Molecular Biology

Pseudoalteromonas haloplanktis TAC125 is a psychrophilic marine bacterium widely used to study cold adaptation and increasingly exploited as a non-conventional platform for biotechnological applications. The strain harbors the endogenous megaplasmid pMEGA (64.7 kb), whose presence may limit its exploitation as a cell factory, making its elimination advantageous to strain engineering. Traditional plasmid-curing approaches based on chemical and physical agents are often inefficient and unsuitable for stable endogenous replicons, such as pMEGA. Here, we describe a targeted protocol for pMEGA curing in P. haloplanktis TAC125 that combines homologous recombination with paired-termini antisense RNA (PTasRNA) gene silencing. First, a selectable marker cassette is inserted into pMEGA by homologous recombination using a suicide vector, enabling selective discrimination between plasmid-positive and plasmid-cured bacteria. Next, PTasRNA gene silencing technology is applied to target a gene essential for the replication of pMEGA, thereby transiently interfering with its replication and promoting its loss. This approach provides a specific method to cure a highly stable endogenous megaplasmid in a psychrophilic non-conventional bacterium, enabling improved functional studies and strain optimization, establishing a broadly applicable framework for targeted curing across diverse bacterial systems.

Transfecting neurons remains technically challenging due to their sensitivity. Conventional methods, such as Lipofectamine 2000 or Lipofectamine RNAiMAX, often result in significant cytotoxicity, which limits their utility. Although lentiviral transfection offers high efficiency, it is hindered by high costs and complex procedures. This experiment employs a small interfering RNA (siRNA)-specific transfection reagent from the Kermey company. This reagent is a novel nanoparticle-based lipid material designed for the efficient delivery of oligonucleotides, including siRNA, into a wide range of cell types. Its efficacy in achieving high transfection efficiency in neurons, however, has not yet been established. After several days of in vitro neuronal culture, researchers can perform a simple transfection procedure using this reagent to achieve robust transfection efficiency. Notably, the protocol does not require medium replacement 6–8 h post-transfection, streamlining the workflow and minimizing cellular stress.

Synthetic trans-acting small interfering RNAs (syn-tasiRNAs) are 21-nucleotide small RNAs designed to induce highly specific and efficient gene silencing in plants. Traditional approaches rely on the transgenic expression of ~1 kb TAS precursors, which limits their use in non-model species, under strict GMO regulations, and in size-constrained expression or delivery systems. This protocol describes a rapid workflow for the design, assembly, and delivery of syn-tasiRNAs derived from much shorter precursors, referred to as minimal precursors. The pipeline includes in silico design of highly specific syn-tasiRNA sequences, cloning of minimal precursors into plant expression or potato virus X (PVX)-based viral vectors through Golden Gate or Gibson assembly, and delivery to plants through Agrobacterium-mediated expression or by spraying crude extracts containing recombinant PVX expressing the minimal precursors. These methodologies make syn-tasiRNA-based tools more accessible and broadly applicable for plant research and biotechnology across diverse species and experimental contexts.

Long noncoding RNAs (lncRNAs) are increasingly understood to play important roles in cell biology, development, and disease, though the vast majority of annotated lncRNAs have yet to be functionally characterized. Disrupting lncRNAs is often challenging owing to their tolerance for mutations (e.g., single-nucleotide polymorphisms and short indels) along with the limitations of other genetic knockdown strategies such as RNA interference (RNAi). Here, we describe a protocol to achieve robust knockdown of lncRNAs in the fruit fly Drosophila using a self-cleaving ribozyme. The 111-bp ribozyme cassette, which consists of the N79 hammerhead ribozyme flanked by flexible linker sequences, is inserted into transcript regions of lncRNA genes using CRISPR/Cas9-mediated homology-directed repair (HDR). The fluorescent eye transformation marker is then removed using a piggyBac transposase, leaving no other modifications at the lncRNA locus save the ribozyme cassette insertion. When transcribed as part of the lncRNA, the ribozyme folds and catalyzes its own self-cleavage, resulting in two RNA cleavage fragments. The efficacy of lncRNA knockdown is then evaluated using reverse transcription quantitative PCR (RT-qPCR) and single-molecule RNA fluorescence in situ hybridization (smFISH). This approach has resulted in efficient knockdown of both nuclear and cytoplasmic lncRNAs in Drosophila, with knockdown of steady-state RNA levels in 3' cleavage fragments typically exceeding 90% and no evidence of off-target effects. The method can also be applied to protein-coding genes in order to knock down specific mRNA isoforms. Thus, self-cleaving ribozymes are a valuable addition to the genetic toolkit in Drosophila.

Advances in imaging technology offer new opportunities in developmental biology. To observe the development of internal structures, microtome cross-sectioning followed by H&E staining on glass slides is a common procedure; however, this technique can be destructive, and artifacts can be introduced during the process. In this protocol, we describe a less invasive procedure with which we can stain insect samples and obtain reconstructed three-dimensional images using micro-computed tomography, or micro-CT (µCT). Specifically, we utilize the fungus-farming ambrosia beetle species Euwallacea validus to observe the morphology of mycangia, a critical internal organ with which beetles transport fungal symbionts. Not only this protocol is ideal to observe mycangia, our staining/scanning procedure can also be applied to observe other delicate tissues and small organs in arthropods.

Graphical abstract

In the Japanese rhinoceros beetle Trypoxylus dichotomus, various candidate genes required for a specific phenotype of interest are listed by next-generation sequencing analysis. Their functions were investigated using RNA interference (RNAi) method, the only gene function analysis tool for T. dichotomus developed so far. The summarized procedure for the RNAi method used for T. dichotomus is to synthesize double-stranded RNA (dsRNA), and inject it in larvae or pupae of T. dichotomus. Although some dedicated materials or equipment are generally required to inject dsRNA in insects, the advantage of the protocol described here is that it is possible to inject dsRNA in T. dichotomus with one syringe.

The co-stimulatory molecule CD40 and its ligand CD40L play a key role in the regulation of immunological processes and are involved in the pathophysiology of autoimmune and inflammatory diseases. Inhibition of the CD40-CD40L axis is a promising therapy, and a number of strategies and techniques have been designed to hinder its functionality. Our group has broad experience in silencing CD40 using RNAi technology, and here we summarize protocols for the systemic administration of a specific anti-CD40 siRNA in different rodents models, in addition to the subsequent quantification of CD40 expression in murine kidneys by immunostaining. The use of RNAi technology with specific siRNAs to silence genes is becoming an essential method to investigate gene functions and is rapidly emerging as a therapeutic tool.

Graphic abstract:

CD40 siRNA mechanism