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While piggyBac transposon-based transgenesis is widely used in various emerging model organisms, its relatively low transposition rate in butterflies and moths has hindered its use for routine genetic transformation in Lepidoptera. Here, we tested the suitability of a codon-optimized hyperactive piggyBac transposase ( hyPBase ) in mRNA form to deliver and integrate transgenic cassettes into the genome of the pantry moth Plodia interpunctella . Co-injection of hyPBase mRNA with donor plasmids successfully integrated 1.5–4.4 kb expression cassettes driving the fluorescent markers EGFP, DsRed, or EYFP in eyes and glia with the 3xP3 promoter. Somatic integration and expression of the transgene in the G 0 injected generation was detectable from 72-h embryos and onward in larvae, pupae and adults carrying a recessive white-eyed mutation. Overall, 2.5% of injected eggs survived into transgene-bearing adults with mosaic fluorescence. Subsequent outcrossing of fluorescent G 0 founders transmitted single-insertion copies of 3xP3::EGFP and 3xP3::EYFP and generated stable isogenic lines. Random in-crossing of a small cohort of G 0 founders expressing 3xP3::DsRed yielded a stable transgenic line segregating for more than one transgene insertion site. We discuss how hyPBase can be used to generate stable transgenic resources in Plodia and other moths.

With the advances in genomic sequencing, many organisms with novel biological properties are ripe for use as emerging model organisms. However, to make full use of them, transformation methods need to be developed to permit genome editing. Here, we present the development of transformation for the fungus fly Bradysia (Sciara) coprophila; this may serve as a paradigm for the development of transformation for other emerging systems, especially insects. Bradysia (Sciara) has a variety of unique biological features, including locus-specific developmentally regulated DNA amplification, chromosome imprinting, a monopolar spindle in male meiosis I, non-disjunction of the X chromosome in male meiosis II, X chromosome elimination in early embryogenesis, germ-line-limited (L) chromosomes and high resistance to radiation. Mining the unique biology of Bradysia (Sciara) requires a transformation system to test mutations of DNA sequences that may play roles for these features. We describe a Bradysia (Sciara) transformation system using a modified piggyBac transformation vector and detailed protocols we have developed to accommodate Bradysia (Sciara) specific requirements. This advance will provide a platform for us and others in the growing Bradysia (Sciara) community to take advantage of this unique biological system. In addition, the versatile piggyBac vectors described here and transformation methods will be useful for other emerging model systems.

Diapause is an important biological characteristic for many insect species to adapt to adverse environmental conditions and maintain the continuity of the race. Compared with the traditional hydrochloric acid or/and cold storage treatment methods, the artificial corona incubation technology of silkworm ( Bombyx mori ) eggs has many advantages including, the absence of pollution, easy operation and safety. However, this technology has not yet been applied in sericulture. In this study, we developed a novel artificial corona instrument to successfully disrupt the diapause of newly laid and refrigerated eggs from various Chinese and Japanese lineage silkworm strains. Subsequently, we invented a very early corona treatment (VECT) strategy to prevent the diapause of newly laid silkworm eggs within 4 h of oviposition. The hatching rates of the larvae were more than 95% in all diapause silkworm strains, which was comparable to the effect of the traditional HCl treatment strategy. In addition, we developed a combination strategy of VECT and pre-blastoderm microinjection and successfully created transgenic silkworms in various diapause strains. The results of the current study can aid in improving the corona artificial incubation technology and promote its application in sericulture.

KEY MESSAGE : Agrobacterium tumefaciens mediates high frequency of germline transformation of cotton meristem explants. The meristem transformation system we developed is rapid, high throughput and genotype-flexible. We have developed a high throughput cotton transformation system based on direct Agrobacterium inoculation of mechanically isolated meristem explants of cotton (Gossypium hirsutum L.). The explants were inoculated with a disarmed A. tumefaciens strain, AB33 harboring a 2 T-DNA binary vector pMON114908. This vector contained a gene of interest, an intron-disrupted β-glucuronidase gene in one T-DNA, and a selectable marker gene, aadA in the other T-DNA. Critical factors, such as method of co-culture, culture temperature during selection, composition of selection medium, and selection scheme were found to influence transformation frequency. The cycle time from initial inoculation to the transplanting of transgenic plants to soil was 7–8 weeks. Stable integration of transgenes and their transmission to progeny were confirmed by molecular and genetic analyses. Transgenes segregated in the expected Mendelian fashion in the T1 generation for most of the transgenic events. It was possible to recover marker-free events in the T1 generation when utilizing a binary vector that contained the selectable marker and gene of interest expression cassettes on independent T-DNAs. The procedure presented here has been used to regenerate thousands of independent transgenic events from multiple varieties with numerous constructs, and we believe it represents a major step forward in cotton transformation technology.

The efficiency of transposon-mediated germline transformation is dependent on the transposon mobility in the host embryo, and on the detectability of the used transformation marker. Therefore, high susceptibility of the transformation marker to position effect suppression is a disadvantage. Here we present data that the eye-specific expression of green fluorescent protein, driven by the 3xP3-EGFP marker, outperforms the commonly used ”mini”-white transformation marker in Drosophila germline transformation experiments: 3xP3-EGFP is more sensitive than ”mini”-white in identifying transgenic individuals and reacts differently to position effect suppression. Therefore, 3xP3-EGFP offers an ideal marker for applications in functional genomics where as many gene loci as possible should be targeted in the genome of a specific organism, for example, as intended in the Drosophila gene disruption project. Furthermore, we give a detailed description of the embryonic and larval expression mediated by the 3xP3-EGFP marker. These pre-adult expression patterns, and the potentially universal applicability of the transformation marker also offer additional advantages for selecting transgenic individuals in organisms other than Drosophila. This will be of great interest to the field of evolutionary developmental biology and to modern pest management programs.

Ecological and evolutionary theory has frequently been inspired by the diversity of colour patterns on the wings of butterflies. More recently, these varied patterns have also become model systems for studying the evolution of developmental mechanisms. A technique that will facilitate our understanding of butterfly colour-pattern development is germline transformation. Germline transformation permits functional tests of candidate gene products and of cis-regulatory regions, and provides a means of generating new colour-pattern mutants by insertional mutagenesis. We report the successful transformation of the African satyrid butterfly Bicyclus anynana with two different transposable element vectors, Hermes and piggyBac, each carrying EGFP coding sequences driven by the 3XP3 synthetic enhancer that drives gene expression in the eyes. Candidate lines identified by screening for EGFP in adult eyes were later confirmed by PCR amplification of a fragment of the EGFP coding sequence from genomic DNA. Flanking DNA surrounding the insertions was amplified by inverse PCR and sequenced. Transformation rates were 5% for piggyBac and 10.2% for Hermes. Ultimately, the new data generated by these techniques may permit an integrated understanding of the developmental genetics of colour-pattern formation and of the ecological and evolutionary processes in which these patterns play a role.

We have previously reported that Bmdsx, a homologue of the sex-determining gene doublesex (dsx), was sex-specifically expressed in various tissues of the silkworm. The primary transcript of Bmdsx is alternatively spliced in males and females to yield sex-specific mRNAs that encode male-specific (BmDSXM) and female-specific (BmDSXF) polypeptides. In the studies reported here, we expressed BmDSXF in males from a ubiquitous promoter and examined its regulatory activities. We show that BmDSXF functions as a positive regulator of the hexameric storage protein termed SP1 and vitellogenin genes that are predominantly expressed in females. We also show that expression of BmdsxF in males results in the repression of the pheromone-binding protein gene that is preferentially expressed in males. Gel-mobility shift assays demonstrated that BmDSX proteins bind to the sequence (ACATTGT) between −95 and −89 nt relative to the transcriptional initiation site of the vitellogenin gene. These results strongly suggest that Bmdsx is a final regulatory gene in the hierarchy of regulatory genes controlling the expression of female-specific protein in Bombyx mori.

Pair‐rule genes in Drosophila integrate the positional information provided by the transcription factor gradients of gap genes and set the framework for the expression of the segment polarity genes. Pair‐rule genes are also expressed during the segmentation of the short germ‐band embryo of the flour beetle Tribolium , although this occurs under cellular conditions that should not allow the generation of transcription factor gradients by passive diffusion. To analyse the regulation of the pair‐rule gene hairy in Tribolium , we have used germline transformation with reporter gene constructs based on the piggyBac vector. We can identify an upstream fragment that drives the full expression of the eight pair‐rule stripes, which are sequentially generated during embryogenesis. Further experiments with smaller fragments reveal separable regions driving stripes three to five, and one region driving stripe three only. Our results suggest that the generation of pair‐rule stripes in Tribolium is comparable to that in Drosophila despite the cellular versus syncytial mode of embryogenesis.

Germline transformation with new transposon vectors now enables causal tests of gene function via ectopic protein expression or RNA interference in non-drosophilid insects. The problem remains of how to drive the transgene expression in vivo. We employed germline transformation using the piggyBac 3xP3-EGFP vector to test whether the Drosophila heat shock hsp70 promoter will be active in the live silkworm. We modified the original vector by cloning the coding sequence for Bombyx nuclear receptor Ftz-F1 between the hsp70 promoter and the terminator. Three independent transgenic lines expressing the Pax-6-driven EGFP marker in larval and adult photoreceptors were obtained with efficiencies of up to 1.7% of fertile G0 adults that gave GFP-positive progeny. Chromosomal integration of the transposon was confirmed with inverse PCR. Heat induction of the transgenic BmFtz-F1 was proven at both the mRNA and protein levels. RT-PCR data showed that the Drosophila heat shock promoter was functional in all three transgenic lines. Although basal activity was apparent at 25°C, 1 h at 42°C induced BmFtz-F1 mRNA at different stages of development and in diverse tissues. The relative levels of induction differed among the transgenic lines. Northern blot hybridization detected transgenic BmFtz-F1 only after heat shock and low levels of the mRNA were still present 6 h after the heat treatment. Immunostaining of epidermis using anti-BmFtz-F1 antibody showed a clear increase of nuclear signal 90 min after a heat shock.

Objectives Spermatogonial stem cells (SSCs), the germline stem cells (GSCs) committed to spermatogenesis in niche, can transform into pluripotent state in long‐term culture without introduction of exogenous factors, typically in p53 deficiency condition. As the guardian for genomic stability, p53 is associated with epigenetic alterations during SSCs transformation. However, the mechanism is still unknown, since complicated roles of p53 baffle our understanding of the regulating process. Materials and Methods The chromatin accessibility and differentially expressed genes (DEGs) were analysed in p53+/+ and p53−/− SSCs using the Assay for Transposase‐Accessible Chromatin with high‐throughput Sequencing (ATAC‐seq) and RNA‐sequencing (RNA‐seq), to explore the connection of p53 and cell fate at chromosomal level. Results Several transcription factors (TFs), such as CTCF, SMAD3 and SOX2, were predicted as important factors mediating the transformation. Molecular evidence suggested that SMAD3 efficiently promoted pluripotency‐associated gene expression both in fresh and long‐term cultured SSCs. However, p53 knockout (KO) is insufficient to induce SMAD3 expression in SSCs. Conclusions These observations indicate that SMAD3 is a key factor for SSCs transformation, and an unknown event is required to activate SMAD3 as the prerequisite for SSCs reprogramming, which may occur in the long‐term culture of SSCs. This study demonstrates the connection of p53 and pluripotency‐associated factors, providing new insight for understanding the mechanisms of SSCs reprogramming and germline tumorigenesis. This study unveils that p53 deficiency is insufficient for spermatogonial stem cells (SSCs) to obtain pluripotency during long‐term culture, and activation of SMAD3 is a key event required for SSCs transformation, which combined with p53 deficiency has a synergic effect in mediating transformation. These observations answered why p53 deficiency accelerated SSCs transformation and facilitated us for further understanding of the mechanism of reprogramming and tumorigenesis in germline cells.