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Open Access Publications from the University of California

Scholarly Works (5 results)

  • Article
  • Peer Reviewed
UC Merced Previously Published Works (2014)

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.
    Cover page: Development of immortalized mouse aortic endothelial cell lines
    • Thesis
    • Peer Reviewed
    UC Merced Electronic Theses and Dissertations (2023)

    Lab-on-a-chip (LOC) technologies have revolutionized the fields of quantitative biology, tissue engineering, diagnostics, etc. However, the LOC implementations in the studies of small model organisms remain relatively low and are constrained in a small research community due to the complexity and high cost in the device development. For zebrafish research in particular, similar zebrafish-on-a-chip (ZOC) designs have been repeatedly used and some of the needs in zebrafish studies are far from fulfilled. The goal for this dissertation is to develop novel ZOCs and an overall zebrafish testing system to assist and accelerate zebrafish studies.In this dissertation, I reported the development of novel ZOC systems for automated zebrafish embryo positioning and flowthrough assays. The ZOCs are fabricated by a rapid and inexpensive 3D LCD stereolithography assisted prototyping method which is suitable for the labs with limited budgets. A simple computational fluid dynamic based analytical model was developed and validated to estimate ZOC’s embryo trapping potential as well as to guide future ZOC designs. Moreover, the operational conditions in the ZOC were evaluated and optimized via both simulations and experiments. To examine the ZOC’s feasibility in zebrafish studies, I showed the zebrafish embryo can be cultured in ZOC’s flowthrough environment and the embryonic development as well as embryo behavior can be affected by alternating the fluidic conditions. To fill the gap for the ZOC-based whole mount zebrafish immunohistochemistry, the whole mount zebrafish Caspase-3 antibody staining procedure was streamlined and accelerated using the ZOC. At last, the versatile ZOC design can be easily modified into a concentration gradient generator and was used for high resolution embryo caffeine dose-response screening. I foresee the developments, and applications of ZOCs will be accelerated in zebrafish research due to the growing needs for the laboratory automation as well as the increasing accessibility of LOC and 3D printing technologies. In addition, the continued input efforts from both academia and industry will facilitate the emergence of highly integrated zebrafish testing systems for fully automated zebrafish procedures.

      Cover page: Zebrafish-on-a-Chip: Milli Fluidic Systems for Zebrafish Studies
      • Article
      • Peer Reviewed
      UC Merced Previously Published Works (2023)
      Whole mount zebrafish antibody staining (ABS) is a common staining technique used to localize protein information in a zebrafish embryo or larva. Like most biological assays, the whole mount zebrafish ABS is still largely conducted manually through labor intensive and time-consuming steps which affect both consistency and throughput of the assay. In this work, we develop a milli fluidic device that can automatically trap and immobilize the fixed chorion-less zebrafish embryos for the whole mount ABS. With just a single loading step, the zebrafish embryos can be trapped by the milli fluidic device through a chaotic hydrodynamic trapping process. Moreover, a consistent body orientation (i.e., head point inward) for the trapped zebrafish embryos can be achieved without additional orientation adjustment device. Furthermore, we employed a consumer-grade SLA 3D printer assisted method for device prototyping which is ideal for labs with limited budgets. Notably, the milli fluidic device has enabled the optimization and successful implementation of whole mount zebrafish Caspase-3 ABS. We demonstrated our device can accelerate the overall procedure by reducing at least 50% of washing time in the standard well-plate-based manual procedure. Also, the consistency is improved, and manual steps are reduced using the milli fluidic device. This work fills the gap in the milli fluidic application for whole mount zebrafish immunohistochemistry. We hope the device can be accepted by the zebrafish community and be used for other types of whole mount zebrafish ABS procedures or expanded to more complicated in situ hybridization (ISH) procedure.
        Cover page: A multi-depth spiral milli fluidic device for whole mount zebrafish antibody staining.
        • Article
        • Peer Reviewed
        UC Merced Previously Published Works (2014)
        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.
          Cover page: Flow-dependent regulation of genome-wide mRNA and microRNA expression in endothelial cells in vivo
          • Article
          • Peer Reviewed
          UC Merced Previously Published Works (2020)
          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.
            Cover page: Efficient Nonviral Stable Transgenesis Mediated by Retroviral Integrase
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