Lab Meeting Bo Shi 2015-09-22

Outlines 1 Dpr and DIP primer testing 2 Confocus imaging 3 Single cell dissociation 4 Transgenetic flies (just starting) 5 Dprs and DIPs recombining flies (just starting)

1 Dpr and DIP primer testing The primers of 12 Dprs have been tested, and can be used for the next step. (The other 8 or 20 Dprs has been tested before my last lab meeting.) 10 of 11 DIPs also can be used. (exception: CG40378)

2nd round PCR images No. 1 2 3 4 5 6 Genes Dpr1 Dpr 3 Dpr 7 Dpr 8 Dpr 10-new cDNA 201 248 270 220 457 225 gDNA N/A 8661 5056 888 1 2 3 4 5 6

No. 1 2 3 4 5 6 Genes Dpr11-new Dpr15-new Dpr16-new Dpr17-new Dpr 19 Dpr 20 cDNA 242 208 370 273 X gDNA 826 3500 801 2124 1 2 3 4 5 6

No. 1 2 3 4 5 6 Genes Dpr19-new Dpr 20-new DIP-α(1) DIP-γ(3) DIP-δ(4) DIP-ε(5) cDNA 202 337 288 385 318 311 gDNA N/A 1332 841 1732 5272 1 2 3 4 5 6

No. 1 2 3 4 5 6 Genes DIP-ζ(6) DIP-η(7) DIP-θ(8) DIP-ι(9) cDNA 274 X CG31814 CG40378 cDNA 274 X 320 253 gDNA N/A 3225 1 2 3 4 5 6

No. 1 2 3 Genes DIP-β(2) cDNA 309 /224 324 X gDNA 15049 N/A 1 2 3 DIP-η(7)-new CG40378-new cDNA 309 /224 324 X gDNA 15049 N/A 1 2 3

2 Confocus imaging ort-c1a-lv Tm20: YW ; 17D01-Flp ; lot-stop-mCD8-GFP + ; ♂ 122 ; C1-Gal4, UAS-mCD8-Cherry + FRT82B, Gal80 FRT82B, Tsc1 Tsc mutation, Tm20

3 Single cell dissociation FH1 ♀ YW ; 17D01-Flp Cyo ; + Tm2 or Tm6B X ♂ YW ; ort-c1a-lv lot-stop-mCD8-GFP ort-c1a-lv F1: YW ; 17D01-Flp ; lot-stop-mCD8-GFP +

2nd round PCR images mCD8 1 2 3 4 5 6 7 8 vGlut 1 2 3 4 5 6 7 8 Primers cDNA gDNA mCD8 266 vGlut 339 807

4 Transgenetic flies Rh3-syb-spGFP1-10 Rh4-syb-spGFP1-10 UAS-YFP-sp1-10cyto

5 Dprs and DIPs recombining flies 57B1-57B2 FRT42 Dpr20 3L 61D2-61D2 FRT2A Dpr3 2L 22C1-22C3 FRT40 CG14010 25F1-25F1 CG31708 29F8-30A1

Future Work 1 Complete CG40378 primers design and test. 2 Test DIPs and Dprs expression in the medulla. 3 Test DIPs and Dprs expression in Tm20, Dm8 & Tm5C. 4 Inditify which Ach receptor expresses in Tm20. 5 Screen some adhesion molecule mutations in Tm20.

6 Continue to pick Transgenetic flies 7 Continue to recombine Dprs and DIPs flies with FRT flies 8 Obtain the specificity of Tm20 in the line of ort-c1a-lv F1: YW ; 17D01-Flp ; lot-stop-mCD8-GFP +

Transgenetic flies X YW F1: 1♂ red eye fly X YW; ; Cyo Tm6B Tm2 B1 3♀ If transgne is on the ChX, all females are red eyes and males are white eyes in the F2 geneation. If Transgene is on the on the Ch2, Ch3 or Ch4, then: F2: 1♂ red eye fly X YW ; ; Cyo Tm6B Tm2 B1 3♀

3 Single cell dissociation 17Dol-Flp + ort-cla-lv lot-stop-mCD8-GFP ♀ YW; ; X ♂ YW; ; Cyo Tm2 or Tm6B Cyo Tm2 or Tm6B ; ♂ 122 ; C1-Gal4, UAS-mCD8-Cherry + FRT82B, Gal80 FRT82B, Tsc1