1. 2015-08-11 Bo Shi

2.  Clonings (What I have done.)  Projects (What I am starting to do.)

3.  The purposes of cloning ◦ 1 Inject into xenopus oocyte to test the GFP signals (Suntag) ◦ 2 Build up Drosophila tools  Suntag  Photoreceptors ◦ 3 For in vitro reconstitution

4.  pT7Ts_GFP_sp1-10  pT7Ts_CG3822_DkaiRID_HA_GFP_1Xsp11  pT7Ts_CG3822_DkaiRID_HA_GFP_3Xsp11  pT7Ts_CG3822_DkaiRID_HA_GFP_10Xsp11

5.  Suntag  pUAST_CG3822_DkaiRID_HA_GFP_1Xsp11  pUAST_CG3822_DkaiRID_HA_GFP_3Xsp11  pUAST_CG3822_DkaiRID_HA_GFP_10Xsp11

6.  Photoreceptors  pCaST_Rh3_syb_spGFP1-10 (pR7)  pCaST_RH4_syb_spGFP1-10 (yR7)  pCaST_RH5_syb_spGFP1-10 (pR8)  pCaST_RH6_syb_spGFP1-10 (yR8)

7.  pT7Ts_CG3822_DkaiRID_QR  pT7Ts_CG5621_QR  pT7Ts_CG9935_QR  pT7Ts_Clumsy_GR  pT7Ts_GluRIA

8. 1 What make Tm20 cells as functional neurons? 2 How neurons contect to others via Dprs & DIPs?

9.  1 Functions– receptors ◦ L3 to Tm20 by Ach ◦ R8 to Tm20 by His

10.  1 Crossing to obtain Tm20 ◦ Female: ◦ Yw; 17Dol-Flp/Cyo; +/Tm2 or Tm6B ◦ Male: ◦ Yw; ort-cla-lv/Cyo; lot-stop-mCD8-GFP/ Tm2 or Tm6B ◦ F1: ◦ Yw; 17Dol-Flp/ort-c1a-lv; lot-stop-mCD8-GFP/+  2 Dissect

11.  3 Ensure GFP cells in the F1  4 Dissociation  5 Pick single cells

12.  2 Development—screening ◦ Screen the possible genes related to Tm20 development

13.  1 Crossing ◦ Tm20, TSC1 ◦ Tm20, Abl1 ◦ Dm8, Abl1 ◦ Dm8, Wts  2 Fixation and Dissection  3 Staining  4 Imaging

14.  Defective proboscis extensive response (Dpr)  Dpr interacting protein (DIP) Engin Ozkan, et al. Cell 154, 228–239. 2003

15.  What we know: ◦ Dm8 expresses DIP- ϒ ◦ yR7 expresses Dpr11 ◦ Dpr 11 is a parter of DIP- ϒ ◦ So? ◦ Actually, we know R7 is connected to Dm8.

16.  Shall we find out the Dpr and DIP expression in our interested cells?  Can we use the partnership information of Dpr and DIP to indentify the connections between neurons?

17.  1 Design primers  2 Nesting PCR ◦ i extract mRNA from 50 fly heads ◦ ii synthesize cDNA ◦ iii use 0.5 ul undiluted and 1:100 diluted cDNA as templates to run 1 st round PCR ◦ iv dilute the 1 st round PCR production 1:10 as templates to run nested PCR ◦ v load 5 ul product from the 2 nd round, and run 1.5% gel

18. 123123  From left to right  Image 1 ◦ Dpr2 (271/804), Dpr4 (223/874), Dpr5 (357/871), Dpr6 (257/744), Dpr10 (265/928), Dpr11 (295/1041)  Image 2 ◦ Dpr10 (265/928) (ND, D), ◦ Dpr11 (295/1041) (ND, D), ◦ Dpr12 (405/1335) (ND, D),  Image 3 ◦ Dpr13 (327/1226), Dpr14 (295/605), Dpr15 (334/733), Dpr17 (323/518), Dpr18 (268/934)

19.  1 The primers can be used in the single cell experiment ◦ Dpr2, Dpr4, Dpr5, Dpr6, Dpr12, Dpr13, Dpr14, Dpr18  2 The primers need to be re-design ◦ Dpr10, Dpr11, Dpr15, Dpr17  3 The primers have not been tested ◦ Dpr1, Dpr3, Dpr7, Dpr8, Dpr9, Dpr16, Dpr19, Dpr20

20. cDNAgDNATaqmanPhusion Tm CG33507Dpr2Dpr2-FACCGACGGACCTCTATGTCA5221150Dm01810588_g165.0 Dpr2-RGTGATTTTCGCACGCAGGTT 67.5 neDpr2-FCGTAACGCTCACTTGTCACG271804 64.9 neDpr2-RGATGACATTGACCACCACGC 67.0 CG33512Dpr4Dpr4-FCGGAGCAAATACAACAGCCG327978Dm02147102_s167.9 Dpr4-RCGCTCGATCGCCCAGATT 69.1 neDpr4-FAATACAACAGCCGGCAGGA223874 65.9 neDpr4-RTGCGAGTACGGGGTTTCC 66.4 CG5308Dpr5Dpr5-FTGTCCTGGATACGACAACGG404918Dm02139115_g166.3 Dpr5-RGTTGCTGGTCTTCATCTGCTG 65.3 neDpr5-FTCGGCATCATGACCTACACG357871 67.5 neDpr5-RTCTTCATCTGCTGCTCGGAC 66.1 CG14162Dpr6Dpr6-FGGATCCGGCATCGAGACAT311798Dm01796885_g167.8 Dpr6-RGATGTAGGCCGGCGGTT 66.5 neDpr6-FCCGGCATCGAGACATCCAC257744 69.5 neDpr6-RTGGTGCTGCCTTTATCCACG 68.5 CG32057Dpr10Dpr10-FATCATCAAGTTCAGCCCGGA5011638Dm01836228_g167.1 Dpr10-RTATCTTGGGGTCAAGCAGGG or Dm01836229_m166.4 neDpr10-FGAACACGGAGATAGCCAGCAT265928 65.6 neDpr10-RGACTCGAGTGGGCATTCCTC 66.5 CG33202Dpr11Dpr11-FTTCCTCGCACGGAGATCCTA5171263Dm02153590_g167.3 Dpr11-RCAGAGTGTTGGGATTCCCCC or Dm02153591_g168.1 neDpr11-FCGCAACTGGATCCCAACCT2951041 67.7 neDpr11-RTTTGGCTTGCTGGCATTGAC 68.6

21. CG34385Dpr12Dpr12-FGGCGCGACATAACCATTGAG5301460Dm01815340_m167.9 Dpr12-RAAACCGGTGTGGATGGATGT 66.7 neDpr12-FGACTCCGGCAACTACACCTG4051335 65.1 neDpr12-RTCGAGTACACAGCGACGATTG 66.7 CG33996Dpr13Dpr13-FTCTAACCCCAGGTTCAACGC5101482Dm01826766_g166.4 Dpr13-RACTGGAAATGGGTAGCCTGTG 65.4 neDpr13-FCTTACCATTCAGCGGACACG3271226 65.9 neDpr13-RGACTTCGAACGGCTGATGAC 65.2 CG10946Dpr14Dpr14-FAAGCGAAGTGAAGTCCGTGG587897Dm01833725_g166.9 Dpr14-RTCGCCGCTATATGTGTGCTG 67.4 neDpr14-FTGCAGTGATGAGATCGAGGC295605 67.1 neDpr14-RGCAGTGCAGATAAACGCTGG 66.1 CG10095Dpr15Dpr15-FTTTCAGTCCGTGTTCTCGCC6181017Dm02148869_g167.9 Dpr15-RCGAAGACGTTGCCTCCACTT 66.9 neDpr15-FGCCAGTGCCATTGTCCATCT334733 67.7 neDpr15-RTTGATCCTGTGGCCGTTGTC 68.7 CG31361Dpr17Dpr17-FTTATCAGCGTGGACGAAACGA478673Dm02148862_g167.9 Dpr17-RACGGAGACGCTGTTCGATG 66.9 neDpr17-FAGCCAAAGCTGAGTGCCAAA323518 67.4 neDpr17-RGTTCGATGGCTGGCACGTAT 67.5 CG14948Dpr18Dpr18-FCCGTGCAAAGGATGAGGCT4071074Dm01839317_m167.9 Dpr18-RACCCCGACTCATTTTCGTGG 68.5 neDpr18-FCCTTCTTCGGCTGCTCACAA268934 68.1 neDpr18-RAGCGTTAGACGCCTCGATTG 66.8