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

发布时间：2019-02-28 19:20

【摘要】：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。
[Abstract]:Objective to establish a method for down-regulating lmna gene of zebrafish by morpholine oligonucleotide technique. Methods the morpholine oligonucleotide sequence (lmna-MO) targeting the zebrafish lmna gene was designed to construct the recombinant plasmid lmna-EGFP-pCS~ (2), which could specifically indicate the expression of the lmna gene in zebrafish lmna gene sequence, and designed the morpholine oligonucleotide sequence (lmna-MO) targeting the zebrafish lmna gene. Both of them were co-injected into zebrafish embryos by microinjection. The expression of lmna gene was observed by observing the green fluorescence expression in embryos, and the expression of lamin protein in embryos was detected by Western blotting. Results the expression of lamin protein in zebrafish was detected by Western blot. The expression of two proteins was 69 KD and 62 KD, respectively. Lmna-MO and recombinant plasmid lmnaEGFP-pCS~ (2) were designed and constructed. Green fluorescent protein expression was observed from 6 hpf to 96 hpf embryos after single injection of lmna-EGFP-pCS~ (2) plasmid. Compared with the control group, the expression of green fluorescent protein (GFP) decreased or disappeared in 6 hpf to 30 hpf embryos in the experimental group, and the expression of lamin protein in the embryos of the experimental group was significantly decreased as compared with the control group. The results showed that the expression of lmna gene in zebrafish embryos had been down-regulated successfully. Conclusion the expression of lmna gene in zebrafish can be down-regulated by co-injection of lmna-MO and recombinant plasmid lmna-EGFP-pCS~ (2), and the down-regulation effect can be reflected by the expression of green fluorescent protein. This method can provide a good animal model for further study of human nuclear lamellar disease.
【作者单位】：贵州医科大学免疫学教研室;贵州医科大学组织工程与干细胞实验中心;贵州医科大学附属医院儿科学教研室;贵州医科大学实验动物中心;
【基金】：国家自然科学基金项目资助(NO.31260284)
【分类号】：R-332

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