An Efficient Vector-Based CRISPR/Cas9 System in Zebrafish Cell Line.

The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has been widely applied in animals as an efficient genome editing tool. However, the technique is difficult to implement in fish cell lines partially due to the lack of efficient promoters to drive the expression of both sgRNA and the Cas9 protein within a single vector. In this study, it was indicated that the zebrafish U6 RNA polymerase III (ZFU6) promoter could efficiently induce tyrosinase (tyr) gene editing and lead to loss of retinal pigments when co-injection with Cas9 mRNA in zebrafish embryo. Furthermore, an optimized all-in-one vector for expression of the CRISPR/Cas9 system in the zebrafish fibroblast cell line (PAC2) was constructed by replacing the human U6 promoter with ZFU6 promoter, basing on the lentiCRISPRV2 system that widely applied in mammal cells. This new vector could successfully target the cellular communication network factor 2a (ctgfa) gene and demonstrated its function in the PAC2 cell. Notably, the vector could also be used to edit the endogenous EMX1 gene in the mammal 293 T cell line, implying its wide application potential. In conclusion, we established a new gene editing tool for zebrafish cell line, which could be a useful in vitro platform for high-throughput analyzing gene function in fish.

Characterization and role of connective tissue growth factor gene in collagen synthesis in swim bladder of chu's croaker (Nibea coibor).

Connective tissue growth factor (Ctgf) is a matricellular protein with diverse biological function. It is regarded as a central regulator of tissue fibrosis and collagen synthesis in mammals. However, its roles in fish remain elusive. Here, a ctgf gene was cloned (NcCtgf), characterized and functionally studied in the chu's croaker (Nibea coibor). NcCtgf encoded a protein containing 346 amino acids, 38 conserved cysteine residues, 4 functional domains and a signal peptide. NcCtgf shared highest identity (99.4 %) to the Larimichthys crocea Ctgf protein. Phylogenetic tree revealed that NcCtgf clustered with the teleost Ctgfa and Ctgf of higher vertebrates, instead of teleost Ctgfb. NcCtgf was expressed with higher level in gonad, spleen, gill and swimming bladder than other tissues, and was up-regulated in swim bladder synchronously with collagen I genes by hydroxyproline and TGF-ß1 treatment. NcCtgf knockdown/overexpression inhibited/promoted collagen synthesis in swim bladder cell, respectively. Notably, NcCtgf protein could be secreted to cell culture medium and up-regulated collagen I expression in swim bladder cell. These findings indicate NcCtgf plays vital roles in collagen synthesis in swim bladder of Nibea coibor, and provide basis for further understanding of ctgf evolution and exploring new approach for enhancing collagen deposition in fish products during aquaculture.

Serum proteomic analysis reveals possible mechanism underlying physiological hemostasis of swim bladder.

The hemostatic effect of isinglass (dried swim bladder) in traditional Chinese medicine is well known. But its mechanism of action remains unclear. Here, mice were gavaged with the dried swim bladder of the chu's croaker (Nibea coibor). The hemostatic effect of swim bladder was investigated, tandem mass tag (TMT)-based quantitative proteomics analysis was performed to screen differentially abundant proteins associated with hemostasis in mouse serum. Results indicated that isinglass significantly shorten bleeding time and promoted coagulation after acute trauma (cut out mouse tail). In total, 57 differentially expressed proteins were identified in the sera between control and swim bladder group, of which 31 were up-regulated and 26 were down-regulated in swim bladder group. KEGG pathway enrichment analysis further demonstrated that the Neutrophil extracellular trap formation pathway was significantly affected. Combined with RT-qPCR verification, our findings further suggested that five candidate proteins in the pathway may be involved in the onset of hemostasis after swim bladder gavage, indicating their important role during the hemostasis process promoting by swim bladder. SIGNIFICANCE: Serum proteomics after swim bladder gavage described differentially enriched proteins related to hemostasis, and enriched pathways were validated. This study revealed the possible pathways involved in the hemostatic effect of swim bladder, which may provide a new effector target for the development of new hemostatic drugs.

Physicochemical properties and potential beneficial effects of porphyran from Porphyra haitanensis on intestinal epithelial cells.

This study examined the beneficial effects of porphyran from Porphyra haitanensis (PHP) on intestinal epithelial cells, in terms of cell proliferation and migration and elucidated the potential molecular mechanism of action of PHP. Purified PHP is a homogenous polysaccharide with a molecular weight of 2.01 × 105 Da, intrinsic viscosity [η] of 463.76 mL/g, and radius of gyration of 61.2 nm. When the intestinal epithelial wound healing activity of PHP was investigated in vitro using the IEC-6 cell line (intestinal epithelial cells-6), it was found that PHP could promote cell migration and proliferation. PHP enhanced the protein expression of cell division control protein 42, paxillin, and focal adhesion kinase, which suggest that PHP might modulate the expression of these proteins to improve intestinal epithelial healing. Thus, this study indicated that PHP could serve as a potential source of functional food constituents for intestinal epithelial protection and restoration.