Lab Meeting2015-08-11

Bo Shi

Clonings (What I have done.)

Projects (What I am starting to do.)

Outline

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

Clonings

1 Inject into xenopus oocyte to test the GFP signals (Suntag)

pT7Ts_GFP_sp1-10

pT7Ts_CG3822_DkaiRID_HA_GFP_1Xsp11

pT7Ts_CG3822_DkaiRID_HA_GFP_3Xsp11

pT7Ts_CG3822_DkaiRID_HA_GFP_10Xsp11

Suntag

pUAST_CG3822_DkaiRID_HA_GFP_1Xsp11

pUAST_CG3822_DkaiRID_HA_GFP_3Xsp11

pUAST_CG3822_DkaiRID_HA_GFP_10Xsp11

2 Build up Drosophila tools

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)

2 Build up Drosophila tools

pT7Ts_CG3822_DkaiRID_QR

pT7Ts_CG5621_QR

pT7Ts_CG9935_QR

pT7Ts_Clumsy_GR

pT7Ts_GluRIA

3 For in vitro reconstitution

Projects

1 What make Tm20 cells as functional neurons?

2 How neurons contect to others via Dprs & DIPs?

1 Functions– receptors

◦ L3 to Tm20 by Ach

◦ R8 to Tm20 by His

What make Tm20 cells as functional neurons?

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

What I am learning

3 Ensure GFP cells in the F1

4 Dissociation

5 Pick single cells

2 Development—screening

◦ Screen the possible genes related to Tm20 development

What make Tm20 cells as functional neurons?

1 Crossing◦ Tm20, TSC1◦ Tm20, Abl1◦ Dm8, Abl1◦ Dm8, Wts

2 Fixation and Dissection

3 Staining

4 Imaging

What I am learning

Defective proboscis extensive response (Dpr)

Dpr interacting protein (DIP)

How neurons contect to others via Dprs & DIPs?

Engin Ozkan, et al. Cell 154, 228–239. 2003

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.

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?

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 1st round PCR◦ iv dilute the 1st round PCR production 1:10 as

templates to run nested PCR◦ v load 5 ul product from the 2nd round, and run

1.5% gel

What I am doing

2nd round PCR product 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)

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

The results of the primer testing

　 　 　 　 cDNA gDNA Taqman Phusion Tm

CG33507 Dpr2 Dpr2-F ACCGACGGACCTCTATGTCA 522 1150Dm01810588_g1 65.0

　 　 Dpr2-R GTGATTTTCGCACGCAGGTT 　 　 　 67.5

　 　 neDpr2-F CGTAACGCTCACTTGTCACG 271 804　 64.9

　 　 neDpr2-R GATGACATTGACCACCACGC 　 　 　 67.0

　 　 　 　 　 　 　 　CG33512 Dpr4 Dpr4-F CGGAGCAAATACAACAGCCG 327 978Dm02147102_s1 67.9

　 　 Dpr4-R CGCTCGATCGCCCAGATT 　 　 　 69.1

　 　 neDpr4-F AATACAACAGCCGGCAGGA 223 874　 65.9

　 　 neDpr4-R TGCGAGTACGGGGTTTCC 　 　 　 66.4

　 　 　 　 　 　 　 　CG5308 Dpr5 Dpr5-F TGTCCTGGATACGACAACGG 404 918Dm02139115_g1 66.3

　 　 Dpr5-R GTTGCTGGTCTTCATCTGCTG 　 　 　 65.3

　 　 neDpr5-F TCGGCATCATGACCTACACG 357 871　 67.5

　 　 neDpr5-R TCTTCATCTGCTGCTCGGAC 　 　 　 66.1

　 　 　 　 　 　 　 　CG14162 Dpr6 Dpr6-F GGATCCGGCATCGAGACAT 311 798Dm01796885_g1 67.8

　 　 Dpr6-R GATGTAGGCCGGCGGTT 　 　 　 66.5

　 　 neDpr6-F CCGGCATCGAGACATCCAC 257 744　 69.5

　 　 neDpr6-R TGGTGCTGCCTTTATCCACG 　 　 　 68.5

　 　 　 　 　 　 　 　CG32057 Dpr10 Dpr10-F ATCATCAAGTTCAGCCCGGA 501 1638Dm01836228_g1 67.1

　 　 Dpr10-R TATCTTGGGGTCAAGCAGGG 　 　 or Dm01836229_m1 66.4

　 　 neDpr10-F GAACACGGAGATAGCCAGCAT 265 928　 65.6

　 　 neDpr10-R GACTCGAGTGGGCATTCCTC 　 　 　 66.5

　 　 　 　 　 　 　 　CG33202 Dpr11 Dpr11-F TTCCTCGCACGGAGATCCTA 517 1263Dm02153590_g1 67.3

　 　 Dpr11-R CAGAGTGTTGGGATTCCCCC 　 　 or Dm02153591_g1 68.1

　 　 neDpr11-F CGCAACTGGATCCCAACCT 295 1041　 67.7

　 　 neDpr11-R TTTGGCTTGCTGGCATTGAC 　 　 　 68.6

CG34385 Dpr12 Dpr12-F GGCGCGACATAACCATTGAG 530 1460Dm01815340_m1 67.9

　 　 Dpr12-R AAACCGGTGTGGATGGATGT 　 　 　 66.7

　 　 neDpr12-F GACTCCGGCAACTACACCTG 405 1335　 65.1

　 　 neDpr12-R TCGAGTACACAGCGACGATTG 　 　 　 66.7

　 　 　 　 　 　 　 　CG33996 Dpr13 Dpr13-F TCTAACCCCAGGTTCAACGC 510 1482Dm01826766_g1 66.4

　 　 Dpr13-R ACTGGAAATGGGTAGCCTGTG 　 　 　 65.4

　 　 neDpr13-F CTTACCATTCAGCGGACACG 327 1226　 65.9

　 　 neDpr13-R GACTTCGAACGGCTGATGAC 　 　 　 65.2

　 　 　 　 　 　 　 　CG10946 Dpr14 Dpr14-F AAGCGAAGTGAAGTCCGTGG 587 897Dm01833725_g1 66.9

　 　 Dpr14-R TCGCCGCTATATGTGTGCTG 　 　 　 67.4

　 　 neDpr14-F TGCAGTGATGAGATCGAGGC 295 605　 67.1

　 　 neDpr14-R GCAGTGCAGATAAACGCTGG 　 　 　 66.1

　 　 　 　 　 　 　 　CG10095 Dpr15 Dpr15-F TTTCAGTCCGTGTTCTCGCC 618 1017Dm02148869_g1 67.9

　 　 Dpr15-R CGAAGACGTTGCCTCCACTT 　 　 　 66.9

　 　 neDpr15-F GCCAGTGCCATTGTCCATCT 334 733　 67.7

　 　 neDpr15-R TTGATCCTGTGGCCGTTGTC 　 　 　 68.7

　 　 　 　 　 　 　 　CG31361 Dpr17 Dpr17-F TTATCAGCGTGGACGAAACGA 478 673Dm02148862_g1 67.9

　 　 Dpr17-R ACGGAGACGCTGTTCGATG 　 　 　 66.9

　 　 neDpr17-F AGCCAAAGCTGAGTGCCAAA 323 518　 67.4

　 　 neDpr17-R GTTCGATGGCTGGCACGTAT 　 　 　 67.5

　 　 　 　 　 　 　 　CG14948 Dpr18 Dpr18-F CCGTGCAAAGGATGAGGCT 407 1074Dm01839317_m1 67.9

　 　 Dpr18-R ACCCCGACTCATTTTCGTGG 　 　 　 68.5

　 　 neDpr18-F CCTTCTTCGGCTGCTCACAA 268 934　 68.1

　 　 neDpr18-R AGCGTTAGACGCCTCGATTG 　 　 　 66.8