Flp-out gal4 system 被廣泛的使用在果蠅中，選擇性的調控特定基因來形成etopic clone。熱休克會啟動Flp-out gal4 system，進而產生flippase，使marker gene被剪出，表達出下游的Gal4，Gal4會結合到染色體上的UAS序列，表達出特定的基因，並同時表達綠螢光蛋白，方便觀察幼蟲的翅膀成蟲盤上，etopic clone的位置。到目前為止，我們已經發現(1)抑制Atg5(2)過表達Toll pathway中的cactus，可以增加clone誘導的頻率，這個機制與細胞分裂和細胞凋亡沒有相關。且atg5-RNAi是誘導clone的負調控物，cactus則是正調控物。另外，我們利用RNA-seq發現，抑制了atg5的細胞，有許多核醣體蛋白的基因表現量提高；且atg5被抑制的細胞，與cactus過表達的細胞，有相同的基因表現量下降。目前，我們正在測試核醣體蛋白基因表現量是否是clone誘導增加的原因。

The FLP-out Gal4 system is widely used to generate ectopic clones of cells in Drosophila. A short pulse of heat shock induces FLP-out event that allows the clonal expression of Gal4 and the co-expression of UAS-GFP and UAS-gene-of interest (GOI). Upon specific GOI overexpression, we quantified the number of GFP-positive clones generated at 24 h after heat shock in the wing discs, we can identify the genes positively or negatively regulate clonal induction. Thus far, we have identified that (1) knockdown autophagy related 5 (atg5) and (2) inhibition of Toll pathway by cactus overexpression greatly increase the frequency of clonal induction, with a mechanism independent of cell division and cell death. Thus, atg5 is a negative regulator whereas cactus is a positive regulator of clonal induction. Moreover, through RNA-seq of atg5 knockdown cells, we have identified that many ribosomal protein genes are up-regulated. Currently, we are testing whether increased translation might contribute to increased clonal induction.

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