CRISPR-mediated knock-in (KI) technology opens a new era of fluorescent-protein labeling in zebrafish, a preferred model organism for in vivo imaging. We described here an optimized zebrafish gene-tagging strategy, which enables easy and high-efficiency KI, ensures high odds of obtaining seamless KI germlines and is suitable for wide applications. Plasmid donors for 3'-labeling were optimized by shortening the microhomologous arms and by reducing the number and reversing the sequence of the consensus Cas9/sgRNA binding sites. To allow for scar-less KI across the genome, linearized dsDNA donors with 5'-chemical modifications were generated and successfully incorporated into our method. To refine the germline screen workflow and expedite the screen process, we combined fluorescence enrichment and caudal-fin junction-PCR. Furthermore, to trace proteins expressed at a low abundance, we developed a fluorescent signal amplifier using the transcriptional activation strategy. Together, our strategies enable efficient gene-tagging and sensitive expression detection for almost every gene in zebrafish.
CRISPR-Cas Systems , Zebrafish , Animals , Zebrafish/genetics , RNA, Guide, CRISPR-Cas Systems , Genome , Fluorescence
Superoxide dismutase (SOD) is an enzyme found in most food sources, might be a candidate to reduce oxidative damage to intestinal barrier, thereby ameliorating the vicious circle between hyperglycemia and the oxidative damage. Here we report the oral administration of SOD, liposome-embedded SOD (L-SOD), and SOD hydrolysate to type 2 diabetic model rats to confirm this hypothesis. Oxidative damage severity in model rat intestine was indicated by malondialdehyde level, GSSG/GSH ratio, and antioxidant enzyme activity. The damage was significantly repaired by L-SOD. Furthermore, blood glucose and related indexes correlated well not only with oxidative damage results but also with indexes indicating physical intestinal damage such as colon density, H&E staining, immunohistochemical analysis of the tight junction proteins occludin and ZO-1 in the colon, as well as lipopolysaccharide and related inflammatory cytokine levels. The order of the magnitude of the effects of these SOD preparations was L-SOD > SOD > SOD hydrolysate. These data indicate that orally administered SOD can exhibit glucose-lowering effect via targeting the intestine of diabetic rats and systemic lipopolysaccharide influx.
Recent studies have focused on capillary pruning in various organs and species. However, the way in which large-diameter vessels are pruned remains unclear. Here we show that pruning of the zebrafish caudal vein (CV) from ventral capillaries of the CV plexus in different transgenic embryos is driven by endothelial cell (EC) rearrangement, which involves EC nucleus migration, junction remodeling, and actin cytoskeleton remodeling. Further observation reveals a growing difference in blood flow velocity between the two vessels in CV pruning in zebrafish embryos. With this model, we identify the critical role of Kruppel-like factor 6a (klf6a) in CV pruning. Disruption of klf6a functioning impairs CV pruning in zebrafish. klf6a is required for EC nucleus migration, junction remodeling, and actin cytoskeleton dynamics in zebrafish embryos. Moreover, actin-related protein transgelin 2 (tagln2) is a direct downstream target of klf6a in CV pruning in zebrafish embryos. Together these results demonstrate that the klf6a-tagln2 axis regulates CV pruning by promoting EC rearrangement.
Blood Circulation/physiology , Microfilament Proteins/physiology , Muscle Proteins/physiology , Nerve Tissue Proteins/physiology , Zebrafish Proteins/physiology , Actin Cytoskeleton/metabolism , Actin Cytoskeleton/physiology , Animals , Animals, Genetically Modified , Capillaries/metabolism , Cell Movement , Endothelial Cells/metabolism , Endothelial Cells/physiology , Kruppel-Like Transcription Factors/genetics , Microfilament Proteins/genetics , Microfilament Proteins/metabolism , Morphogenesis , Muscle Proteins/genetics , Muscle Proteins/metabolism , Nerve Tissue Proteins/metabolism , Zebrafish/metabolism , Zebrafish/physiology , Zebrafish Proteins/metabolism
Cardiovascular stent restenosis remains a major challenge in interventional treatment of cardiovascular occlusive disease. Although the changes in arterial mechanical environment due to stent implantation are the main causes of the initiation of restenosis and thrombosis, the mechanisms that cause this initiation are still not fully understood. In this article, we reviewed the studies on the issue of stent-induced alterations in arterial mechanical environment and discussed their roles in stent restenosis and late thrombosis from three aspects: (i) the interaction of the stent with host blood vessel, involve the response of vascular wall, the mechanism of mechanical signal transmission, the process of re-endothelialization and late thrombosis; (ii) the changes of hemodynamics in the lumen of the vascular segment and (iii) the changes of mechanical microenvironment within the vascular segment wall due to stent implantation. This review has summarized and analyzed current work in order to better solve the two main problems after stent implantation, namely in stent restenosis and late thrombosis, meanwhile propose the deficiencies of current work for future reference.
