FLP-out Gal4 system是果蠅實驗中被廣泛使用的genetic tool，它能夠誘導果蠅的細胞表達GFP和特定的基因並形成ectopic clones。在經過短時間的加熱後我們能夠誘導heat shock flippase(hsflp)表現並使FLP-out Gal4 cassette的FRT片段產生loop-out recombination，之後Gal4會被表現並和Upstream Activating Sequence (UAS)結合而促進gene of interest (GOI)和GFP的表現。藉由FLP-out Gal4 system的研究我們一開始發現cactus的過表達能夠使果蠅翅膀的imaginal discs產生很多的ectopic clones.在實驗中我們使用的cactus stock是yw;;UAS-Cactus(也表示為yw;;cactus)。我在測試了在cactus過表達的情況下產生ectopic clones的機轉時發現cactus的過表達和clone的產生無關，我們還發現在ectopic clones產生的過程不需要flippase的參與但是需要進行heat shock，因此我們認為在yw;;cactus中存在FLP-like activity。而這個Flp-like activity 能讓不同的FLP-out cassette產生很多的ectopic clones。在接下來的實驗中我發現FLP-like activity是位在yw;;cactus的X染色體上，在使用polymarker chromosome 進行meiotic mapping後發現Flp-like activity位於forked基因(X染色體15的位置)的後面，最後我發現在yw;;cactus stock的X染色體上有一個flippase 的P-element插入而使其能夠誘導許多ectopic clones的產生，因此我的結論是我們所使用的yw;;UAS-Cactus stock 其實是ywhsflp;;UAS-Cactus.

FLP-out Gal4 system is a widely used genetic tool in drosophila and it can induce GFP-labelled cells to form ectopic clones and express certain genes. With a short time of heat shock, we can induce the expression of heat shock flippase (hsflp) that processes loop-out recombination at the FRT site of the FLP-out Gal4 cassette. After the FRT is looped out, Gal4 expressed and binds to Upstream Activating Sequence (UAS) and promotes the expression of the gene of interest (GOI) and GFP.
By using the FLP-out Gal4 system, we previously found that the overexpression of cactus can generate many ectopic clones in the wing imaginal discs. The cactus stock we used is yw;;UAS-Cactus (referred to as yw;;cactus). Here, I investigated the mechanism of ectopic clone formation upon cactus overexpression. Interestingly, I found that the overexpression of cactus is not required for the ectopic clone generation. However, we found that it required heat shock but not flippase to generate ectopic clones. This suggests the presence of Flippase-like activity in the yw;;cactus stock. This Flippase-like activity can efficiently induce ectopic clone generation in several different FLP-out cassettes. I further identified that the X chromosome of yw;;cactus stock is required for clone generation. Through meiotic mapping by using polymarker chromosome, I mapped this FLP-like activity to a region distal to forked gene (15 of the X chromosome). Finally, we found that a heat shock flippase P-element is inserted in 18E of yw;;cactus stock that promotes clone induction. I conclude that the yw;;UAS-Cactus we used is indeed ywhsflp;;UAS-Cactus.

1. Introduction----------------------------------------------------------------------1
2. Materials-------------------------------------------------------------------------3
3. Method----------------------------------------------------------------------------4
4. Results---------------------------------------------------------------------------5
4-1.The effect of Cactus and other Toll pathway factors on the clone number of AYG cassette. -----------------------------------------------------------------------------5
4-2.To test if yw;;cactus can promote clone induction on the FLP-out cassette without the overexpression of cactus and the existence of heat shock flippase------------6
4-3.The factor that leads to high clone number is on the X chromosome of yw;; cactus --------------------------------------------------------------------------------------9
4-4.Mapping the factor on the yw that leads to a high clone number-----------------------------------10
4-5.The FLP-like activity of yw;;cactus is caused by a heat shock flippase inserted in 18E of the X chromosome---------------------------------------------------------11
5. Summary and Discussion----------------------------------------------------------------------------12
6. References----------------------------------------------------------------------------------------14
7. Figures-------------------------------------------------------------------------------------------15

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Federico Germani, Cora Bergantinos, and Laura A. Johnston
GENETICS February 1, 2018
vol. 208 no.2 473-490; https://doi.org/10.1534/genetics.117.300256
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4. Drosophila - Mutanten (uni-halle.de)