摘要
目的观察hand2在斑马鱼胚胎内的表达情况,验证显微注射吗啡啉修饰的反义寡核苷酸(Morpholi-no oligonucleotides,MO)使hand2表达下调的有效性。观察hand2表达下调后斑马鱼胚胎发育的异常表型,初步探讨hand2在斑马鱼胚胎发育中的作用,为利用斑马鱼开展hand2的功能研究提供线索。方法采用胚胎整体原位杂交的方法观察hand2在斑马鱼胚胎内的表达情况。利用向斑马鱼受精卵显微注射MO的方法使hand2表达下调,并进行hand2表达下调的效率验证。将斑马鱼单细胞受精卵分为4组进行显微注射,即对照MO(Con MO)注射组、hand2MO注射组、hand2-GFP mRNA注射组和hand2-GFP mRNA+hand2MO注射组。在荧光显微镜下观察hand2-GFP mRNA注射组和hand2-GFP mRNA+hand2MO注射组的荧光表达情况以验证hand2表达下调的效率。在显微镜下观察hand2MO注射组斑马鱼胚胎的各器官发育情况并进行评定。结果hand2在斑马鱼胚胎的侧板中胚层、咽弓、心脏和鳍有较强表达。hand2-GFP mRNA注射组在8hpf(hours post fertilization)时胚胎有较强的荧光表达,而hand2-GFP mRNA+hand2MO注射组胚胎几乎无荧光表达。与Con MO注射组相比,hand2MO注射组斑马鱼胚胎的心脏、血管、血细胞、鳍以及体节有明显的发育异常。结论向斑马鱼受精卵显微注射hand2MO的方法可有效的使hand2表达下调。hand2在斑马鱼胚胎的心脏、血管、血细胞、鳍以及体节的发育过程中有重要作用。
Objective To detect the hand2 expressing pattern in zebrafish embryos, and to demonstrate that microinjecting morpholino oligonucleotides （MO） would be an effective method to block the hand2 expression and observe the abnormal phenotypes induced by hand2 knock-down. The present study is expected to provide the clue for the functional study on hand2. Methods Whole-mount in si- tu hybridization was performed to detect the expression pattern of hand2. Zebrafish fertilized eggs were divided into four groups （ Con MO microinjected group, hand2 MO microinjected group, hand2-GFP mRNA microinjected group and hand2-GFP mRNA ＋ hand2 MO microinjected group）. The GFP ex- pression of hand2-GFP mRNA microinjected group and hand2-GFP mRNA ＋ hand2 MO microinjected group were detected by fluorescent microscope to evaluate the efficiency of hand2 knock-down. The de- velopment of Con MO microinjected embryos and hand2 MO microinjected embryos were observed un- der microscope. Results The hand2 expression were observed in lateral mesoderm, pharyngeal ar- ches, heart and fins. Strong GFP expression was observed in hand2-GFP mRNA microinjected group at 8 hpf（hours post fertilization）. Compared with hand2-GFP mRNA microinjected group, the expres- sion of GFP in hand2-GFP mRNA ＋ hand2 MO microinjected group was obviously reduced. Compared with Con MO group, the hand2 MO injected group showed apparent malformation at the heart, the vessel, the blood cells, the fin and the somite. Conclusions Microinjecting hand2 MO can effectively knock-down hand2, hand2 plays an important role in the development of the heart, the vessel, the blood cell, the fin and the somite in zebrafish.
出处
《复旦学报（医学版）》
CAS
CSCD
北大核心
2008年第3期325-329,共5页
Fudan University Journal of Medical Sciences
基金
上海市科委专项课题“先心病产前诊断技术的多中心研究和评价”项目(054119512)
