Insect transformation with piggyBac: getting the number of injections just right

Abstract

The insertion of exogenous genetic cargo into insects using transposable elements is a powerful research tool with potential applications in meeting food security and public health challenges facing humanity. piggyBac is the transposable element most commonly utilized for insect germline transformation. The described efficiency of this process is variable in the published literature, and a comprehensive review of transformation efficiency in insects is lacking. This study compared and contrasted all available published data with a comprehensive data set provided by a biotechnology group specializing in insect transformation. Based on analysis of these data, with particular focus on the more complete observational data from the biotechnology group, we designed a decision tool to aid researchers' decision‐making when using piggyBac to transform insects by microinjection. A combination of statistical techniques was used to define appropriate summary statistics of piggyBac transformation efficiency by species and insect order. Publication bias was assessed by comparing the data sets. The bias was assessed using strategies co‐opted from the medical literature. The work culminated in building the Goldilocks decision tool, a Markov‐Chain Monte‐Carlo simulation operated via a graphical interface and providing guidance on best practice for those seeking to transform insects using piggyBac.

Discovering genes responsible for silk synthesis in Bombyx mori by piggyBac‐based random insertional mutagenesis

“…However, such an application has rarely been adopted in non‐Drosophilid insects. Recently, more and more transgenic researches using piggyBac transposon system were successfully carried out in non‐model insects (Gregory et al ., ). In the present study, a piggyBac random insertion library was constructed in B. mori , and at least two mutants with obvious phenotypes, “thin cocoon” (Fig.…”

“…It recognizes the tetranucleotide sequence "TTAA" (Elick et al, 1996) and integrates itself into the target sites by a "cut-and-paste" mechanism (Yusa, 2015;Zhao et al, 2016). Unlike P-element, piggyBac is active in a variety of organisms, including yeast, protozoa, fish, birds, mammals, plants (Yusa, 2015) and insects, such as D. melanogaster, Aedes aegypti, Anopheles gambitae, Musca domestica, Apis mellifera, Triboium castaneum, Plutella xylostella, Ostrinia furnacalis and Bombyx mori (Handler, 2002;Yusa, 2015;Gregory et al, 2016). Further, piggyBac is less susceptible to hotspots, which makes it better for saturation mutagenesis of the genome (Häcker et al, 2003).…”

CRISPR/Cas9‐mediated ebony knockout results in puparium melanism in Spodoptera litura

“…Genetic transformation techniques are becoming more widely used in pest control (Robinson et al, 2004;Black et al, 2011). Several species of lepidopteran pests have been genetically modified (Gregory et al, 2016). Marker genes are critical in genome editing applications.…”

“…Genome engineering achieved by customized Zincfinger nuclease, transcription activator-like effector nucleases, or clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) systems has been exploited in a variety of model organisms. The CRISPR/Cas9 system is an effective genome editing tool in insects and has potential for use in pest management (Wang et al, 2013;Gantz et al, 2015;Alphey, 2016). CRISPR/Cas9 has been used to edit the genomes of numerous eukaryotic organisms (Cho et al, 2013;Cong et al, 2013;Hwang et al, 2013;Jinek et al, 2013) and model insect species in the orders of Diptera (Bassett et al, 2013;Hall et al, 2015) and Coleoptera (Gillies et al, 2015).…”

BAPC-assistedCRISPR/Cas9 System: Targeted Delivery into Adult Ovaries for Heritable Germline Gene Editing (Arthropoda: Hemiptera)

“…CRISPR/Cas systems have demonstrated important applications in agriculture, increasing the options for the management of arthropod pests, insect vectors, and treatments against pathogens of plants, animals and humans (Chen et al, 2016; Chen et al, 2017; Cui et al, 2017; Taning et al, 2017; Sun et al, 2017; Gantz & Akbari 2018; Gundersen-Rindal et al, 2017; Sinisterra-Hunter & Hunter 2018). However, one of the hurdles for rapid adoption in arthropods has been the reliance on embryo injections (Li et al, 2017), which is often unsuccessful in many arthropod species (Bortesi & Fischer 2015; Boettcher & McManus, 2015; Chaverra-Rodriguez et al, 2018; Gregory et al, 2016). Thus, an improved delivery method is needed if gene editing is to be realized for many arthropod species.…”