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Biology

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

Published: September 23rd, 2021

DOI:

10.3791/62714

1Department of Entomology, College of Agriculture, Food and Environment, University of Kentucky

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

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.

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 no....

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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' UTR: hyPBase: polyhedrin-3' UTR: poly (A). The full sequence of the pTD1-hyPBase construct is provided in the Supplementary Material.

  1. Preparatio.......

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A construct for the expression of hyPBase-containing T7 promoter: polyhedrin-5'UTR: hyPBase: polyhedrin-3'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).

.......

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

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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).

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NameCompanyCatalog NumberComments
1.5" Dental Cotton RollsPlastCare USA8542025591REARING
1 oz Souffle Cup LidsDARTPL1N
1 oz Souffle CupsDARTP100NREARING
48 oz Plastic Deli ContainersGenpackAD48REARING
Add-on Filter Set (Green)NightSea LLCSFA-BLFS-GRSCREENING
Borosilicate GlassSutter InstrumentsBF100-50-10INJECTION
Borosilicate GlassSUTTER INSTRUMENTBF-100-50-10
Dissecting ScopeNikonSMZ745TSCREENING
Featherweight ForcepsBioQuip4750REARING
Gutter GuardThermWell ProductsVX620REARING
Inverted MicroscopeOlympusIX71INJECTION
MicroinjectorNarishigeIM-300INJECTION
Micropipette PullerSutter InstrumentsP-1000INJECTION
Microscope SlidesVWR16004-22INJECTION
NightSea Full SystemNightSea LLCSFA-RB-DIMSCREENING
Nitrogen GasAWG/Scott-GrossNI 225INJECTION
Paper TowelsBounty 43217-45074REARING
Spodoptera frugiperda Artificial DietSouthland Products, IncN/A [Request Species/Quantity]REARING
Spodoptera frugiperda EggsBenzon Research, IncN/A [Request Species/Quantity]REARING
Taq MasterMixpolymerase mixture

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Hyperactive PiggyBac Transposase

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