Since its discovery in 1989, RNA interference (RNAi) has become a widely used tool for the in vitro downregulation of specific gene expression in molecular biological research. This basically involves a complementary RNA that binds a target sequence to affect its transcription or translation process. Currently, various small RNAs, such as small interfering RNA (siRNA), micro RNA (miRNA), small hairpin RNA (shRNA), and PIWI interacting RNA (piRNA), are available for application on in vitro cell culture, to regulate the cells’ gene expression by mimicking the endogenous RNAi-machinery. In addition, several biochemical, physical, and viral methods have been established to deliver these RNAs into the cell or nucleus. Since each RNA and each delivery method entail different off-target effects, limitations, and compatibilities, it is crucial to understand their basic mode of action. This review is intended to provide an overview of different nucleic acids and delivery methods for planning, interpreting, and troubleshooting of RNAi experiments.

/pdf/basic-principles-of-rna-interference-nucleic-acid-types-and-11kpfdch.pdf

Basic Principles of RNA Interference: Nucleic Acid Types and In Vitro Intracellular Delivery Methods

Advancements in technology and science have facilitated the development of various materials for use in different industries. These materials can range from large sizes to even nanometer dimensions. Nanomaterials or nanoparticles, which fall within the size range of 1 to 100 nanometers, have emerged as a prominent aspect of nanotechnology. Nanotechnology, known as one of the fastest-growing technologies today, has found applications in a wide array of fields such as health, energy, metallurgy, physics, chemistry, and engineering. Nanomaterials can be produced using diverse methods, including chemical synthesis and the utilization of biological structures. Biocompatible nanomaterials, as they are commonly referred to, have particularly found significance in the medical science, including applications in oncology, tissue engineering, and drug delivery. While certain areas have made remarkable progress in terms of nanobiotechnology, there are other areas that are comparatively new to the realm of nanotechnology. Reproductive biotechnologies are among the fields that are slowly and cautiously exploring the use of nanomaterials. Similar to a baby's crawling, progress in this field is being made gradually, and each day brings forth surprising developments through ongoing studies. In this book, which is prepared for our esteemed readers, the uses of nanotechnology in reproduction have been compiled. It covers various bioengineering studies and explores its functions in embryo manipulation, semen purification, infertility treatments, as well as the effects of different nanomaterials on mammalian semen and aquatic organisms. I would like to express my gratitude to all my colleagues who have contributed to this book, aimed at explaining the applications of nanotechnology in reproductive biotechnologies. With the expectation that it would be beneficial to the scientific community... Assist. Prof. Ali Erdem ÖZTÜRK 

Nanotechnology in Reproduction

Recent advances in genome editing technologies have redefined our ability to probe and precisely edit the human genome and epigenome in vitro and in vivo. More specifically, RNA-guided CRISPR/Cas systems have revolutionized the field due to their simplicity in design and adaptability across biological systems. This Collection highlights results in CRISPR/Cas technology that increase the efficiency of precision genome editing, and allow genetic manipulation in model systems traditionally intractable to site-directed gene modification. 

Genome editing

Gene therapy has become an important modality for a wide range of therapeutic indications with a rapid increase in the number of therapeutic candidates being developed in this field. Understanding the molecular biology underlying the gene therapy is often critical to develop appropriate safety assessment strategies. We aimed to discuss some of the commonly used gene therapy modalities and common preclinical toxicology testing considerations when developing gene therapies. Non-viral gene delivery methods such as electroporation, microinjection, peptide nanoparticles and lipid nanoparticles are deployed as innovative molecular molecular construct which are included in the design of novel gene therapies and the associated molecular biology mechanisms have become relevant knowledge to non-clinical toxicology. Viral gene delivery methodologies including Adenovirus vectors, Adeno-Associated virus vectors and Lentivirus gene therapy vectors have also advanced considerably across numerous therapeutic areas, raising unique non-clinical toxicology and immunological considerations. General toxicology, biodistribution and tumorigenicity are the pillars of non-clinical safety testing in gene therapies. Evaluating the tumorigenicity potential of a gene editing therapy often leverages molecular pathology while some translational challenges remain. Toxicology study design is entering a new era where science-driven customized approaches and program specific considerations have become the norm.

An Overview of Gene Editing Modalities and Related Non-clinical Testing Considerations

Abstract Transgenic animals are invaluable tools in genetic studies, disease modeling, drug discovery, and biotechnology. However, the low efficiency of transgenic animal generation can be an obstacle to their application. Here, we report the generation of transgenic mice using PBatase, the piggyBat transposase from the little brown bat (Myotis lucifugus). PBatase exhibits detectable transposition activity in fertilized mouse eggs within a limited concentration range, although the overall activity was lower than that of PBase, the piggyBac transposase from the cabbage looper moth (Trichoplusia ni). Transgenic animals carrying low transgene copy numbers were successfully generated with high efficiency using PBatase, and the transgene was subsequently transmitted to the next generation. This technique will be useful for the generation of transgenic animals carrying single copies of a transgene. 

Efficient generation of single-copy transgenic mice using piggyBat transposase from the little brown bat Myotis lucifugus

Genetically modified mice are commonly used in biologic, medical, and drug discovery research, but conventional microinjection methods used for genetic modification require extensive training and practical experience. Here we present a fully automated system for microinjection into the pronucleus to facilitate genetic modification. We first developed software that automatically controls the microinjection system hardware. The software permits automatic rotation of the zygote to move the pronucleus to the injection pipette insertion position. We also developed software that recognizes the pronucleus in 3-dimensional coordinates so that the injection pipette can be automatically inserted into the pronucleus, and achieved a 94% insertion rate by linking the 2 pieces of software. Next, we determined the optimal solution injection conditions (30 hPa, 0.8–2.0 s) by examining the survival rate of injected zygotes. Finally, we produced transgenic (traditional DNA injection and piggyBac Transposon system) and knock-in (genomic editing) mice using our newly developed Integrated Automated Embryo Manipulation System (IAEMS). We propose that the IAEMS will simplify highly reproducible pronuclear stage zygote microinjection procedures.

/pdf/establishment-of-an-integrated-automated-embryonic-5dg2cbg1v7.pdf

Establishment of an integrated automated embryonic manipulation system for producing genetically modified mice.

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Establishment of an integrated automated embryonic manipulation system for producing genetically modified mice.