Recent advances in genome editing using programmable nucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system, have facilitated reverse genetics in Xenopus tropicalis. To establish a practical workflow for analyzing genes of interest using CRISPR-Cas9, we examined various experimental procedures and conditions. We first compared the efficiency of gene disruption between Cas9 protein and mRNA injection by analyzing genotype and phenotype frequency, and toxicity. Injection of X. tropicalis embryos with Cas9 mRNA resulted in high gene-disrupting efficiency comparable with that produced by Cas9 protein injection. To exactly evaluate the somatic mutation rates of on-target sites, amplicon sequencing and restriction fragment length polymorphism analysis using a restriction enzyme or recombinant Cas9 were performed. Mutation rates of two target genes (slc45a2 and ltk) required for pigmentation were estimated to be over 90% by both methods in animals exhibiting severe phenotypes, suggesting that targeted somatic mutations were biallelically introduced in almost all somatic cells of founder animals. Using a heteroduplex mobility assay, we also showed that off-target mutations were induced at a low rate. Based on our results, we propose a CRISPR-Cas9-mediated gene disruption workflow for a rapid and efficient analysis of gene function using X. tropicalis founders.

Keratin genes belong to the intermediate filament superfamily and their expression is altered following morphological and physiological changes in vertebrate epithelial cells. Keratin genes are divided into two groups, type I and II, and are clustered on vertebrate genomes, including those of Xenopus species. Various keratin genes have been identified and characterized by their unique expression patterns throughout ontogeny in Xenopus laevis; however, compilation of previously reported and newly identified keratin genes in two Xenopus species is required for our further understanding of keratin gene evolution, not only in amphibians but also in all terrestrial vertebrates. In this study, 120 putative type I and II keratin genes in total were identified based on the genome data from two Xenopus species. We revealed that most of these genes are highly clustered on two homeologous chromosomes, XLA9_10 and XLA2 in X. laevis, and XTR10 and XTR2 in X. tropicalis, which are orthologous to those of human, showing conserved synteny among tetrapods. RNA-Seq data from various embryonic stages and adult tissues highlighted the unique expression profiles of orthologous and homeologous keratin genes in developmental stage- and tissue-specific manners. Moreover, we identified dozens of epidermal keratin proteins from the whole embryo, larval skin, tail, and adult skin using shotgun proteomics. In light of our results, we discuss the radiation, diversification, and unique expression of the clustered keratin genes, which are closely related to epidermal development and terrestrial adaptation during amphibian evolution, including Xenopus speciation.

-A large number of chemicals are routinely detected in aquatic environments, and these chemicals may adversely affect aquatic organisms. Accurate risk assessment requires understanding drugmetabolizing systems in aquatic organisms because metabolism of these chemicals is a critical determinant of chemical bioaccumulation and related toxicity. In this study, we evaluated mRNA expression levels of nuclear receptors and drug-metabolizing enzymes as well as cytochrome P450 (CYP) activities in pro-metamorphic tadpoles, froglets, and adult frogs to determine how drug-metabolizing systems are altered at different life stages. We found that drug-metabolizing systems in tadpoles were entirely immature, and therefore, tadpoles appeared to be more susceptible to chemicals compared with metamorphosed frogs. On the other hand, cyp1a mRNA expression and CYP1A-like activity were higher in tadpoles. We found that thyroid hormone (TH), which increases during metamorphosis, induced CYP1A-like activity. Because endogenous TH concentration is significantly increased during metamorphosis, endogenous TH would induce CYP1A-like activity in tadpoles.

BackgroundDespite recent impressive successes in chimeric antigen receptor (CAR)-T cell therapy, there are still considerable clinical challenges. To improve T cell persistence and antitumor effect, which are critical for clinical responses, various efforts have been made to optimize the CAR design such as the inclusion of a costimulatory domain(s). It is known that non-specific activation of CAR-T cells is greatly influenced by the CAR design, and excessive T cell activation leads exhaustion of T cells and depletion of naïve/memory subsets important for durable clinical responses. Thus, the CAR construct needs to be optimized so that transduced T cells persist and induce potent antigen-specific response with reduced non-specific activation. For optimal T cell activation and proliferation, three signals including TCR (signal 1), co-stimulatory (signal 2), and cytokine (signal 3) signals, are essential. The conventional second and third generation CARs containing CD3ζ and a co-stimulatory domain such as a signal domain of CD28 and 4-1BB can conduct signal 1 and 2, but not signal 3. Recently, we have developed a new generation JAK-STAT CAR composed of a truncated cytoplasmic domain of the IL-2 receptor β chain and STAT3/5 binding motifs, CD28 co-stimulatory domain, and CD3ζ domain. The novel anti-CD19 JAK-STAT CAR-T cells showed antigen-specific activation of the JAK-STAT signaling pathway, enhanced proliferation, and limited terminal differentiation in vitro compared to second generation 28ζ CAR or 4-1BBζ CAR-transduced T cells. Furthermore, the anti-CD19 JAK-STAT CAR-T cells demonstrated superior in vivo persistence and antitumor effect in mouse models.1 In addition, we previously showed that a hinge region and the composition of a single chain variable fragment (scFv) such as the order of VH and VL regions critically influence not only antigen-dependent activation but also undesired antigen-independent activation known as tonic signaling.2MethodsIn this study, we have optimized the scFv design in 28ζ CAR and JAK-STAT CAR constructs to show superior antigen-specific activation and reduced tonic signaling for several targets (CD19, CD20, Mesothelin, and GD2). And we have evaluated the feature of JAK-STAT CAR-T cells compared to 28ζ CAR-T cells.ResultsJAK-STAT CAR-T cells showed superior antigen-specific proliferation with less differentiated status, whereas 28ζ CAR-T cells showed antigen-independent proliferation and displayed higher exhaustion marker expression after repetitive stimulations.ConclusionsThese results suggest that our JAK-STAT-CARs with enhanced antigen-specific response with minimized tonic signaling targeting various antigens has the potential to demonstrate improved clinical efficacy.ReferencesKagoya Y, et al. A novel chimeric antigen receptor containing a JAK–STAT signaling domain mediates superior antitumor effects. Nat Med 2018;24:p352–359.Okamoto S, et al. Detail analysis of non-specific activation of CD19 CAR-T cells caused by CAR design. ASGCT ( 2015)