斑馬魚scratch基因的選殖，表現模式與功能分析

Cloning,expression pattern and functional assay of zebrafish scratch gene

簡寬穎(KUAN-YING JIAN)

指導教授 ： 許立松

中山醫學大學/醫學院/生化暨生物科技研究所/碩士(2013年)

摘要

Scratch基因是調控神經發育中重要的角色之一。在最近研究中發現，降低內生性Scratch基因於小鼠模式中，會影響中樞神經的分化[1]。在本實驗中想了解scratch基因對斑馬魚的發育情形的影響，在一開始的實驗中發現了在斑馬魚中scratch基因與人類和老鼠有高度的保守性。在PCR結果發現，scratch在受精36個小時後才有表現，而在48小時後明顯表現。而在成魚的組織中，主要是表現於腦部。透過全胚胎原位雜交實驗加以確定各時期表現位置。之後透過MO抑制scratch蛋白合成後發現會使胚胎在24小時以後在頭部呈現細胞凋亡的現象，在36小時眼睛部位亦有凋亡發生。同時，在抑制scratch後會使頭部與眼睛的外觀上呈現變小的趨勢，其中，在抑制scrt 1b時效果更顯著。而在後續的實驗中發現，抑制scratch導致的眼睛發育不正常，可以藉由共同注射scratch mRNA達到回復的效果。綜合以上的結果，在本實驗中顯示出scratch在神經發育中扮演著重要的角色，特別是針對頭部與眼睛。

關鍵字

scratch基因

並列摘要

Scratch gene play an important role in the regulation of neuron development. Recent study showed that downregulation of Scratch gene in mouse model impaired CNS differentiation. In present article, we want to examine whether knockdown of Scratch gene also cause the deficient in neurogenesis via zerbrafish model. According to the phylogenetic tree suggested zerbarfish scrt gene is high conserve with human and mouse counterpart. Based on PCR analysis and in situ hybridization assay we also founded that scrt gene is expressed from 36 hpf through 48 hpf, with relatively enrichment in adult zerbrafish brain. Knocking down of scrt gene by injection of anti-sense morpholino resulting in apoptosis in head at 24 hpf. In addition, there are massive apoptosis signal in eye region at 36 hpf. Moreover, disruption of scrt1b appeared more severe phenotype than scrt1a morphant. However, this effect could be restore via co-injection with scrt mRNA, implying the specificity of morpholino. Hence, these finding demonstrated a critical role of scrt gene not only in CNS but also in eye development.

並列關鍵字

zebrafish scratch gene

參考文獻

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