fly-genetics-homework (2)

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1、1．Readthepaperprovided,anddescribehowthefollowingexperimentsareaccomplished:(1)ToreplacewildtypeWgwithmembrane-tethereda.Injecttheposteriorregionofpre-cellularwhite1118embryoswithaplasmidencodingCas9andanotherplasmidencodingawingless–specifictargetegRNA,alongwiththepTVCherry[wingless

2、]andaplasmidencodingFLPandI-Scel.b.Theresultingadultswerebackcrossedtowhite1118flies.c.ScreeningthesubsequentF2progenyforredeyes.Thegenotypesofthesefliesareasfollows:Theabovethreestepsareasfollows:d.Cre-mediatedexcisiontoproducewg[KO]：e.ConfirmfaithfulexpressionofthereintegratecDNAat

3、thetargetedlocus：Insertawild-typeWgcDNAintotheattPsite,alongwithmini-whiteasageneticmarker,andtheresultingflies(wg{KO;Wg})wereindistinguishablefromwild-typeflies,confirmingfaithfulexpressionofthereintegratedcDNAa.ThereintegrationoftheNRT–WgcDNAinwg{KO}toproducewg[KO；NRT-Wg](1)Togener

4、atewg-/-clonesinwildtypebackgroundorwg-/-clonesintetheredwgbackgrounda.Togeneratewg-/-clonesinwildtypebackgroundFRT40wgCX4FRT40wgCX4FRT40ubiquitin-GFPFRT40ubiquitin-GFPFRT40ubiquitin-GFPFlipaseFRT40wgCX4FRT40ubiquitin-GFPFRT40wgCX4FRT40ubiquitin-GFPMitosisFRT40wgCX4FRT40wgCX4FRT40ubi

5、quitin-GFPFRT40ubiquitin-GFPa.Togeneratewg-/-clonesintetheredwgbackgroundFRT40wgCX4FRT40wgCX4FRT40ubiquitin-GFPNRT-WgFRT40ubiquitin-GFPNRT-WgFlipaseFRT40wgCX4FRT40ubiquitin-GFPNRT-WgFRT40wgCX4FRT40ubiquitin-GFPNRT-WgmitosisFRT40wgCX4FRT40wgCX4FRT40ubiquitin-GFPNRT-WgFRT40ubiquitin-GF

6、PNRT-Wga.Togenerateflieswithwild-typewginthebodyandtetheredwginthewings,orflieswithtetheredwginthebodyandwildtypewginthewings(a).Togeneratewg{KO;WTBody;NRT–WgDiscs}wg{FRTWgFRTNRT–Wg}vestigial-gal4,UAS-Flpa.wg{KO;WTBody;NRT–WgDiscs}(b).Togeneratewg{KO;NRT–WgBody;WTDiscs}wg{FRTNRT–WgFR

7、TWg}vestigial-gal4,UAS-Flpwg{KO;NRT–WgBody;WTDiscs}Pesg-Gal4/Cyo;Gal80ts♀ｘUAS-IR/TM6♂2.DesignasimpleandefficientgeneticscreenfortumorsuppressorsintheDrosophiladigestivetract.18℃WildtypeF1♀:esg-Gal4/UAS-IR;Gal80tsShiftto29℃for7d解剖F1肠子，经固定、脱水、染色、制片，最后在荧光显微镜下观察肠道细胞的组成情况，如果有tumour产生，则说明相

8、应的基因是tumours