利用吗啉代寡核苷酸技术下调早期斑马鱼胚胎lmna基因的初步研究

目的利用吗啉代寡核苷酸技术建立下调斑马鱼lmna基因的技术方法。方法在斑马鱼lmna基因序列中选择靶点,设计针对斑马鱼lmna基因的吗啉代寡核苷酸序列(lmna-MO),构建能特异指示lmna基因表达的lmna-EGFP-pCS~(2+)重组质粒,并通过显微注射方式将二者共注射入斑马鱼胚胎中,通过观察胚胎中绿色荧光表达量反应lmna基因表达量,并通过蛋白质印迹法检测胚胎中lamin蛋白表达量。结果蛋白质印迹法检测斑马鱼体内lamin蛋白的表达,分别有大小为69 KD和62 KD两种蛋白表达。设计并构建了lmna-MO和重组质粒lmnaEGFP-pCS~(2+),单独注射lmna-EGFP-pCS~(2+)质粒后观察到从6 hpf到96 hpf胚胎均有绿色荧光蛋白表达;二者共注射后观察到,与对照组相比,实验组从6 hpf至30 hpf胚胎中绿色荧光蛋白表达量均不同程度下降或消失;蛋白质印迹实验结果显示实验组胚胎内lamin蛋白表达量明显下降。表明已成功下调了斑马鱼胚胎lmna基因表达。结论可通过lmna-MO和重组质粒lmna-EGFP-pCS~(2+)共注射方法下调斑马鱼lmna基因表达,并通过绿色荧光蛋白表达量反映下调效果。该方法可为深入研究人核纤层病提供良好的动物模型。

Study on the lmna gene knockdown in zebrafish embryo with morpholino oligonucleotides

Objective Lamins are the major components of nuclear lamina underneath the inner nuclear membrane (INM). Lamins express in most cells and are involved in the whole process of growth, also play a major role in cell stability and embryonic development. Mutant in human LMNA gene may lead to a series of disorders, which are similar to progeria or other aging-associate syndrome. In this study, we report a new lmna knockdown animal model generated in our laboratory in order to provide a useful tool for studying laminopathies. Methods Two plasmids tagged to zebrafish lmna gene were designed based on morpholino oligonucleotides technology. Co-microinjected the plasmids into zebrafish embryos to knockdown lmna gene. Imagining and western blot detection were used to identify the mutants. Results Two different proteins, Lamin A/C, were expressed in the zebrafish embryos. Two plasmids lmna-MO and lmna-EGFP-pCS²⁺ were generated and co-microinjected into embryos. The results of imagining and western blot showed that the expression of lmna gene was downregulated in the zebrafish embryos. Conclusions Lamin A/C are expressed in zebrafish. lmna gene can be knocked down by the injection of lmna-MO and lmna-EGFP-pCS²⁺. This new animal model may be a powerful tool for study on laminopathies.