From: Kathy Matthews <matthewk@XXXX> Personal communication from: J.-M.Reichhart and D. Ferrandon, UPR CNRS, Strasbourg To: Bloomington Drosophila Stock Center Subject: Green Balancers Date: 4 June 1998 Information communicated: We have used the S65T green fluorescent protein (GFP) as a vital reporter to introduce a dominant innocuous marker onto the balancers of the three major chromosomes of D. melanogaster. Construction : The drosomycin promoter contained in pJM802 was replaced by the distal actin 5C promoter as an EcoRI-NheI fragment originating from pPac in which an NheI linker was inserted into the polylinker. The P element mediated transformation plasmid derived from pCaSpeR contained the actin 5C promoter, followed by the S65T version of the GFP and the drosomycin terminator. The nucleotide sequence of the transformation vector is available upon request. Transgenic fly lines were established as described. One of the P element insertion obtained was remobilized using a Delta(2-3) source of transposase. Insertions in FM7 (FM7i : , CyO and TM3 balancer chromosomes were selected. The following stocks were sent to the Bloomington stock center : - FM7i-pAct-GFP : C(1)DX,f/FM7,y[93j],sc[8],w,oc,ptg,B,P[w+mC act::GFP =pActGFP] - CyO-pAct-GFP: w; In(2LR)noc[4L],Sco[rv9R],b / In(2LR)O,Cy,dp[lvI],pr,cn[1],P[w+mC act::GFP =pActGFP] - TM3-pAct-GFP : w; Sb[1] / In(3LR)TM3, ri,pp,sep,l(3)89Aa,bx34eSer,P[w+mC act::GFP =pActGFP] Expression pattern : Since their cuticle is transparent, third instar larvae carrying the marked balancers are easy to score under the fluorescent dissecting microscope. The main GFP expression pattern consists of a strong fluorescence in the salivary duct, the copper cells, the proventriculus and the visceral musculature of the midgut. A weaker signal can be detected in imaginal disks. In first instar larvae, the fluorescence appears to be restricted to the midgut . Adult flies carrying GFP balancers can be recognized by a deep pseudopupil type of expression in the eye, a mild fluorescence in the proboscis and a strong signal in the abdomen. Upon dissection, it appears that the abdominal fluorescence is due to : - GFP expression in the reproductive tract of the male ; - GFP expression in ovaries (yolk of mature stages and musculature of the ovary sheath) and in the seminal receptacle in females. In many animals, the visceral musculature of the midgut is also fluorescent. In the embryo, there is a strong maternal contribution which masks the zygotic expression until about stage 15 of development, when a weak signal can be detected in the midgut, as in first instar larvae. In the absence of this maternal contribution, the expression of GFP can first be detected around 12h after egg laying. Selected pictures showing these expression patterns can be viewed at http://ibmc.u-strasbg.fr/upr9022/GreenBalancers.html In conclusion, these green balancers constitute a highly useful tool to score living larvae, pupae, and adult flies, especially when working with mutations on the second chromosome.
