- 學位論文
果蠅atg5-RNAi過度表達是否影響克隆細胞產生？
Can atg5-RNAi overexpression affect clonal induction in drosophila?
摘要
Flip-out Gal4 system是一種遺傳工具，常用於過度表達感興趣的基因，以及用來追蹤細胞譜系。當幼蟲熱休克處理後，Flippase重組酶(Flp)被表達，誘導兩個FRT中間的標識基因和終止密碼子切除，下游的Gal4會持續地被表達，再活化下游的UAS-GFP（報導基因）及感興趣的基因過度表達。我們主要研究的對象是果蠅翅膀的假想盤，在假想盤中細胞分裂後會製造出flip-out clones。 在過去，我們實驗室發現過表達atg5-RNAi會產生大量的clone。我們所使用的atg5-RNAi果蠅基因型是yw,UAS-atg5-RNAi（簡稱atg5-RNAi）。在這篇論文我探討了過表達atg5-RNAi時ectopic clone形成的機制。首先，我發現將atg5-RNAi跳耀子移除，此時不會產生很多ectopic clone，因此增加clone頻率的原因，是因為在 X 染色體上插入了atg5-RNAi跳耀子。然而相較於atg5突變，atg5-RNAi下產生ectopic clone的頻率很高，而在atg5突變下mitotic clone產生的頻率較低，這個結果說明了atg5在ectopic clone形成中所扮演的角色，與我發現的一致，過表達atg5-RNAi不是clone產生頻率上升所必需的。 此外， atg5-RNAi insertion-mediated system促進clone產生，是需要熱休克的，但不需要Flp。總而言之，我們的數據表明在yw, UAS-atg5-RNAi果蠅中，存在類似Flp的活性。 有趣的是，我觀察到這種類似Flp的活性和hsflp很像，可以產生mitotic clone; 但與hsflp不同的是，它不能在唾液腺的多線染色體中產生ectopic clone。接著我使用meiotic mapping，將這種類似Flp的活性在X 染色體上定位，最終定位出這種類似Flp的活性在forked基因（X 染色體的15）之後的區域。最後，我發現在yw,UAS-atg5-RNAi果蠅，在18E中插入了一個heat-shock flippase 12的跳耀子（hsflp12），clone生成的高頻率是因為hsflp12造成的。得出結論，我們使用的yw,UAS-atg5-RNAi果蠅基因型其實是yw,hsflp12,UAS-atg5-RNAi。
並列摘要
The flip-out Gal4 system is a genetic tool commonly used for gene overexpression and lineage tracing. After heat-shock treatment of larvae, Flippase (Flp) recombinase is expressed to induce the excision of marker gene flanked by FRTs , which causes the constitutive expression of Gal4 to activate UAS-GFP reporter or other gene of interest in the flip-out clones after cell division in the wing disc. Our lab previously found that overexpression of atg5-RNAi generated a large number of clones. The atg5-RNAi stock we used is yw, UAS-atg5-RNAi (referred to as atg5-RNAi). Here, I explored the mechanism of ectopic clone formation when atg5-RNAi is overexpressed. First, I found that atg5-RNAi P-element revertant cannot generate many ectopic clones. Therefore, the increase in the frequency of clone induction is due to the insertion of the atg5-RNAi P-element on the X chromosome. However, the frequency to generate atg5 mutant mitotic clones is low when compared to the high frequency of atg5-RNAi ectopic clones. This data argued the role of atg5 in ectopic clone formation. Consistent with this, I found that atg5-RNAi overexpression is not required to promote clonal induction. Moreover, this atg5-RNAi insertion-mediated system required heat-shock but not Flp to promote clonal induction. Together, our data suggests the presence of Flp-like activity in yw,UAS-atg5-RNAi stock. Interestingly, I observed that this Flp-like activity, similar to hsflp, can generate mitotic clones but, unlike hsflp, it cannot generate ectopic clones in the polytene chromosomes of salivary gland. Next, I mapped this Flp-like activity on X chromosome using meiotic mapping. This Flp-like activity was mapped to a region distal to forked gene (15 of X chromosome). Finally, I found that there is a heat-shock flippase12 P-element (hsflp12) inserted in 18E of yw,UAS-atg5-RNAi stock and hsflp12 is responsible for the high frequency of clone induction. I conclude that the yw,UAS-atg5-RNAi stock we used was indeed yw,hsflp12,UAS-atg5-RNAi.