NF-κB信号对细胞存活有广泛的影响,组织生长,和扩散活动。它控制许多参与炎症的基因,因此是许多炎症性疾病的关键参与者。NF-κB激活剂的升高与死亡率升高有关。尤其是癌症和心血管疾病。斑马鱼已成为转化研究中全生物体体内建模的重要模型。在脊椎动物中,由于皮肤和真皮下结构的正常混浊,体内空间分辨率受到限制。对于体内成像,通过化学抑制阻止色素沉着的皮肤透明度是必需的,保持这种透明度是至关重要的。卡斯珀(roy-/-,斑马鱼的nacre-/-)突变体在其整个生命周期中都保持这种透明度,并且是体内干细胞分析和成像的灵敏度和分辨率的理想组合。我们开发了NF-kB:GFP/Casper透明转基因斑马鱼细胞表型,以研究体内炎症过程。我们概述了通过Casper和NF-kB转基因成年鱼的杂交育种产生斑马鱼透明转基因NF-kB/Casper菌株的实验设置,并以杂合后代的形式产生了F01。将转基因F01后代进一步近交以产生F1至F4代的杂合后代。此外,继续成功开发纯合菌株Tg(6xNF-kB:EGFP);Casper(roy-/-,nacre-/-)在F05代。这种F05代的新菌株在Tg(6xNF-kB:EGFP)的转基因透明后代中显示出100%的纯合性;Casper(roy-/-,nacre-/-)。通过产生纯合子代的F06代证实了该菌株,并在F07代中再次验证和验证了其同质性。新开发的NF-kB报告系的新型透明转基因菌株已被创造为“Tg(6xNF-kB:EGFP);Casper(roy-/-,nacre-/-)gmc1\"。我们已经建立了一种新产生的透明转基因斑马鱼表型,用于延时体内共聚焦显微镜,以研究细胞水平随时间变化的细胞表型和病理。这将允许量化NF-kB功能活性随时间的变化,并允许对照和心脏肿瘤学实验治疗剂的比较。我们验证了新开发的Tg(6xNF-kB:EGFP);Casper(roy-/-,nacre-/-)gmc1纯合斑马鱼菌株通过研究细菌脂多糖(LPS)暴露的炎症反应,容忍度,以及潜在的新型候选药物对LPS诱导的炎症的抑制作用。结果通过确定用于实验疗法的命中和前导药物候选物,建立了新开发菌株的独特应用。
NF-κB signaling has broad effects on cell survival, tissue growth, and proliferation activities. It controls many genes that are involved in inflammation and thus is a key player in many inflammatory diseases. The elevation of NF-κB activators is associated with elevated mortality, especially in cancer and cardiovascular diseases. The zebrafish has emerged as an important model for whole-organism in vivo modeling in translational research. In vertebrates, in-vivo spatial resolution is limited due to normal opacification of skin and subdermal structure. For in vivo imaging, skin transparency by blocking the pigmentation via chemical inhibition is required and the maintenance of this transparency is vital. The Casper(roy-/-, nacre-/-) mutant of zebrafish maintains this transparency throughout its life and serves as an ideal combination of sensitivity and resolution for in vivo stem cell analyses and imaging. We developed an NF-kB:GFP/Casper transparent
transgenic zebrafish cellular phenotype to
study inflammatory processes in vivo. We outline the experimental setup to generate a transparent
transgenic NF-kB/Casper strain of zebrafish through the cross-breeding of Casper and NF-kB transgenic adult fish and have generated F01 in the form of heterozygous progeny. The transgenic F01 progeny was further inbred to generate heterozygous progenies from F1 to F4 generations. Furthermore, it continued to successfully develop the homozygous strain Tg(6xNF-kB:EGFP); Casper(roy-/-, nacre-/-) in the F05 generation. This novel strain of F05 generation showed 100% homozygosity in the
transgenic transparent progeny of Tg(6xNF-kB:EGFP); Casper(roy-/-, nacre-/-). The strain has been confirmed by generating the F06 generation of homozygous progeny and again verified and validated for its homogeneity in the F07 generation. The newly developed novel transparent
transgenic strain of the NF-kB reporter line has been coined as \"Tg(6xNF-kB:EGFP); Casper(roy-/-, nacre-/-)gmc1\". We have established a newly generated phenotype of transparent transgenic zebrafish for time-lapse in vivo confocal microscopy to
study the cellular phenotype and pathologies at the cellular level over time. This will allow for quantifying the changes in the NF-kB functional activities over time and allow the comparison of control and cardiac-oncology experimental therapeutics. We validated the newly developed Tg(6xNF-kB:EGFP); Casper(roy-/-, nacre-/-)gmc1 homozygous strain of zebrafish by studying the inflammatory response to bacterial lipopolysaccharide (LPS) exposure, tolerance, and the inhibitory role of a potential novel drug candidate against LPS-induced inflammation. The results establish the unique application of newly developed strains by identifying hit and lead drug candidates for experimental therapeutics.