male pupae in a 35 cm x 35 cm x 35 cm cage. Hang several pieces of paper towels (~30 cm x 15 cm) in the cage to collect the eggs.</li>
	<li>Collect the paper towels with eggs daily, and keep the eggs at 27 &#177; 1 &#176;C, 60 &#177; 10% relative humidity until hatching.</li>
	<li>Keep the neonate larvae at 4 &#176;C for 5 min to immobilize them, and then transfer them onto an ice-cold plate.</li>
	<li>Screen the immobilized neonate larvae under a fluorescence stereomicroscope. Select the EGFP-positive neonates, raise them to the pupal stage in ~2 weeks at 27 &#177; 1 &#176;C, and separate them by sex according to the phenotype differences at the ventral abdomen segments. 
	NOTE: The female pupa has a slit-like genital opening on the eighth abdominal segment and the cephalic margins of the ninth and tenth segments curving toward the genital opening. The genital opening of the male pupa has a slight elevation on the ninth abdominal segment.</li>
	<li>Place the EGFP-positive pupae and the wild-type pupae of the opposite sex in a male: female ratio of 1:1 in a cage. Sib-cross the EGFP-expressing adults to produce the following generations.</li>
</ol>
4. PCR detection of transgenic insertion
<ol>
	<li>Extract genomic DNA from the wild-type and EGFP-positive individual larvae using any commercial genomic DNA extraction kit following the manufacturer&#39;s instructions.</li>
	<li>Perform a PCR reaction using the genomic DNA as a template; a forward primer, 5&#39;- acgtacgctcctcgtgttccgttc-3&#39; located in the ie1 promoter region; and a reverse primer, 5&#39;- aagcactgcacgccgtaggtcag-3&#39; located in the EGFP region of the transformation vector.</li>
	<li>Set up each PCR reaction (final volume of 40 &#181;L) to contain 20 &#181;L of the polymerase mixture, 1.0 &#181;L of genomic DNA (40 &#181;g/&#181;L), 1.0 &#181;L each of 10 &#181;M forward and reverse primers, and 17 &#181;L of nuclease-free water. Use the following settings: an initial incubation of 95 &#176;C for 3 min, followed by 35 cycles of 95 &#176;C for 20 s, 56 &#176;C for 20 s, and 68 &#176;C for 40 s settings.</li>
	<li>Check the PCR products on a 1% agarose gel at 120 V for 30 min.</li>
</ol>
5. Determination of the transgenic insertion site
<ol>
	<li>Perform high-efficiency TAIL-PCR using genomic DNA as a template to determine the integration site of transgene insertion in the EGFP-positive insects.
	<ol>
		<li>Perform the PCR reactions following the steps described elsewhere9.
		<ol>
			<li>Use three primers, P1: 5&#39;-atcagtgacacttaccgcattgacaagcacgcc-3&#39;, P2: 5&#39;-tcacgggagctccaagcggcgactg-3&#39;, and P3: 5&#39;-atgtcctaaatgcacagcgacggattcgcgct-3&#39;, which are specific to the piggyBac vector, in the 3 rounds of PCR reaction, respectively.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Sequence the final PCR products to determine the integration site.</li>
</ol>

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	<li>Gui, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+and+transcriptomic+analysis+unveils+population+evolution+and+development+of+pesticide+resistance+in+fall+armyworm+Spodoptera+frugiperda.">Genomic and transcriptomic analysis unveils population evolution and development of pesticide resistance in fall armyworm Spodoptera frugiperda.</a> Protein Cell. , (2020).</li><li>Montezano, D. 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N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperactive+piggyBac+transposase+improves+transformation+efficiency+in+diverse+insect+species.">Hyperactive piggyBac transposase improves transformation efficiency in diverse insect species.</a> Insect Biochemistry and Molecular Biology. 98, 16-24 (2018).</li><li>Chen, X., Koo, J., Gurusamy, D., Mogilicherla, K., Palli, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+systemic+RNA+interference+defective+protein+1+enhances+RNAi+efficiency+in+a+lepidopteran+insect,+the+fall+armyworm,+in+a+tissue-specific+manner.">Caenorhabditis elegans systemic RNA interference defective protein 1 enhances RNAi efficiency in a lepidopteran insect, the fall armyworm, in a tissue-specific manner.</a> RNA Biology. , 1-9 (2020).</li><li>Liu, Y. G., Chen, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+thermal+asymmetric+interlaced+PCR+for+amplification+of+unknown+flanking+sequences.">High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences.</a> Biotechniques. 43 (5), 649-650 (2007).</li><li>Yusa, K., Zhou, L., Li, M. A., Bradley, A., Craig, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hyperactive+piggyBac+transposase+for+mammalian+applications.">A hyperactive piggyBac transposase for mammalian applications.</a> Proceedings of the National Academy of Sciences of the Unites States of America. 108 (4), 1531-1536 (2011).</li><li>Xu, H., O'Brochta, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+technologies+for+genetically+manipulating+the+silkworm+Bombyx+mori,+a+model+Lepidopteran+insect.">Advanced technologies for genetically manipulating the silkworm Bombyx mori, a model Lepidopteran insect.</a> Proceedings, Biological Sciences. 282 (1810), 20150487 (2015).</li><li>Wu, S. C. -. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=piggyBac+is+a+flexible+and+highly+active+transposon+as+compared+to+sleeping+beauty,+Tol2,+and+Mos1+in+mammalian+cells.">piggyBac is a flexible and highly active transposon as compared to sleeping beauty, Tol2, and Mos1 in mammalian cells.</a> Proceedings of the National Academy of Sciences of the Unites States of America. 103 (41), 15008-15013 (2006).</li><li>Tamura, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germline+transformation+of+the+silkworm+Bombyx+mori+L.+using+a+piggyBac+transposon-derived+vector.">Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector.</a> Nature Biotechnology. 18 (1), 81-84 (2000).</li><li>Handler, A. M., Harrell, R. 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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 insect silkworm, Bombyx mori11. However, conducting transgenesis work in other lepidopteran insects, including some major agriculture pests, is still challenging.
The major limitations and the key steps of establishing a transgenic FAW line using this protocol are synthesizing high-quality hyPBase mRNA and delivering piggyBac transposition components into the early embryos. The two core components of the traditional piggyBac system are a donor plasmid containing the cargo to be inserted into the genome and a helper plasmid providing the transposase12. To increase the transformation efficiency, highly active promoters are used in the helper plasmid to drive the expression of transposase13,14. As these promoters are usually obtained from model insects, their activity in other insects may not be high as in the species from which the promoter was identified. Thus, transposase mRNA is more effective than the helper plasmid, which may improve germline transformation in most non-model insects.
In this protocol, hyPBase mRNA was prepared in vitro by constructing a plasmid containing the hyPBase-expressing cassette (T7 promoter: polyhedrin-5&#39;UTR: hyPBase: polyhedrin-3&#39;UTR: poly (A) signal). The T7 promoter allows in vitro synthesis of mRNA using commercially available mRNA synthesis kits. Both polyhedrin-5&#39;UTR and polyhedrin-3&#39;UTR could help in improving transcription of hyPBase mRNA in vitro and translation of hyPBase protein in vivo. The presence of the poly (A) signal helps enhance the stability of hyPBase mRNA in vivo15. Additionally, injection of the piggyBac components into the very early embryos with a limited number of dividing cells is also critical to improve the chances of delivering piggyBac system component progenitors of germline cells. The transposition events occurring in the germline cells could be inherited. In this protocol, FAW embryos less than 2 hours old were collected and kept on ice to slow down their development before injection. For most insects, collecting embryos as early as possible and finding a way to slow down their development could also help in successful germline transformation.
Using fluorescent proteins in insect transgenesis helps in the identification of transgenic animals. In this protocol, the IE1 promoter, which has been reported to be an efficient and ubiquitous promoter in lepidopteran insects11, derived from the Autographa california multicapsid nuclear polyhedrosis virus (AcNPV) immediate early gene, was fused with the hr5 enhancer to drive the expression of EGFP. In transgenic individuals in which EGFP was inserted into the FAW genome, the EGFP signal could be observed at all stages (data not shown). Molecular confirmation of piggyBac-mediated insertion was usually performed by southern blot or inverse PCR and sequencing. In this protocol, PCR amplification of a fragment in the transgenic cargo was used to confirm the transgenic insertion rapidly. In addition, a PCR-based high-efficiency TAIL-PCR method was employed to identify the integration site of transgenic insertion9, which is easy to manipulate compared with the widely used inverse PCR method. The high-efficiency TAIL-PCR method was developed to identifying the transgenic integration sites in plants. In this study, this method is used for the first time in insects. The presented protocol provides an effective way to generate transgenic FAW and could also be further applied to many other model and non-model insects.

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td>1.5&#34; Dental Cotton Rolls</td>
			<td>PlastCare USA</td>
			<td>8542025591</td>
			<td>REARING</td>
		</tr>
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			<td>1 oz Souffle Cup Lids</td>
			<td>DART</td>
			<td>PL1N</td>
			<td></td>
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			<td>1 oz Souffle Cups</td>
			<td>DART</td>
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			<td>REARING</td>
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			<td>48 oz Plastic Deli Containers</td>
			<td>Genpack</td>
			<td>AD48</td>
			<td>REARING</td>
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			<td>Add-on Filter Set (Green)</td>
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			<td>SCREENING</td>
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			<td>Borosilicate Glass</td>
			<td>Sutter Instruments</td>
			<td>BF100-50-10</td>
			<td>INJECTION</td>
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			<td>Borosilicate Glass</td>
			<td>SUTTER INSTRUMENT</td>
			<td>BF-100-50-10</td>
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			<td>Dissecting Scope</td>
			<td>Nikon</td>
			<td>SMZ745T</td>
			<td>SCREENING</td>
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			<td>Featherweight Forceps</td>
			<td>BioQuip</td>
			<td>4750</td>
			<td>REARING</td>
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			<td>Gutter Guard</td>
			<td>ThermWell Products</td>
			<td>VX620</td>
			<td>REARING</td>
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			<td>Inverted Microscope</td>
			<td>Olympus</td>
			<td>IX71</td>
			<td>INJECTION</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>INJECTION</td>
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			<td>NightSea LLC</td>
			<td>SFA-RB-DIM</td>
			<td>SCREENING</td>
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			<td>Nitrogen Gas</td>
			<td>AWG/Scott-Gross</td>
			<td>NI 225</td>
			<td>INJECTION</td>
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			<td>Paper Towels</td>
			<td>Bounty</td>
			<td>&#160;43217-45074</td>
			<td>REARING</td>
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			<td>Spodoptera frugiperda Artificial Diet</td>
			<td>Southland Products, Inc</td>
			<td>N/A [Request Species/Quantity]</td>
			<td>REARING</td>
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			<td>Spodoptera frugiperda Eggs</td>
			<td>Benzon Research, Inc</td>
			<td>N/A [Request Species/Quantity]</td>
			<td>REARING</td>
		</tr>
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			<td>Taq MasterMix</td>
			<td></td>
			<td></td>
			<td>polymerase mixture</td>
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</table>

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).
Then, a piggyBac-based EGFP expression construct was prepared (Figure 2A). A mixture of this plasmid and hyPBase mRNA, or PBS solution, was injected into embryos within 2 h after oviposition. At 24 h after injection, a transient signal of EGFP expression was observed in the injected embryos. The PBS-injected embryos did not show EGFP signal (Figure 2B), whereas the EGFP-expressing plasmid and hyPBase mRNA-injected embryos showed EGFP fluorescence (Figure 2C), indicating that the piggyBac construction was successful. Thus, the hr5ie1 promoter can function during the early embryonic development.
Approximately 2,000 eggs were injected, and ~700 of them developed into pupae. The G0 adults were self-crossed, and eggs were collected. The G1 larvae hatched from these eggs were examined for the EGFP signal under a fluorescence stereomicroscope. As shown in Figure 3A, the transgenic larvae showed a strong EGFP signal. To test the insertion of the EGFP expression cassette into the genomes of EGFP-positive insects, a fragment containing the promoter and EGFP fragment was amplified using the genomic DNA isolated from the EGFP-positive larvae as a template (Figure 3B). This fragment was not detected when genomic DNA was isolated from the wild-type larvae as the template (Figure 3B). By conducting high-efficiency TAIL-PCR and sequencing of the PCR products, the integration site of transgene insertion was determined. As shown in Figure 4, the transgene integration site lies in the 81st intron of the Twinchin-like gene in chromosome 26.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62714/62714fig01.jpg" /> 
Figure 1: Production of hyPBase mRNA. (A)&#160;The hyPBase-expressing cassette containing the T7 promoter: polyhedrin-5&#39;UTR: hyPBase: polyhedrin-3&#39;UTR: poly (A) signal was constructed. (B) The ~2.2 kb DNA template for synthesizing hyPBase mRNA was amplified from the hyPBase construct. The left lane shows the 1-kb ladder run on the same gel. (C) A ~1.1 kb hyPBase mRNA was synthesized from the amplified DNA template. The left lane shows the 1-kb ladder run on the same gel. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62714/62714fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62714/62714fig02.jpg" /> 
Figure 2:&#160;Expression of EGFP in injected embryos. (A) Schematic diagram of the piggyBac vector used in this study. (B) No EGFP signal was detected in embryos injected with 1x PBS. (C) EGFP signal was detected in embryos injected with the piggyBac vector and hyPBase mRNA. Scale bars = 50 &#181;m. Abbreviations: EGFP = enhanced green fluorescent protein and PBS = phosphate-buffered saline. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62714/62714fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62714/62714fig03.jpg" /> 
Figure 3: Characterization of transgenic FAW. (A)&#160;EGFP expression in transgenic but not in wild-type FAW larvae. Scale bars = 0.5 mm. (B) PCR analysis of genomic insertion of the transgenes. A pair of primers targeting the promoter and EGFP regions were used to amplify a ~0.5 kb fragment using the genomic DNA isolated from transgenic or wild-type FAW larvae as a template. Abbreviations: EGFP = enhanced green fluorescent protein and FAW = Fall armyworm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62714/62714fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62714/62714fig04.jpg" /> 
Figure 4: Genomic insertion of piggyBac construct. Genomic insertion of the piggyBac construct in transgenic FAW was determined by TAIL-PCR and sequencing. Chromosome localization and partial genomic DNA sequences flanking the boundaries of the piggyBac construct are shown. Abbreviations: FAW = Fall armyworm and TAIL-PCR = thermal asymmetric interlaced PCR. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62714/62714fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Material. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62714/Supplimentary_Info.docx" target="_blank">Please click here to download this File.</a>

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 ...

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4.1K Views.  University of Kentucky. Successful germline transformation in the fall armyworm, Spodoptera frugiperda, was achieved using mRNA of hyperactive piggyBac transposase.

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

Transformation of Plasmid DNA into E. coli Using the Heat Shock Method

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign plasmid or ligation product into bacteria. This video protocol describes the traditional method of transformation using commercially available chemically competent bacteria from Genlantis. After a short incubation in ice, a mixture of chemically competent bacteria and DNA is placed at 42&deg;C for 45 seconds (heat shock) and then placed back in ice. SOC media is added and the transformed cells are incubated at 37&deg;C for 30 min with agitation. To be assured of isolating colonies irrespective of transformation efficiency, two quantities of transformed bacteria are plated. This traditional protocol can be used successfully to transform most commercially available competent bacteria. The turbocells from Genlantis can also be used in a novel 3-minute transformation protocol, described in the instruction manual.

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign ...

Brain Slice Stimulation Using a Microfluidic Network and Standard Perfusion Chamber

We have demonstrated the fabrication of a two-level microfluidic device that can be easily integrated with existing electrophysiology setups. The two-level microfluidic device is fabricated using a two-step standard negative resist lithography process 1. The first level contains microchannels with inlet and outlet ports at each end. The second level contains microscale circular holes located midway of the channel length and centered along with channel width. Passive pumping method is used to pump fluids from the inlet port to the outlet port 2. The microfluidic device is integrated with off-the-shelf perfusion chambers and allows seamless integration with the electrophysiology setup. The fluids introduced at the inlet ports flow through the microchannels towards the outlet ports and also escape through the circular openings located on top of the microchannels into the bath of the perfusion. Thus the bottom surface of the brain slice placed in the perfusion chamber bath and above the microfluidic device can be exposed with different neurotransmitters. The microscale thickness of the microfluidic device and the transparent nature of the materials [glass coverslip and PDMS (polydimethylsiloxane)] used to make the microfluidic device allow microscopy of the brain slice. The microfluidic device allows modulation (both spatial and temporal) of the chemical stimuli introduced to the brain slice microenvironments.

We demonstrate fabrication of a simple microfluidic device that can be integrated with standard electrophysiology setups to expose microscale surfaces of a brain slice in a well controlled manner to different neurotransmitters.

We have demonstrated the fabrication of a two-level microfluidic device that can be easily integrated with existing electrophysiology setups. The ...

Efficient Polyethylene Glycol (PEG) Mediated Transformation of the Moss Physcomitrella patens

A simple and efficient method to transform Physcomitrella pantens protoplasts is described. This method is adapted from protocols for Physocmitrella protonemal protoplast and Arabidopsis mesophyll protoplast transformation1. Due to its capacity to undergo efficient mitotic homologous recombination, Physcomitrella patens has emerged as an important model system in recent years2. This capacity allows high frequencies of gene targeting3-9, which is not seen in other model plants such as Arabidopsis. To take full advantage of this system, we need an effective and easy method to deliver DNA into moss cells. The most common ways to transform this moss are particle bombardment10 and PEG-mediated DNA uptake11. Although particle bombardment can produce a high transformation efficiency12, gene guns are not readily available to many laboratories and the protocol is difficult to standardize. On the other hand, PEG mediated transformation does not require specialized equipments, and can be performed in any laboratory with a sterile hood. Here, we show a simple and highly efficient method for transformation of moss protoplasts. This method can generate more than 120 transient transformants per microgram of DNA, which is an improvement from the most efficient protocol previously reported13. Because of its simplicity, efficiency, and reproducibility, this method can be applied to projects requiring large number of transformants as well as for routine transformation.

Efficient Polyethylene Glycol PEG Mediated Transformation of the Moss Physcomitrella patens

A simple and efficient method to transform Physcomitrella pantens protoplasts is described. This method is adapted from protocols for Physocmitrella protonemal protoplast and Arabidopsis mesophyll protoplast transformation1.

A simple and efficient method to transform Physcomitrella pantens protoplasts is described. This method is adapted from protocols for Physocmitrella ...

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic redundancy affecting simple genetics in higher plants. The novel model organism Ostreococcus tauri is the smallest free-living eukaryote known to date, and possesses a greatly reduced genome size and cellular complexity1,2, manifested by the presence of just one of most organelles (mitochondrion, chloroplast, golgi stack) per cell, and a genome containing only ~8000 genes. Furthermore, the combination of unicellularity and easy culture provides a platform amenable to chemical biology approaches. Recently, Ostreococcus has been successfully employed to study basic mechanisms underlying circadian timekeeping3-6. Results from this model organism have impacted not only plant science, but also mammalian biology7. This example highlights how rapid experimentation in a simple eukaryote from the green lineage can accelerate research in more complex organisms by generating testable hypotheses using methods technically feasible only in this background of reduced complexity. Knowledge of a genome and the possibility to modify genes are essential tools in any model species. Genomic1, Transcriptomic8, and Proteomic9 information for this species is freely available, whereas the previously reported methods6,10 to genetically transform Ostreococcus are known to few laboratories worldwide.
In this article, the experimental methods to genetically transform this novel model organism with an overexpression construct by means of electroporation are outlined in detail, as well as the method of inclusion of transformed cells in low percentage agarose to allow selection of transformed lines originating from a single transformed cell. Following the successful application of Ostreococcus to circadian research, growing interest in Ostreococcus can be expected from diverse research areas within and outside plant sciences, including biotechnological areas. Researchers from a broad range of biological and medical sciences that work on conserved biochemical pathways may consider pursuing research in Ostreococcus, free from the genomic and organismal complexity of larger model species.

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

This article describes genetic transformation of the unicellular marine alga Ostreococcus tauri by electroporation. This eukaryotic organism is an effective model platform for higher plants, possesing greatly reduced genomic and cellular complexity and being readily amenable to both cell culture and chemical biology.

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic ...

Identification of Metabolically Active Bacteria in the Gut of the Generalist Spodoptera littoralis via DNA Stable Isotope Probing Using 13C-Glucose

Guts of most insects are inhabited by complex communities of symbiotic nonpathogenic bacteria. Within such microbial communities it is possible to identify commensal or mutualistic bacteria species. The latter ones, have been observed to serve multiple functions to the insect, i.e. helping in insect reproduction1, boosting the immune response2, pheromone production3, as well as nutrition, including the synthesis of essential amino acids4, among others. &#160; &#160;
Due to the importance of these associations, many efforts have been made to characterize the communities down to the individual members. However, most of these efforts were either based on cultivation methods or relied on the generation of 16S rRNA gene fragments which were sequenced for final identification. Unfortunately, these approaches only identified the bacterial species present in the gut and provided no information on the metabolic activity of the microorganisms.
To characterize the metabolically active bacterial species in the gut of an insect, we used stable isotope probing (SIP) in vivo employing 13C-glucose as a universal substrate. This is a promising culture-free technique that allows the linkage of microbial phylogenies to their particular metabolic activity. This is possible by tracking stable, isotope labeled atoms from substrates into microbial biomarkers, such as DNA and RNA5. The incorporation of 13C isotopes into DNA increases the density of the labeled DNA compared to the unlabeled (12C) one. In the end, the 13C-labeled DNA or RNA is separated by density-gradient ultracentrifugation from the 12C-unlabeled similar one6. Subsequent molecular analysis of the separated nucleic acid isotopomers provides the connection between metabolic activity and identity of the species.
Here, we present the protocol used to characterize the metabolically active bacteria in the gut of a generalist insect (our model system), Spodoptera littoralis (Lepidoptera, Noctuidae). The phylogenetic analysis of the DNA was done using pyrosequencing, which allowed high resolution and precision in the identification of insect gut bacterial community. As main substrate, 13C-labeled glucose was used in the experiments. The substrate was fed to the insects using an artificial diet.

Identification of Metabolically Active Bacteria in the Gut of the Generalist Spodoptera littoralis via DNA Stable Isotope Probing Using 13C-Glucose

The active bacterial community associated with the gut of Spodoptera littoralis, was determined by stable-isotope-probing (SIP) coupled to pyrosequencing. Using this methodology, identification of the metabolically active bacteria species within the community was done with high resolution and precision.

Guts of most insects are inhabited by complex communities of symbiotic nonpathogenic bacteria. Within such microbial communities it is possible to ...

Floral-Dip Transformation of Flax (Linum usitatissimum) to Generate Transgenic Progenies with a High Transformation Rate

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be successful for many plant species. However, flax (Linum usitatissimum) transformation by floral-dip has not been reported. The goal of this protocol is to establish that Agrobacterium and the floral-dip method can be used to generate transgenic flax. We show that this technique is simple, inexpensive, efficient, and more importantly, gives a higher transformation rate than the current available methods of flax transformation.
In summary, inflorescences of flax were dipped in a solution of Agrobacterium carrying a binary vector plasmid (T-DNA fragment plus the Linum Insertion Sequence, LIS-1) for 1 - 2 min. The plants were laid flat on their side for 24 hr. Then, plants were maintained under normal growth conditions until the next treatment. The process of dipping was repeated 2 - 3 times, with approximately 10 - 14 day intervals between dipping. The T1 seeds were collected and germinated on soil. After approximately two weeks, treated progenies were tested by direct PCR; 2 - 3 leaves were used per plant plus the appropriate T-DNA primers. Positive transformants were selected and grown to maturity. The transformation rate was unexpectedly high, with 50 - 60% of the seeds from treated plants being positive transformants. This is a higher transformation rate than those reported for Arabidopsis thaliana and other plant species, using floral-dip transformation. It is also the highest, which has been reported so far, for flax transformation using other methods for transformation.

Floral-Dip Transformation of Flax Linum usitatissimum to Generate Transgenic Progenies with a High Transformation Rate

Here, we present a protocol to transform flax using Agrobacterium-mediated plant transformation via floral-dip. This protocol is simple to perform and inexpensive, yet yields a higher transformation rate than the current available methods for flax transformation.

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be ...

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and develops most often in the setting of immunosuppression. Despite decades of research, optimal treatment of KS remains poorly defined and clinical outcomes are especially unfavorable in resource-limited settings. KS lesions are driven by pathological angiogenesis, chronic inflammation, and oncogenesis, and various in vitro cell culture models have been developed to study these processes. KS arises from KSHV-infected cells of endothelial origin, so EC-lineage cells provide the most appropriate in vitro surrogates of the spindle cell precursor. However, because EC have a limited in vitro lifespan, and as the oncogenic mechanisms employed by KSHV are less efficient than those of other tumorigenic viruses, it has been difficult to assess the processes of transformation in primary or telomerase-immortalized EC. Therefore, a novel EC-based culture model was developed that readily supports transformation following infection with KSHV. Ectopic expression of the E6 and E7 genes of human papillomavirus type 16 allows for extended culture of age- and passage-matched mock- and KSHV-infected EC and supports the development of a truly transformed (i.e., tumorigenic) phenotype in infected cell cultures. This tractable and highly reproducible model of KS has facilitated the discovery of several essential signaling pathways with high potential for translation into clinical settings.

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is a tumor induced by infection with the oncogenic virus human herpesvirus-8/KS herpesvirus (HHV-8/KSHV). The endothelial cell culture model described here is uniquely suited for studying the mechanisms by which KSHV transforms host cells.

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and ...

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by filarial nematodes such as canine heartworm and elephantasis. Recently, CRISPR/Cas9 genome editing has been used to induce site-directed mutations by injecting a Cas9 protein that has been complexed with a guide RNA (gRNA) into freshly laid embryos of several insect species, including mosquitoes that belong to the genera Anopheles and Aedes. Manipulating and injecting Culex mosquitoes is slightly more difficult as these mosquitoes lay their eggs upright in rafts rather than individually like other species of mosquitoes. Here we describe how to design gRNAs, complex them with Cas9 protein, induce female mosquitoes of Culex pipiens to lay eggs, and how to prepare and inject newly laid embryos for microinjection with Cas9/gRNA. We also describe how to rear and screen injected mosquitoes for the desired mutation. The representative results demonstrate that this technique can be used to induce site-directed mutations in the genome of Culex mosquitoes and, with slight modifications, can be used to generate null-mutants in other mosquito species as well.&#160;

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

CRISPR/Cas9 is increasingly used to characterize gene function in non-model organisms. This protocol describes how to generate knock-out lines of Culex pipiens, from preparing injection mixes, to obtaining and injecting mosquito embryos, as well as how to rear, cross, and screen injected mosquitoes and their progeny for desired mutations.

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by ...

Microsurgical Obstruction of Testes Fusion in Spodoptera litura

Instead of using genetic methods like RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease Cas9, a physical barrier was microsurgically inserted between the testes of Spodoptera litura to study the impact of this microsurgery on its growth and reproduction. After inserting aluminum foil between the testes, insect molting during metamorphosis proceeded normally. Insect growth and development were not remarkably altered; however, the number of sperm bundles changed if testes fusion was stopped by the microsurgery. These findings imply that blocking testicular fusion can influence male reproduction capability. The method can be further applied to interrupt communication between organs to study the function of specific signaling pathways. Compared to conventional surgery, microsurgery only requires freezing anesthetization, which is preferable to carbon dioxide anesthetization. Microsurgery also minimizes the surgery site area and facilitates wound healing. However, the selection of materials with specific functions needs further investigation. Avoiding tissue injury is crucial when making incisions during the operation.

Microsurgical Obstruction of Testes Fusion in Spodoptera litura

Aluminum foil was microsurgically inserted between the testes of Spodoptera litura to obstruct the fusion of testis. The procedure includes freezing, fixing, disinfection, incision, placing the barrier, suturing, postoperative feeding, and inspection. This approach provides a method to interfere with tissue formation.

Instead of using genetic methods like RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated ...

Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna

Cyanobacteria are the focus of basic research and biotechnological projects in which solar energy is utilized for biomass production. Phormidium lacuna is a newly isolated filamentous cyanobacterium. This paper describes how new filamentous cyanobacteria can be isolated from marine rockpools. It also describes how DNA can be extracted from filaments and how the genomes can be sequenced. Although transformation is established for many single-celled species, it is less frequently reported for filamentous cyanobacteria. A simplified method for the natural transformation of P. lacuna is described here. P. lacuna is the only member of the order Oscillatoriales for which natural transformation is established. This paper also shows how natural transformation is used to express superfolder green fluorescent protein (sfGFP). An endogenous cpcB promoter induced approximately 5 times stronger expression than cpc560, A2813, or psbA2 promoters from Synechocystis sp. PCC6803. Further, a method for the cryopreservation of P. lacuna and Synechocystis sp. CPP 6803 was established, and methods for assessing motility in a liquid medium and on agar and plastic surfaces are described.

Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna

Phormidium lacuna is a filamentous cyanobacterium that was isolated from marine rockpools. This article describes the isolation of filaments from natural sources, DNA extraction, genome sequencing, natural transformation, expression of sfGFP, cryoconservation, and motility methods.

Cyanobacteria are the focus of basic research and biotechnological projects in which solar energy is utilized for biomass production. Phormidium lacuna is ...

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

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Transformation of Plasmid DNA into E. coli Using the Heat Shock Method

Brain Slice Stimulation Using a Microfluidic Network and Standard Perfusion Chamber

Efficient Polyethylene Glycol (PEG) Mediated Transformation of the Moss Physcomitrella patens

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

Identification of Metabolically Active Bacteria in the Gut of the Generalist Spodoptera littoralis via DNA Stable Isotope Probing Using ¹³C-Glucose

Floral-Dip Transformation of Flax (Linum usitatissimum) to Generate Transgenic Progenies with a High Transformation Rate

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

Microsurgical Obstruction of Testes Fusion in Spodoptera litura

Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna