Cell Biology

In the Japanese rhinoceros beetle Trypoxylus dichotomus, gene function studies have relied mainly on systemic larval RNA interference (RNAi), as gain-of-function techniques remain underdeveloped and germline transgenesis is impractical given the species’ approximately one-year generation time. In addition, because larval RNAi is systemic, it has been difficult to analyze the function of lethal genes. Here, we present a simple and efficient protocol for the direct introduction of exogenous DNA into T. dichotomus larvae via in vivo electroporation. This protocol includes optimized procedures for adult breeding and egg collection, as well as a rigorously parameterized electroporation technique that delivers a piggyBac transposon vector into region-specific larval tissues. Within one day after electroporation, treated larvae exhibit mosaic expression of a reporter gene, enabling rapid tissue-specific functional analysis without the need to establish stable germline transgenic lines. Moreover, the key promoter used in this system (T. dichotomus actinA3 promoter) is effective across diverse insect species, indicating that the method can be readily adapted to other non-model insects. Overall, this electroporation-based approach provides a valuable gain-of-function tool for T. dichotomus and potentially many other insect species.

Aging and wasting of skeletal muscle reduce organismal fitness. Regrettably, only limited interventions are currently available to address this unmet medical need. Many methods have been developed to study this condition, including the intramuscular electroporation of DNA plasmids. However, this technique requires surgery and high electrical fields, which cause tissue damage. Here, we report an optimized protocol for the electroporation of small interfering RNAs (siRNAs) into the tibialis anterior muscle of mice. This protocol does not require surgery and, because of the small siRNA size, mild electroporation conditions are utilized. By inducing target mRNA knockdown, this method can be used to interrogate gene function in muscles of mice from different strains, genotypes, and ages. Moreover, a complementary method for siRNA transfection into differentiated myotubes can be used for testing siRNA efficacy before in vivo use. Altogether, this streamlined protocol is instrumental for basic science and translational studies in muscles of mice and other animal models.

This protocol describes the technique of ex-vivo electroporation to target embryonic hippocampal progenitors in an organotypic slice preparation. This technique allows gene perturbation for examining developmental processes in the embryonic hippocampus while retaining the environment and connectivity of the cells. Gene perturbation can include Cre-mediated recombination, RNAi-mediated knockdown, gene overexpression, or a combination of any of these. Ex-vivo electroporation can be performed at a wide range of embryonic stages, giving temporal control to the experimenter. Spatial control can be achieved more easily by preparing the brain in a Petri dish to target particular regions of the hippocampus. The electroporated explant cultures provide a highly tractable system for the study of developmental processes that include progenitor proliferation, migration and cell fate acquisition.

In utero electroporation (IUE) of mouse cerebellar Purkinje cells allows high expression levels of transgenes without toxicity (Nishiyama et al., 2012). This technique is suitable for co-transfection of multiple plasmid genes. Therefore, it is useful to express various sets of genes such as drug-inducible Cre/loxP constructs and CRISPR/Cas9 genome editing constructs (Takeo et al., 2015). Murine Purkinje cells arise from subventricular zone of fourth ventricle at embryonic day (E) 10-12. IUE at E11.5 into fourth ventricle results the most efficient transfection into Purkinje cells.