Electroporation-mediated somatic transgenesis for rapid functional analysis in insects

Abstract

Transgenesis is a powerful technique for determining gene function; however, it is time-consuming. It is virtually impossible to carry out in non-model insects in which egg manipulation and screening are difficult. We have established a rapid genetic functional analysis system for non-model insects using a low-cost electroporator (costing under US$200) designed for somatic transformation with the piggyBac transposon. Using this system, we successfully generated somatic transgenic cell clones in various target tissues (e.g. olfactory neurons, wing epidermis, larval epidermis, muscle, fat body and trachea) of the silkworm Bombyx mori during development. We also induced stable and transient RNA interference (RNAi) using short hairpin RNA (shRNA)-mediating DNA vectors and direct transfer of small interfering RNAs (siRNAs), respectively. We found that these electroporation-mediated approaches could also be applied to the swallowtail butterfly Papilio xuthus and the red flour beetle Tribolium castaneum. Thus, this method could be a powerful genetic tool for elucidating various developmental phenomena in non-model insects.

Positional cloning of a Bombyx pink-eyed white egg locus reveals the major role of cardinal in ommochrome synthesis

“…The sequences of the siRNA are as follows: pe-1 sense strand, 5′-GCGAUUACAGCACUUUC AAUG-3′; antisense strand, 5′-UUGAAAGUGCUGUAAUCGCUU-3′. We used the following siRNA for Bm-white as reported in Ando and Fujiwara, 2013; sense strand, 5′-CAUUUAUGGCCCAAAACGUUA-3′; antisense strand 5′-ACGUUUUGGGCCAUAAAUGAA-3′. To produce templates for double stranded RNA (dsRNA) synthesis the following primers were used for T. castaneum cardinal: Tccd-F3 5′-GACGATGTCAGGGGGTGAAC-3′ and Tccd-R2 5′-GTTATAGCCAACTAATCCATGGTC-3′.…”

“…For embryonic RNAi of B. mori, 2-3 nl of 100 μM siRNA was injected into eggs 3-5 h after laying as reported previously (Osanai-Futahashi et al, 2012). For larval and pupal RNAi of B. mori, an electroporation-mediated method was performed as reported by Ando and Fujiwara, 2013. The larval epidermis of the third-or fourth-instar p S strain and the compound eye region of day 1-3 pupae were injected with 0.25-0.5 μl of 100 μM siRNA solution.…”

“…We next analyzed the effect of RNAi on adult compound eyes. Because simple systemic RNAi by injection of dsRNA does not work in the larval and pupal period in Lepidopteran species, including B. mori, in general (Terenius et al, 2011), we conducted recently developed electroporationmediated RNAi as reported by Ando and Fujiwara (2013). We found that one side of compound eyes became reddish by electroporation of Bm-cardinal siRNA, like the pe mutant, whereas it became white by electroporation of Bm-white siRNA like w-3 (Bm-white) mutant (Figure 4b).…”

A genetic mechanism for female-limited Batesian mimicry in Papilio butterfly

“…To verify the function of dsx in mimetic wing pattern formation, we performed electroporation-mediated small interfering RNA (siRNA) incorporation, enabling mosaic analysis by knocking down the expression of target genes 11,12 . First, we optimized our methods to minimize side effects on pigmentation patterns 13 (Online Methods and Supplementary Fig.…”

Molecular Mechanisms Underlying Color Vision and Color Formation in Dragonflies

“…Molecular bases underlying color pattern formation and its evolution in dragonflies are just as fascinating and challenging as in butterflies. Recently, effective RNAi-and genome-editing methods have been developed for gene functional analyses in butterflies (Ando and Fujiwara 2013;Nishikawa et al 2015;Li et al 2015;Perry et al 2016;Zhang and Reed 2016;Beldade and Peralta 2017). Applying these methods to dragonflies will be an important step toward future studies in this field (Okude et al 2017).…”