Protocol for high-resolution 3D visualization of insect regenerating legs through micro-computed tomography.

Appendage regeneration occurs within the opaque exoskeleton in arthropods, making it challenging to visualize the regenerative processes dynamically. In this protocol, we present a strategy to scan and capture the high-resolution details of microstructural tissues at certain regeneration points through micro-computed tomography (micro-CT). We describe steps for tissue preparation, fixation, critical point drying, micro-CT scanning, and 3D visualization.

ERK-activated CK-2 triggers blastema formation during appendage regeneration.

Appendage regeneration relies on the formation of blastema, a heterogeneous cellular structure formed at the injury site. However, little is known about the early injury-activated signaling pathways that trigger blastema formation during appendage regeneration. Here, we provide compelling evidence that the extracellular signal-regulated kinase (ERK)-activated casein kinase 2 (CK-2), which has not been previously implicated in appendage regeneration, triggers blastema formation during leg regeneration in the American cockroach, .

Two transcriptional cascades orchestrate cockroach leg regeneration.

The mystery of appendage regeneration has fascinated humans for centuries, while the underlying regulatory mechanisms remain unclear. In this study, we establish a transcriptional landscape of regenerating leg in the American cockroach, Periplaneta americana, an ideal model in appendage regeneration studies showing remarkable regeneration capacity. Through a large-scale in vivo screening, we identify multiple signaling pathways and transcription factors controlling leg regeneration.

5mC modification orchestrates choriogenesis and fertilization by preventing prolonged ftz-f1 expression.

DNA methylation at the fifth position of cytosine (5-methylcytosine, 5mC) is a crucial epigenetic modification for regulating gene expression, but little is known about how it regulates gene expression in insects. Here, we pursue the detailed molecular mechanism by which DNMT1-mediated 5mC maintenance regulates female reproduction in the German cockroach, Blattella germanica. Our results show that Dnmt1 knockdown decreases the level of 5mC in the ovary, upregulating numerous genes during choriogenesis, especially the transcription factor ftz-f1.

Nutrition- and hormone-controlled developmental plasticity in Blattodea.

Blattodea, which includes cockroaches and termites, possesses high developmental plasticity that is mainly controlled by nutritional conditions and insect hormones. Insulin/insulin-like growth factor signaling (IIS), target of rapamycin complex 1 (TORC1), and adenosine monophosphate-activated protein complex are the three primary nutrition-responsive signals. Juvenile hormone (JH) and 20-hydroxyecdysone (20E) constitute the two most vital insect hormones that might interact with each other through the Met, Kr-h1, E93 (MEKRE93) pathway.

Physiological and molecular mechanisms of insect appendage regeneration.

Regeneration, as a fascinating scientific field, refers to the ability of animals replacing lost tissue or body parts. Many metazoan organisms have been reported with the regeneration phenomena, but showing evolutionarily variable abilities. As the most diverse metazoan taxon, hundreds of insects show strong appendage regeneration ability.

Identification of a novel collagen-like peptide by high-throughput screening for effective wound-healing therapy.

Tissue regeneration and wound healing are still serious clinical complications globally and lack satisfactory cures. Inspired by the impressive regeneration ability of the post-injury earthworms and their widely accepted medicinal properties, we screened and identified a novel collagen-like peptide from the amputated earthworms using high-throughput techniques, including transcriptomics, proteomics, and mass spectrum. The identified collagen-like peptide col4a1 was cloned and expressed to comprehensively investigate the wound healing effect and underlying mechanism.

Grainy head signaling regulates epithelium development and ecdysis in Blattella germanica.

The transcription factor grainy head (Grh) functions in the protection of the epithelium against the external environment by generating strongly adhesive layers, and this function is conserved in vertebrates and invertebrates. In Drosophila, the top model for holometabolous insects, Grh is necessary during embryonic development, epidermal differentiation, central nervous system specification and epithelial repair. However, the function of this gene in hemimetabolous insect epithelia remains unknown.

Alteration of insulin and nutrition signal gene expression or depletion of Met reduce both lifespan and reproduction in the German cockroach.

In insects, nutrition and hormones coordinately regulate lifespan and reproduction, which might affect each other. We here investigated how nutrition, insulin, and juvenile hormone (JH) signal genes affect lifespan and reproduction in the German cockroach, Blattella germanica, a serious urban pest throughout the world. Starvation as well as altering insulin and nutrition signal genes by RNA interference (RNAi) knockdown of the expression of either positive or negative components in the two pathways simultaneously reduced lifespan and ootheca number of the mated female cockroaches.

In Vivo Applications of Cell-Penetrating Zinc-Finger Transcription Factors.

Artificial transcription factors based on zinc finger, TALE, and CRISPR/Cas9 programmable DNA-binding platforms have been widely used to regulate the expression of specific genes in cultured cells, but their delivery into organs such as the brain represents a critical challenge to apply such tools in live animals. In previous work, we developed a zinc-finger-based artificial transcription factor harboring a cell-penetrating peptide (CPP) that could be injected systemically, cross the blood-brain barrier, and alter expression of a specific gene in the brain of an adult mouse. Importantly, our mode of delivery produced widespread distribution throughout the brain.

Strategies for the Enrichment and Selection of Genetically Modified Cells.

Programmable artificial nucleases have transitioned over the past decade from ZFNs and TALENs to CRISPR/Cas systems, which have been ubiquitously used with great success to modify genomes. The efficiencies of knockout and knockin vary widely among distinct cell types and genomic loci and depend on the nuclease delivery and cleavage efficiencies. Moreover, genetically modified cells are almost phenotypically indistinguishable from normal counterparts, making screening and isolating positive cells rather challenging and time-consuming.

Unexpected binding behaviors of bacterial Argonautes in human cells cast doubts on their use as targetable gene regulators.

Prokaryotic Argonaute proteins (pAgos) have been proposed as an alternative to the CRISPR/Cas9 platform for gene editing. Although Argonaute from Natronobacterium gregoryi (NgAgo) was recently shown unable to cleave genomic DNA in mammalian cells, the utility of NgAgo or other pAgos as a targetable DNA-binding platform for epigenetic editing has not been explored. In this report, we evaluated the utility of two prokaryotic Argonautes (NgAgo and TtAgo) as DNA-guided DNA-binding proteins.

The genomic and functional landscapes of developmental plasticity in the American cockroach.

Many cockroach species have adapted to urban environments, and some have been serious pests of public health in the tropics and subtropics. Here, we present the 3.38-Gb genome and a consensus gene set of the American cockroach, Periplaneta americana.

Gene editing as a promising approach for respiratory diseases.

Respiratory diseases, which are leading causes of mortality and morbidity in the world, are dysfunctions of the nasopharynx, the trachea, the bronchus, the lung and the pleural cavity. Symptoms of chronic respiratory diseases, such as cough, sneezing and difficulty breathing, may seriously affect the productivity, sleep quality and physical and mental well-being of patients, and patients with acute respiratory diseases may have difficulty breathing, anoxia and even life-threatening respiratory failure. Respiratory diseases are generally heterogeneous, with multifaceted causes including smoking, ageing, air pollution, infection and gene mutations.

dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression.

Distinct epigenomic profiles of histone marks have been associated with gene expression, but questions regarding the causal relationship remain. Here we investigated the activity of a broad collection of genomically targeted epigenetic regulators that could write epigenetic marks associated with a repressed chromatin state (G9A, SUV39H1, Krüppel-associated box (KRAB), DNMT3A as well as the first targetable versions of Ezh2 and Friend of GATA-1 (FOG1)). dCas9 fusions produced target gene repression over a range of 0- to 10-fold that varied by locus and cell type.

Enhancing CRISPR/Cas9-mediated homology-directed repair in mammalian cells by expressing Saccharomyces cerevisiae Rad52.

Precise genome editing with desired point mutations can be generated by CRISPR/Cas9-mediated homology-directed repair (HDR) and is of great significance for gene function study, gene therapy and animal breeding. However, HDR efficiency is inherently low and improvements are necessitated. Herein, we determined that the HDR efficiency could be enhanced by expressing Rad52, a gene that is involved in the homologous recombination process.

Enhanced CRISPR/Cas9-mediated biallelic genome targeting with dual surrogate reporter-integrated donors.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system has recently emerged as a simple, yet powerful genome engineering tool, which has been widely used for genome modification in various organisms and cell types. However, screening biallelic genome-modified cells is often time-consuming and technically challenging. In this study, we incorporated two different surrogate reporter cassettes into paired donor plasmids, which were used as both the surrogate reporters and the knock-in donors.

Multiplex CRISPR/Cas9-based genome engineering enhanced by Drosha-mediated sgRNA-shRNA structure.

The clustered regularly interspaced short palindromic repeats (CRISPR) system has recently been developed into a powerful genome-editing technology, as it requires only two key components (Cas9 protein and sgRNA) to function and further enables multiplex genome targeting and homology-directed repair (HDR) based precise genome editing in a wide variety of organisms. Here, we report a novel and interesting strategy by using the Drosha-mediated sgRNA-shRNA structure to direct Cas9 for multiplex genome targeting and precise genome editing. For multiplex genome targeting assay, we achieved more than 9% simultaneous mutant efficiency for 3 genomic loci among the puromycin-selected cell clones.

Generation of VDR Knock-Out Mice via Zygote Injection of CRISPR/Cas9 System.

CRISPR/Cas9 system has become a new versatile technology for genome engineering in various species. To achieve targeted modifications at the same site in both human and mice genomes by a CRISPR/Cas9 nuclease, we designed two target sites in conserved regions of vitamin D receptor (VDR) gene, which cover more than 17 kb of chromosome region depending on the species. We first validated the efficacy of single sgRNA mediated gene specific modifications were 36% and 31% in HEK293T cells.