. Y. Chiang C, Ligunas GD, Chin WC, Ni CW. Efficient Nonviral Stable Transgenesis Mediated by Retroviral Integrase. Mol Ther Methods Clin Dev. 2020;17:1061-1070.
Efficient transgene delivery is critical for genetic manipulation and therapeutic intervention of target cells. Two well-characterized integrative systems have been described that rely on viral and nonviral vectors. However, use of viral vectors for gene therapy has been associated with several safety concerns. Here, we report a virus-free method for stable transgenesis based on the reaction of retroviral integrase. We constructed a gateway cloning compatible vector containing two truncated long terminal repeat (LTR) sequences (dLTR) that flank the transgene cassette. Notably, 5’-ACTG-3’ and blunt-end restriction cutting sites were also embedded at the end of dLTR to be recognized by HIV-1 integrase. When performing coinjection of transgene cassette and integrase mRNA into zebrafish embryos at one cell stage, there were 50% to 55% of injected embryos expressing a marker gene in a desired pattern. When applying our method in mammalian cells, there were 42% of cultured human epithelial cell lines showing stable integration. These results demonstrated that our method can successfully insert an exogenous gene into the host genome with highly efficient integration. Importantly, this system operates without most of the viral components while retaining effective stable transgenesis. We anticipate this method will provide a convenient, safe, and highly efficient way for applications in transgenesis and gene therapy.
Shiu R-F, Vazquez CI, Chiang C-Y, Chiu M-H, Chen C-S, Ni C-W, Gong G-C, Quigg A, Santschi PH, Chin W-C. Nano- and microplastics trigger secretion of protein-rich extracellular polymeric substances from phytoplankton. Sci Total Environ. 2020;748:141469.
The substantial increase in plastic pollution in marine ecosystems raises concerns about its adverse impacts on the microbial community. Microorganisms (bacteria, phytoplankton) are important producers of exopolymeric substances (EPS), which govern the processes of marine organic aggregate formation, microbial colonization, and pollutant mobility. Until now, the effects of nano- and micro-plastics on characteristics of EPS composition have received little attention. This study investigated EPS secretion by four phytoplankton species following exposure to various concentrations of polystyrene nano- and microplastics (55 nm nanoparticles; 1 and 6 μm microparticles). The 55 nm nanoparticles induced less growth/survival (determined on a DNA basis) and produced EPS with higher protein-to-carbohydrate (P/C) ratios than the exposure to microplastic particles. The amount of DNA from the four marine phytoplankton showed a higher negative linear correlation with increasing P/C ratios, especially in response to nanoplastic exposure. These results provide evidence that marine phytoplankton are quite sensitive to smaller-sized plastics and actively modify their EPS chemical composition to cope with the stress from pollution. Furthermore, the release of protein-rich EPS was found to facilitate aggregate formation and surface modification of plastic particles, thereby affecting their fate and colonization. Overall, this work offers new insights into the potential harm of different-sized plastic particles and a better understanding of the responding mechanism of marine phytoplankton for plastic pollution. The data also provide needed information about the fate of marine plastics and biogenic aggregation and scavenging processes.


Butko E, Distel M, Pouget C, Weijts B, Kobayashi I, Ng K, Mosimann C, Poulain FE, McPherson A, Ni CW, et al. Gata2b is a restricted early regulator of hemogenic endothelium in the zebrafish embryo. Development. 2015;142:1050-61.
The adult blood system is established by hematopoietic stem cells (HSCs), which arise during development from an endothelial-to-hematopoietic transition of cells comprising the floor of the dorsal aorta. Expression of aortic runx1 has served as an early marker of HSC commitment in the zebrafish embryo, but recent studies have suggested that HSC specification begins during the convergence of posterior lateral plate mesoderm (PLM), well before aorta formation and runx1 transcription. Further understanding of the earliest stages of HSC specification necessitates an earlier marker of hemogenic endothelium. Studies in mice have suggested that GATA2 might function at early stages within hemogenic endothelium. Two orthologs of Gata2 exist in zebrafish: gata2a and gata2b. Here, we report that gata2b expression initiates during the convergence of PLM, becoming restricted to emerging HSCs. We observe Notch-dependent gata2b expression within the hemogenic subcompartment of the dorsal aorta that is in turn required to initiate runx1 expression. Our results indicate that Gata2b functions within hemogenic endothelium from an early stage, whereas Gata2a functions more broadly throughout the vascular system.
Kok FO, Shin M, Ni CW, Gupta A, Grosse AS, van Impel A, Kirchmaier BC, Peterson-Maduro J, Kourkoulis G, Male I, et al. Reverse genetic screening reveals poor correlation between morpholino-induced and mutant phenotypes in zebrafish. Dev Cell. 2015;32:97-108.
The widespread availability of programmable site-specific nucleases now enables targeted gene disruption in the zebrafish. In this study, we applied site-specific nucleases to generate zebrafish lines bearing individual mutations in more than 20 genes. We found that mutations in only a small proportion of genes caused defects in embryogenesis. Moreover, mutants for ten different genes failed to recapitulate published Morpholino-induced phenotypes (morphants). The absence of phenotypes in mutant embryos was not likely due to maternal effects or failure to eliminate gene function. Consistently, a comparison of published morphant defects with the Sanger Zebrafish Mutation Project revealed that approximately 80% of morphant phenotypes were not observed in mutant embryos, similar to our mutant collection. Based on these results, we suggest that mutant phenotypes become the standard metric to define gene function in zebrafish, after which Morpholinos that recapitulate respective phenotypes could be reliably applied for ancillary analyses.


Ni CW, Kumar S, Ankeny CJ, Jo H. Development of immortalized mouse aortic endothelial cell lines. Vasc Cell. 2014;6:7.
BACKGROUND: The understanding of endothelial cell biology has been facilitated by the availability of primary endothelial cell cultures from a variety of sites and species; however, the isolation and maintenance of primary mouse aortic endothelial cells (MAECs) remain a formidable challenge. Culturing MAECs is difficult as they are prone to phenotypic drift during culture. Therefore, there is a need to have a dependable in vitro culture system, wherein the primary endothelial cells retain their properties and phenotypes. METHODS: Here, we developed an effective method to prepare immortalized MAEC (iMAEC) lines. Primary MAECs, initially isolated from aortic explants, were immortalized using a retrovirus expressing polyoma middle T-antigen. Immortalized cells were then incubated with DiI-acetylated-low density lipoprotein and sorted via flow cytometry to isolate iMAECs. RESULTS: iMAECs expressed common markers of endothelial cells, including PECAM1, eNOS, VE-cadherin, and von Willebrand Factor. iMAECs aligned in the direction of imposed laminar shear and retained the ability to form tubes. Using this method, we have generated iMAEC lines from wild-type and various genetically modified mice such as p47phox-/-, eNOS-/-, and caveolin-1-/-. CONCLUSION: In summary, generation of iMAEC lines from various genetically modified mouse lines provides an invaluable tool to study vascular biology and pathophysiology.
Atherosclerosis preferentially occurs in arterial regions exposed to disturbed blood flow (d-flow), in part, due to alterations in gene expression in the endothelium. While numerous in vitro studies have shown how anti-atherogenic flow and pro-atherogenic flow differently regulate gene expression of cultured endothelial cells, similar in vivo studies have been scarce. Recently, we developed a mouse model of atherosclerosis that rapidly develops robust atherosclerosis by partially ligating the left carotid artery (LCA) branches, while using the contralateral right carotid (RCA) as control. We also developed a novel method to collect endothelial-enriched RNAs from the carotids of these animals, which enabled us to perform genome-wide expression analyses of mRNAs and miRNAs in the arterial endothelium exposed to either d-flow or s-flow. These microarray results were used to identify novel mechanosensitive genes such as DNA methyltransferase-1 and miR-712 that play key roles in atherosclerosis. Here, we report these endothelial mRNA and miRNA expression profiles with in-depth information on experimental procedures along with an example of usage of these data.


Son DJ, Kumar S, Takabe W, Kim CW, Ni C-W, Alberts-Grill N, Jang I-H, Kim S, Kim W, Kang SW, et al. The atypical mechanosensitive microRNA-712 derived from pre-ribosomal RNA induces endothelial inflammation and atherosclerosis. Nat Commun. 2013;4:3000.
MicroRNAs (miRNAs) regulate cardiovascular biology and disease, but the role of flow-sensitive microRNAs in atherosclerosis is still unclear. Here we identify miRNA-712 (miR-712) as a mechanosensitive miRNA upregulated by disturbed flow (d-flow) in endothelial cells, in vitro and in vivo. We also show that miR-712 is derived from an unexpected source, pre-ribosomal RNA, in an exoribonuclease-dependent but DiGeorge syndrome critical region 8 (DGCR8)-independent manner, suggesting that it is an atypical miRNA. Mechanistically, d-flow-induced miR-712 downregulates tissue inhibitor of metalloproteinase 3 (TIMP3) expression, which in turn activates the downstream matrix metalloproteinases (MMPs) and a disintegrin and metalloproteases (ADAMs) and stimulate pro-atherogenic responses, endothelial inflammation and permeability. Furthermore, silencing miR-712 by anti-miR-712 rescues TIMP3 expression and prevents atherosclerosis in murine models of atherosclerosis. Finally, we report that human miR-205 shares the same ’seed sequence’ as murine-specific miR-712 and also targets TIMP3 in a flow-dependent manner. Targeting these mechanosensitive ’athero-miRs’ may provide a new treatment paradigm in atherosclerosis.


Fazeli G, Stopper H, Schinzel R, Ni CW, Jo H, Schupp N. Angiotensin II induces DNA damage via AT1 receptor and NADPH oxidase isoform Nox4. Mutagenesis. 2012;27:673-81.
Epidemiological studies revealed increased renal cancer incidences and higher cancer mortalities in hypertensive individuals. Activation of the renin-angiotensin-aldosterone system leads to the formation of reactive oxygen species (ROS). In vitro, in renal cells, and ex vivo, in the isolated perfused mouse kidney, we could show DNA-damaging potential of angiotensin II (Ang II). Here, the pathway involved in the genotoxicity of Ang II was investigated. In kidney cell lines with properties of proximal tubulus cells, an activation of NADPH oxidase and the production of ROS, resulting in the formation of DNA strand breaks and micronuclei induction, was observed. This DNA damage was mediated by the Ang II type 1 receptor (AT1R), together with the G protein G ( alpha-q/11 ) . Subsequently, phospholipase C (PLC) was activated and intracellular calcium increased. Both calcium stores of the endoplasmic reticulum and extracellular calcium were involved in the genotoxicity of Ang II. Downstream, a role for protein kinase C (PKC) could be detected, because its inhibition hindered Ang II from damaging the cells. Although PKC was activated, no involvement of its known target, the NADPH oxidase isoform containing the Nox2 subunit, could be found, as tested by small-interfering RNA down-regulation. Responsible for the DNA-damaging activity of Ang II was the NADPH oxidase isoform containing the Nox4 subunit. In summary, in kidney cells the DNA-damaging activity of Ang II depends on an AT1R-mediated activation of NADPH oxidase via PLC, PKC and calcium signalling, with the NADPH subunit Nox4 playing a crucial role.


Rezvan A, Ni CW, Alberts-Grill N, Jo H. Animal, in vitro, and ex vivo models of flow-dependent atherosclerosis: role of oxidative stress. Antioxid Redox Signal. 2011;15:1433-48.
Atherosclerosis is an inflammatory disease preferentially occurring in curved or branched arterial regions, whereas straight parts of the arteries are protected, suggesting a close relationship between flow and atherosclerosis. However, evidence directly linking disturbed flow to atherogenesis is just emerging, thanks to the recent development of suitable animal models. In this article, we review the status of various animal, in vitro, and ex vivo models that have been used to study flow-dependent vascular biology and atherosclerosis. For animal models, naturally flow-disturbed regions such as branched or curved arterial regions as well as surgically created models, including arterio-venous fistulas, vascular grafts, perivascular cuffs, and complete, incomplete, or partial ligation of arteries, are used. Although in vivo models provide the environment needed to mimic the complex pathophysiological processes, in vitro models provide simple conditions that allow the study of isolated factors. Typical in vitro models use cultured endothelial cells exposed to various flow conditions, using devices such as cone-and-plate and parallel-plate chambers. Ex vivo models using isolated vessels have been used to bridge the gap between complex in vivo models and simple in vitro systems. Here, we review these flow models in the context of the role of oxidative stress in flow-dependent inflammation, a critical proatherogenic step, and atherosclerosis.