Snake Venom Gland Organoids.

Wnt dependency and Lgr5 expression define multiple mammalian epithelial stem cell types. Under defined growth factor conditions, such adult stem cells (ASCs) grow as 3D organoids that recapitulate essential features of the pertinent epithelium. Here, we establish long-term expanding venom gland organoids from several snake species. The newly assembled transcriptome of the Cape coral snake reveals that organoids express high levels of toxin transcripts. Single-cell RNA sequencing of both organoids and primary tissue identifies distinct venom-expressing cell types as well as proliferative cells expressing homologs of known mammalian stem cell markers. A hard-wired regional heterogeneity in the expression of individual venom components is maintained in organoid cultures. Harvested venom peptides reflect crude venom composition and display biological activity. This study extends organoid technology to reptilian tissues and describes an experimentally tractable model system representing the snake venom gland.

Development and evaluation of an efficient heterologous gene knock-in reporter system in Lactococcus lactis.

BACKGROUND: Lactococcus lactis is a food grade probiotics and widely used to express heterologous proteins. Generally, target genes are knocked into the L. lactis genome through double-crossover recombination to express heterologous proteins stably. However, creating marker-less heterologous genes knocked-in clones is laborious. In this study, an efficient heterologous gene knock-in reporter system was developed in L. lactis NZ9000. RESULTS: Our knock-in reporter system consists of a temperature-sensitive plasmid pJW and a recombinant L. lactis strain named NZB. The pJW contains homologous arms, and was constructed to knock-in heterologous genes at a fixed locus of NZ9000 genome. lacZ (ß-galactosidase) gene was knocked into the chromosome of NZ9000 as a counter-selective marker through the plasmid pJW to generate NZB. The engineered NZB strain formed blue colonies on X-Gal plate. The desired double-crossover mutants formed white colonies distinctive from the predominantly blue colonies (parental and plasmid-integrated clones) when the embedded lacZ was replaced with the target heterologous genes carried by pJW in NZB. CONCLUSIONS: By using the system, the heterologous gene knocked-in clones are screened by colony phenotype change rather than by checking colonies individually. Our new knock-in reporter system provides an efficient method to create heterologous genes knocked-in clones.

Live imaging YAP signalling in mouse embryo development.

YAP protein is a critical regulator of mammalian embryonic development. By generating a near-infrared fusion YAP reporter mouse line, we have achieved high-resolution live imaging of YAP localization during mouse embryonic development. We have validated the reporter by demonstrating its predicted responses to blocking LATS kinase activity or blocking cell polarity. By time lapse imaging preimplantation embryos, we revealed a mitotic reset behaviour of YAP nuclear localization. We also demonstrated deep tissue live imaging in post-implantation embryos and revealed an intriguing nuclear YAP pattern in migrating cells. The YAP fusion reporter mice and imaging methods will open new opportunities for understanding dynamic YAP signalling in vivo in many different situations.

Characterization of a bicistronic knock-in reporter mouse model for investigating the role of CABLES2 in vivo.

Two members of the CDK5 and ABL enzyme substrate (CABLES) family, CABLES1 and CABLES2, share a highly homologous C-terminus. They interact and associate with cyclin-dependent kinase 3 (CDK3), CDK5, and c-ABL. CABLES1 mediates tumor suppression, regulates cell proliferation, and prevents protein degradation. Although Cables2 is ubiquitously expressed in adult mouse tissues at RNA level, the role of CABLES2 in vivo remains unknown. Here, we generated bicistronic Cables2 knock-in reporter mice that expressed CABLES2 tagged with 3×FLAG and 2A-mediated fluorescent reporter tdTomato. Cables2-3×FLAG-2A-tdTomato (Cables2Tom) mice confirmed the expression of Cables2 in various mouse tissues. Interestingly, high intensity of tdTomato fluorescence was observed in the brain, testis and ovary, especially in the corpus luteum. Furthermore, immunoprecipitation analysis using the brain and testis in Cables2Tom/Tom revealed interaction of CABLES2 with CDK5. Collectively, our new Cables2 knock-in reporter model will enable the comprehensive analysis of in vivo CABLES2 function.

Induction of IL-22-Producing CD4+ T Cells by Segmented Filamentous Bacteria Independent of Classical Th17 Cells.

The intestinal microbiota modulates IL-22 production in the intestine, including the induction of IL-22-producing CD4+ T helper cells. Which specific bacteria are responsible for the induction of these cells is less well understood. Here, we demonstrate through the use of novel gnotobiotic knock-in reporter mice that segmented filamentous bacteria (SFB), which are known for their ability to induce Th17 cells, also induce distinct IL-17A negative CD4+ T cell populations in the intestine. A subset of these cells instead produces IL-22 upon restimulation ex vivo and also during enteric infections. Furthermore, they produce a distinct set of cytokines compared to Th17 cells including the differential expression of IL-17F and IFN-γ. Importantly, genetic models demonstrate that these cells, presumably Th22 cells, develop independently of intestinal Th17 cells. Together, our data identifies that besides Th17, SFB also induces CD4+ T cell populations, which serve as immediate source of IL-22 during intestinal inflammation.

Light up the embryos: knock-in reporter generation for mouse developmental biology.

Developmental biology seeks to understand the sophisticated regulated process through which a single cell - a fertilized egg - generates a highly organized organism. The most effective way to reveal the nature of these processes is to follow single cells and cell lineages in real-time. Recent advances in imaging equipment, fluorescent tags and computational tools have made long term multi-color imaging of cells and embryos possible. However, there is still one major challenging for achieving live imaging of mammalian embryos- the generation of embryos carrying reporters that recapitulate the endogenous expression pattern of marker genes. Recent developments of genome editing technology played important roles in enabling efficient generation of reporter mouse models. This mini review discusses recent developments of technologies for efficiently generate knock-in reporter mice and the application of these models in live imaging development. With these developments, we are starting to realize the long-sought promises of realtime analysis of mammalian development.

CRISPR/Cas9-Mediated Zebrafish Knock-in as a Novel Strategy to Study Midbrain-Hindbrain Boundary Development.

The midbrain-hindbrain boundary (MHB) acts as an organizer and controls the fate of neighboring cells to develop into either mesencephalic (midbrain) or metencephalic (hindbrain) cells by secreting signaling molecules like Wnt1 and Fgf8. The zebrafish is an excellent vertebrate model for studying MHB development due to the ease of gene manipulation and the possibility of following cellular dynamics and morphogenetic processes using live imaging. Currently, only very few reporter and/or Cre-driver lines are available to study gene expression at the MHB, hampering the understanding of MHB development, and traditional transgenic technologies using promoter/enhancer fragments or bacterial artificial chromosome (BAC)-mediated transgenesis often do not faithfully recapitulate endogenous expression patterns. In contrast, CRISPR/Cas9-mediated genome editing technology now provides a great opportunity to efficiently knock-in or knock-out genes. We have generated four CRISPR/Cas9-based knock-in fluorescent reporter lines for two crucial genes involved in MHB development, namely otx2 and pax2a. The coding sequences of the reporters were knocked-in upstream of the corresponding ATG and are, thus, under the control of the endogenous promoter/enhancer elements. Interestingly, this strategy does not disturb endogenous gene expression. Using the fast maturing fluorescent protein reporter, Venus, enabled us to follow MHB development using cell tracking and live imaging. In addition, we show that these reporter lines label various neuronal and glial cell types in the adult zebrafish brain, making them highly suitable for investigating embryonic and adult midbrain, hindbrain, and MHB development.