Post-Integration Silencing of piggyBac Transposable Elements in Aedes aegypti

The piggyBac transposon, originating in the genome of the Lepidoptera Trichoplusia ni, has a broad host range, making it useful for the development of a number of transposon-based functional genomic technologies including gene vectors, enhancer-, gene- and protein-traps. While capable of being used as a vector for the creation of transgenic insects and insect cell lines, piggyBac has very limited mobility once integrated into the genome of the yellow fever mosquito, Aedes aegypti. A transgenic Aedes aegypti cell line (AagPB8) was created containing three integrated piggyBac elements and the remobilization potential of the elements was tested. The integrated piggyBac elements in AagPB8 were transpositionally silent in the presence of functional transposase, which was shown to be capable of catalyzing the movement of plasmid-borne piggyBac elements in the same cells. The structural integrity of one of the integrated elements along with the quality of element-flanking DNA, which is known to influence transposition rates, were tested in D. melanogaster. The element was found to be structurally intact, capable of transposition and excision in the soma and germ-line of Drosophila melanogaster, and in a DNA sequence context highly conducive to element movement in Drosophila melanogaster. These data show that transpositional silencing of integrated piggyBac elements in the genome of Aedes aegypti appears to be a function of higher scale genome organization or perhaps epigenetic factors, and not due to structural defects or suboptimal integration sites

A gene drive is a gene drive: the debate over lumping or splitting definitions

We address a controversy over use of the term “gene drive” to include both natural and synthetic genetic elements that promote their own transmission within a population, arguing that this broad definition is both practical and has advantages for risk analysis

Location of <i>piggyBac</i> integration sites in AegPB8.

*<p>Position of underlined nucleotide shown based on <i>Aedes aegypti</i> genome version 66.1 (AegL1)</p

Plasmids used in this study.

<p>All plasmids are described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068454#s2" target="_blank">Material and Methods</a>. Large arrowheads represent the terminal sequences of <i>piggyBac</i>. Act5C, promoter from <i>D. melanogaster</i> gene <i>Actin5C; Hygromycin^R,</i> coding region for bacterial gene <i>hygromycin B phosphotransferase</i>; ie1, promoter from the baculovirus gene <i>immediate early 1</i>; Amp^R, bacterial gene <i>beta-lactamase</i>; hsp70, promoter from <i>D. melanogaster</i> gene <i>hsp70</i>; PB-transposase, coding region for <i>piggyBac</i> transposase; DsRed, coding region for <i>Discosoma sp</i>. gene <i>red fluorescent protein</i>; pUb, promoter from <i>D. melanogaster</i> gene <i>pUbi-p63e</i>; Kan^R, bacterial gene <i>Neomycin phosphotransferase II</i>; gDNA, refers to <i>Aedes aegypti</i> genomic DNA flanking the 5′ and 3 ends of <i>piggyBac</i> elements integrated in the genome of cell line AagPB8 (in pCL1w+) and in transgenic line 40D (in p40Dw+; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068454#pone.0068454-Sethuraman1" target="_blank">[25]</a>); mini-white, the <i>D. melanogaster</i> gene <i>w^+mW.hs;</i> attB, the bacterial attachment site for phage <i>ΦC31.</i></p

Plasmid-based <i>piggyBac</i> excision assay.

<p>A) Diagrammatic representation of the <i>piggyBac</i>-containing plasmid, <i>piggyBac</i> 3×P3EGFP used in the excision assay described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068454#s2" target="_blank">Material and Methods</a> (donor plasmid), and the same plasmid following precise excision of the <i>piggyBac</i> element (excision plasmid). The <i>piggyBac</i>-containing donor plasmid, <i>piggyBac</i> 3×P3EGFP, and <i>piggyBac</i> transposase expressing helper plasmid, pHspPBtpase:PubDsRed, were co-transfected into AagPB8 cells. Transfected cells were heat-shocked after 12 hrs and collected after 72 hrs. DNA was extracted and used as a template for PCR. Primers 1 and 2 (shown as labeled short half-arrows) were specific to the donor-plasmid backbone (494donorFWD, 494donorREV) and yield a 751 bp product (grey line) in the presence of the donor and excision plasmids. Primers 3 and 4 (494excisionFWD, 494excisionREV and shown as labeled short half-arrows) are specific to the plasmid DNA flanking the <i>piggyBac</i> element, however under the conditions of this experiment PCR products were only detected if donor plasmids missing the <i>piggyBac</i> element through excision were present, yielding a 540 bp PCR product (grey line). The 5′ and 3′ terminal <i>piggyBac</i> sequences are represented by arrows (5′PB, 3′PB). The duplicated TTAA target sequence into which <i>piggyBac</i> integrated is shown as a black diamond and the 3×P3EGFP transgene within the <i>piggyBac</i> element is shown as a black rectangle. The normally circular plasmids are represented as linear molecules. B) The PCR results from two <i>piggyBac</i> excision assays in AagPB8 cells. Lanes 1 and 2: from cells transfected with donor and <i>pHspPBtpase:PubDsRed</i> (2 independent transfections). Lane 3: from cells transfected with donor and control plasmids (pBluescript SKII+). Lane 4 and 5: positive controls for detecting excision events. The DNA used as a template in these reactions was a purified excision plasmid recovered from a previous excision assay (2 independent transfections). Lane 6: negative control for detecting excision events. DNA used as a template in this reaction came from cells transfected with donor plasmid only, without the transposase helper plasmid. Two PCR reactions were performed on each sample using primer combinations indicated above the lanes numbers. Primers 1+2 (same primers referred to in panel A) detected the presence of donor and excision plasmids and yielded a 751 bp reaction product (white arrow). Primers 3+4 (same primers referred to in panel A) yielded a 540 bp reaction product (white arrow) only when the <i>piggyBac</i> element in the donor plasmid had excised. Only the 540 and 751 bp bands are specific reaction products.</p

<i>piggyBac</i> transposable element display results using DNA isolated from cell line AagPB8.

<p>Lanes 1 are the results using DNA as a template isolated from cell line AagPB8 and shows evidence of the 5′ end of one of the two <i>piggyBac</i> elements that had integrated by canonical cut-and-paste transposition –80 bp band. The 5′ end of the second <i>piggyBac</i> element that integrated by canonical cut-and-paste transposition is not visible. This element can be detected when the 3′ ends of integrated <i>piggyBac</i> elements are visualized using transposable element display (not shown). The band at 250 bp is the <i>piggyBac</i> element associated with a copy of the integrated plasmid pBac:Act5cHyg:ie1EGFP. The sample was loaded into two adjacent lanes. Lanes 2 are the results using DNA as a template isolated from non-transgenic Aag-2 cells and this serves as a negative control for this assay since there are no <i>piggyBac</i> elements in <i>Ae. aegypti</i>. The sample was loaded into two adjacent lanes. Lanes 3 are the results using DNA as a template from AagPB8 cells 72 hours after being transfected with <i>piggyBac</i>-transposase-expressing pHspPBtpase:PubDsRed. The sample was loaded into two adjacent lanes. There was no evidence of <i>piggyBac</i> elements in other positions in the genome in Lane 3 as would be expected if <i>piggyBac</i> transposase mobilized the integrated <i>piggyBac</i> elements in AagPB8 cells. The asterisk indicated the position of a non-specific TE display band present in all samples. The positions of molecular weight markers 80 bp and 250 bp in length are shown.</p

An Anopheles stephensi Promoter-Trap: Augmenting Genome Annotation and Functional Genomics

The piggyBac transposon was modified to generate gene trap constructs, which were then incorporated into the genome of the Asian malaria vector, Anopheles stephensi and remobilized through genetic crosses using a piggyBac transposase expressing line. A total of 620 remobilization events were documented, and 73 were further characterized at the DNA level to identify patterns in insertion site preferences, remobilization frequencies, and remobilization patterns. Overall, the use of the tetameric AmCyan reporter as the fusion peptide displayed a preference for insertion into the 5′-end of transcripts. Notably 183 – 44882 bp upstream of the An. stephensi v1.0 ab initio gene models, which demonstrated that the promoter regions for the genes of An. stephensi are further upstream of the 5′-proximal regions of the genes in the ab inito models than may be otherwise predicted. RNA-Seq transcript coverage supported the insertion of the splice acceptor gene trap element into 5′-UTR introns for nearly half of all insertions identified. The use of a gene trap element that prefers insertion into the 5′-end of genes supports the use of this technology for the random generation of knock-out mutants, as well as the experimental confirmation of 5′-UTR introns in An. stephensi

Mosquito midgut stem cell cellular defense response limits Plasmodium parasite infection

Abstract A novel cellular response of midgut progenitors (stem cells and enteroblasts) to Plasmodium berghei infection was investigated in Anopheles stephensi. The presence of developing oocysts triggers proliferation of midgut progenitors that is modulated by the Jak/STAT pathway and is proportional to the number of oocysts on individual midguts. The percentage of parasites in direct contact with enteroblasts increases over time, as progenitors proliferate. Silencing components of key signaling pathways through RNA interference (RNAi) that enhance proliferation of progenitor cells significantly decreased oocyst numbers, while limiting proliferation of progenitors increased oocyst survival. Live imaging revealed that enteroblasts interact directly with oocysts and eliminate them. Midgut progenitors sense the presence of Plasmodium oocysts and mount a cellular defense response that involves extensive proliferation and tissue remodeling, followed by oocysts lysis and phagocytosis of parasite remnants by enteroblasts

Perspectives of African stakeholders on gene drives for malaria control and elimination: a multi-country survey

Abstract Background Gene drive modified mosquitoes (GDMMs) have the potential to address Africa’s persistent malaria problem, but are still in early stages of development and testing. Continuous engagement of African stakeholders is crucial for successful evaluation and implementation of these technologies. The aim of this multi-country study was, therefore, to explore the insights and recommendations of key stakeholders across Africa on the potential of GDMMs for malaria control and elimination in the continent. Methods A concurrent mixed-methods study design was used, involving a structured survey administered to 180 stakeholders in 25 countries in sub-Saharan Africa, followed by 18 in-depth discussions with selected groups and individuals. Stakeholders were drawn from academia, research and regulatory institutions, government ministries of health and environment, media and advocacy groups. Thematic content analysis was used to identify key topics from the in-depth discussions, and descriptive analysis was done to summarize information from the survey data. Results Despite high levels of awareness of GDMMs among the stakeholders (76.7%), there was a relatively low-level of understanding of their key attributes and potential for malaria control (28.3%). When more information about GDMMs was provided to the stakeholders, they readily discussed their insights and concerns, and offered several recommendations to ensure successful research and implementation of the technology. These included: (i) increasing relevant technical expertise within Africa, (ii) generating local evidence on safety, applicability, and effectiveness of GDMMs, and (iii) developing country-specific regulations for safe and effective governance of GDMMs. A majority of the respondents (92.9%) stated that they would support field trials or implementation of GDMMs in their respective countries. This study also identified significant misconceptions regarding the phase of GDMM testing in Africa, as several participants incorrectly asserted that GDMMs were already present in Africa, either within laboratories or released into the field. Conclusion Incorporating views and recommendations of African stakeholders in the ongoing research and development of GDMMs is crucial for instilling stakeholder confidence on their potential application. These findings will enable improved planning for GDMMs in Africa as well as improved target product profiles for the technologies to maximize their potential for solving Africa’s enduring malaria challenge