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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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.

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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).
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 ...

hyperactive-piggybac-transposase-mediated-germline-transformation

Nanyang Technological University

Research

JoVE Journal

Biology

4.2K Views.  University of Kentucky. Dit protocol toont de productie van transgene Fall Armyworm met behulp van piggyBac-gebaseerd transformatiesysteem. We hopen dat deze methoden ten goede zullen komen aan een betere transformatie, niet alleen in de Fall Armyworm, maar ook in lepidoptera-insecten. De hier beschreven methode maakt de kiembaantransformatie van lepidopteran plaaginsecten efficiënt.De procedure wordt gedemonstreerd door Dr. Xien Chen, een postdoctoraal fellow in mijn laboratorium. Begin met het plaatsen van...

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

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. This is due to its reproductive behavior: females damage the external surface of fruits and vegetables when they lay eggs and the hatched larvae feed on their pulp. Wild C. capitata populations are traditionally controlled through insecticide spraying and/or eco-friendly approaches, the most successful being the Sterile Insect Technique (SIT). The SIT relies on mass-rearing, radiation-based sterilization and field release of males that retain their capacity to mate but are not able to generate fertile progeny. The advent and the subsequent rapid development of biotechnological tools, together with the availability of the medfly genome sequence, has greatly boosted our understanding of the biology of this species. This favored the proliferation of new strategies for genome manipulation, which can be applied to population control.
In this context, embryo microinjection plays a dual role in expanding the toolbox for medfly control. The ability to interfere with the function of genes that regulate key biological processes, indeed, expands our understanding of the molecular machinery underlying medfly invasiveness. Furthermore, the ability to achieve germ-line transformation facilitates the production of multiple transgenic strains that can be tested for future field applications in novel SIT settings. Indeed, genetic manipulation can be used to confer desirable traits that can, for example, be used to monitor sterile male performance in the field, or that can result in early life-stage lethality. Here we describe a method to microinject nucleic acids into medfly embryos to achieve these two main goals.

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Diptera: Tephritidae) is a worldwide pest of agriculture. A deeper understanding of its biology is key to control medfly populations and thus reduce economic impact. Embryo microinjection is a fundamental tool allowing both germ-line transformation and reverse genetics studies in this species.

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. ...

Genetics

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

Developmental Biology

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Immunology and Infection

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. The observation of Tribolium embryos with light sheet-based fluorescence microscopy has multiple advantages over conventional widefield and confocal fluorescence microscopy. Due to the unique properties of a light sheet-based microscope, three dimensional images of living specimens can be recorded with high signal-to-noise ratios and significantly reduced photo-bleaching as well as photo-toxicity along multiple directions over periods that last several days. With more than four years of methodological development and a continuous increase of data, the time seems appropriate to establish standard operating procedures for the usage of light sheet technology in the Tribolium community as well as in the insect community at large. This protocol describes three mounting techniques suitable for different purposes, presents two novel custom-made transgenic Tribolium lines appropriate for long-term live imaging, suggests five fluorescent dyes to label intracellular structures of fixed embryos and provides information on data post-processing for the timely evaluation of the recorded data. Representative results concentrate on long-term live imaging, optical sectioning and the observation of the same embryo along multiple directions. The respective datasets are provided as a downloadable resource. Finally, the protocol discusses quality controls for live imaging assays, current limitations and the applicability of the outlined procedures to other insect species.
This protocol is primarily intended for developmental biologists who seek imaging solutions that outperform standard laboratory equipment. It promotes the continuous attempt to close the gap between the technically orientated laboratories/communities, which develop and refine microscopy methodologically, and the life science laboratories/communities, which require 'plug-and-play' solutions to technical challenges. Furthermore, it supports an axiomatic approach that moves the biological questions into the center of attention.

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

Imaging the morphogenesis of insect embryos with light sheet-based fluorescence microscopy has become state of the art. This protocol outlines and compares three mounting techniques appropriate for Tribolium castaneum embryos, introduces two novel custom-made transgenic lines well suited for live imaging, discusses essential quality controls and indicates current experimental limitations.

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. ...

phiC31 Integrase-Mediated Embryo Transgenesis in Drosophila

Embryo Microinjection for Transgenesis in Drosophila

Transgenesis in Drosophila is an essential approach to studying gene function at the organism level. Embryo microinjection is a crucial step for the construction of transgenic flies. Microinjection requires some types of equipment, including a microinjector, a micromanipulator, an inverted microscope, and a stereo microscope.&#160;Plasmids isolated with a plasmid miniprep kit are qualified for microinjection. Embryos at the pre-blastoderm or syncytial blastoderm stage, where nuclei share a common cytoplasm, are subjected to microinjection. A cell strainer eases the process of dechorionating embryos. The optimal time for dechorionation and desiccation of embryos needs to be determined experimentally. To increase the efficiency of embryo microinjection, needles prepared by a puller need to be beveled by a needle grinder. In the process of grinding needles, we utilize a foot air pump with a pressure gauge to avoid the capillary effect of the needle tip. We routinely inject 120-140 embryos for each plasmid and obtain at least one transgenic line for around 85% of plasmids. This article takes the phiC31 integrase-mediated transgenesis in Drosophila as an example and&#160;presents a detailed protocol for embryo microinjection for transgenesis in Drosophila.

Author Spotlight: Optimizing Embryo Microinjection for Transgenesis in Drosophila

This article takes the phiC31 integrase-mediated transgenesis in Drosophila as an example and presents an optimized protocol for embryo microinjection,&#160;a crucial step for creating transgenic flies.

Transgenesis in Drosophila is an essential approach to studying gene function at the organism level. Embryo microinjection is a crucial step for the ...

Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma

An urgent need exists to test the contribution of new genes to the pathogenesis and progression of human glioblastomas (GBM), the most common primary brain tumor in adults with dismal prognosis. New potential therapies are rapidly emerging from the bench and require systematic testing in experimental models which closely reproduce the salient features of the human disease. Herein we describe in detail a method to induce new models of GBM with transposon-mediated integration of plasmid DNA into cells of the subventricular zone of neonatal mice. We present a simple way to clone new transposons amenable for genomic integration using the Sleeping Beauty transposon system and illustrate how to monitor plasmid uptake and disease progression using bioluminescence, histology and immuno-histochemistry. We also describe a method to create new primary GBM cell lines. Ideally, this report will allow further dissemination of the Sleeping Beauty transposon system among brain tumor researchers, leading to an in depth understanding of GBM pathogenesis and progression and to the timely design and testing of effective therapies for patients.

Here we describe an efficient and versatile protocol to induce, monitor and analyze novel glioblastomas (GBM) using transposon DNA injected into the ventricles of neonatal mice. Cells of the subventricular zone, which take up the plasmid, transform, proliferate and generate tumors with histo-pathological characteristics of human GBM.

An urgent need exists to test the contribution of new genes to the pathogenesis and progression of human glioblastomas (GBM), the most common primary ...

Medicine

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

The western corn rootworm (WCR) is an important pest of corn and is well known for its ability to rapidly adapt to pest management strategies. Although RNA interference (RNAi) has proved to be a powerful tool for studying WCR biology, it has its limitations. Specifically, RNAi itself is transient (i.e. does not result in long-term Mendelian inheritance of the associated phenotype), and it requires knowing the DNA sequence of the target gene. The latter can be limiting if the phenotype of interest is controlled by poorly conserved, or even novel genes, because identifying useful targets would be challenging, if not impossible. Therefore, the number of tools in WCR&#39;s genomic toolbox should be expanded by the development of methods that could be used to create stable mutant strains and enable sequence-independent surveys of the WCR genome. Herein, we detail the methods used to collect and microinject precellular WCR embryos with nucleic acids. While the protocols described herein are aimed at the creation of transgenic WCR, CRISPR/Cas9-genome editing could also be performed using the same protocols, with the only difference being the composition of the solution injected into the embryos.

Microinjection of Western Corn Rootworm, Diabrotica virgifera virgifera, Embryos for Germline Transformation, or CRISPR/Cas9 Genome Editing

Here we present protocols for collecting and microinjecting precellular western corn rootworm embryos for the purpose of performing functional-genomic assays such as germline transformation and CRISPR/Cas9-genome editing.

The western corn rootworm (WCR) is an important pest of corn and is well known for its ability to rapidly adapt to pest management strategies. Although ...

Bioengineering

Generating Transgenics and Knockouts in Strongyloides Species by Microinjection

The genus Strongyloides consists of multiple species of skin-penetrating nematodes with different host ranges, including Strongyloides stercoralis and Strongyloides ratti. S.&#160;stercoralis is a human-parasitic, skin-penetrating nematode that infects approximately 610 million people, while the rat parasite S.&#160;ratti is closely related to S.&#160;stercoralis and is often used as a laboratory model for S.&#160;stercoralis. Both S.&#160;stercoralis and S.&#160;ratti are easily amenable to the generation of transgenics and knockouts through the exogenous nucleic acid delivery technique of intragonadal microinjection, and as such, have emerged as model systems for other parasitic helminths that are not yet amenable to this technique.
Parasitic Strongyloides adults inhabit the small intestine of their host and release progeny into the environment via the feces. Once in the environment, the larvae develop into free-living adults, which live in feces and produce progeny that must find and invade a new host. This environmental generation is unique to the Strongyloides species and similar enough in morphology to the model free-living nematode Caenorhabditis elegans that techniques developed for C. elegans can be adapted for use with these parasitic nematodes, including intragonadal microinjection. Using intragonadal microinjection, a wide variety of transgenes can be introduced into Strongyloides. CRISPR/Cas9 components can also be microinjected to create mutant Strongyloides larvae. Here, the technique of intragonadal microinjection into Strongyloides, including the preparation of free-living adults, the injection procedure, and the selection of transgenic progeny, is described. Images of transgenic Strongyloides larvae created using CRISPR/Cas9 mutagenesis are included. The aim of this paper is to enable other researchers to use microinjection to create transgenic and mutant Strongyloides.

Generating Transgenics and Knockouts in Strongyloides Species by Microinjection

The functional genomic toolkit for the parasitic nematodes Strongyloides stercoralis and Strongyloides ratti includes transgenesis, CRISPR/Cas9-mediated mutagenesis, and RNAi. This protocol will demonstrate how to use intragonadal microinjection to introduce transgenes and CRISPR components into S.&#160;stercoralis and S.&#160;ratti.

The genus Strongyloides consists of multiple species of skin-penetrating nematodes with different host ranges, including Strongyloides stercoralis and ...

Electroporation-Based Genetic Modification of Primary Human Pigment Epithelial Cells Using the Sleeping Beauty Transposon System

Our increasingly aging society leads to a growing incidence of neurodegenerative diseases. So far, the pathological mechanisms are inadequately understood, thus impeding the establishment of defined treatments. Cell-based additive gene therapies for the increased expression of a protective factor are considered as a promising option to medicate neurodegenerative diseases, such as age-related macular degeneration (AMD). We have developed a method for the stable expression of the gene encoding pigment epithelium-derived factor (PEDF), which is characterized as a neuroprotective and anti-angiogenic protein in the nervous system, into the genome of primary human pigment epithelial (PE) cells using the Sleeping Beauty (SB) transposon system. Primary PE cells were isolated from human donor eyes and maintained in culture. After reaching confluence, 1 x 104 cells were suspended in 11 &#181;L of resuspension buffer and combined with 2 &#181;L of a purified solution containing 30 ng of hyperactive SB (SB100X) transposase plasmid and 470 ng of PEDF transposon plasmid. Genetic modification was carried out with a capillary electroporation system using the following parameters: two pulses with a voltage of 1,100 V and a width of 20 ms. Transfected cells were transferred into culture plates containing medium supplemented with fetal bovine serum; antibiotics and antimycotics were added with the first medium exchange. Successful transfection was demonstrated in independently performed experiments. Quantitative polymerase chain reaction (qPCR) showed the increased expression of the PEDF transgene. PEDF secretion was significantly elevated and remained stable, as evaluated by immunoblotting, and quantified by enzyme-linked immunosorbent assay (ELISA). SB100X-mediated transfer allowed for a stable PEDF gene integration into the genome of PE cells and ensured the continuous secretion of PEDF, which is critical for the development of a cell-based gene addition therapy to treat AMD or other retinal degenerative diseases. Moreover, analysis of the integration profile of the PEDF transposon into human PE cells indicated an almost random genomic distribution.

We have developed a protocol to transfect primary human pigment epithelial cells by electroporation with the gene encoding pigment epithelium-derived factor (PEDF) using the Sleeping Beauty (SB) transposon system. Successful transfection was demonstrated by quantitative polymerase chain reaction (qPCR), immunoblotting, and enzyme-linked immunosorbent assay (ELISA).

Our increasingly aging society leads to a growing incidence of neurodegenerative diseases. So far, the pathological mechanisms are inadequately ...

PiggyBac Transposon-Mediated Gene Editing in Human iPSCs: A Procedure to Integrate Gene of Interest in Human iPSCs Using PiggyBac Transposon System

Source: Garone, M. G. et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/59321">Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors.</a> J. Vis. Exp. (2019)
This video demonstrates the procedure to generate motor neurons by transfection of human iPSCs with PiggyBac transposon system by the ectopic expression of lineage-specific transcription factors.

Source: Garone, M. G. et al. Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac ...

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

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