Petar V. Todorov N EGATIVE G ENE S ENSOR S YSTEMS FOR MICRO RNA D ETECTION USING C HERRY Carlos Loya Van Vactor Laboratory

W HAT IS MICRO RNA? Non-protein coding ribonucleic acid Single-stranded ~21 nucleotides Important regulator of gene expression Acts by binding mRNA In Drosophila ~156 miRs and ~600+ in humans Shown to be involved in: – Development – Differentiation – Proliferation – Cell death – Metabolic control – Transposon silencing – Antiviral defense V. Narry Kim, Jinju Han & Mikiko C. Siomi, 2009

AAAA AGO2 GW18 2 Inhibition at Initiation of Translation 1 Inhibition of Translation Elongation AAAA AGO2 GW Endonucleolytic cleavage AND Degradation AGO1/2 3 AAAA Carlos Loya

C URRENT M ETHODS FOR MI R D ETECTION : I N SITU HYBRIDIZATION (D ROSOPHILA E MBRYO ) miR-315 miR-124miR-8 miR-13b-2 A. Aboobaker, P. Tomancak, N. Patel, G. Rubin, & E. Lai, 2005

N EGATIVE S ENSOR S YSTEM T UBULIN PROMOTER EGFP miR-x Binding Site x3 FLUORESCENCE!

miR-8 Sensor (EGFP) MUSCLE (PHALLOIDIN) W1118 miR-8 C URRENT M ETHODS FOR MI R D ETECTION : N EGATIVE F LUORESCENCE S ENSOR Motor neuron Brain (Extracts) Muscle (Extracts) ControlmiR-8 ControlmiR-8 α -GFP α - Tub α -GFP α - Tub Western Blots Brain Carlos Loya et al. 2009

C LONING M AP pAc-Cherry KpnI-Cherry-NotI ptub-KpnI-EGFP-NotI-SV40 ptub- -SV40 -EGFP- -Cherry- ptub- -SV40 ptub-Cherry- SV40 PCR & Purify Restriction Digest & Purify Restriction Digest Ligate Run Gel & Extract

PTUB -EGFP-SV40 V ECTOR ptub-EGFP-SV40

R ESTRICTION D IGEST : K PN I & N OT I ptub- -SV40 -EGFP-

ptub- -SV40 & EGFP 10kb Ladder

C LONING M AP pAc- Cherry KpnI-Cherry- NotI ptub-KpnI-EGFP-NotI-SV40 ptub- -SV40 -EGFP- -Cherry- ptub- -SV40 ptub-Cherry- SV40 PCR & Purify Restriction Digest & Purify Restriction Digest Ligate Run Gel & Extract

95°C DENATURE 60°C ANNEAL PRIMERS 72°C REPLICATE DNA Cherry

95°C DENATURE 60°C ANNEAL PRIMERS 72°C REPLICATE DNA

95°C DENATURE 60°C ANNEAL PRIMERS 72°C REPLICATE DNA

CherryNotIKpnI

ptub-Cherry-SV40 ptub- -SV40

F ROM V ECTOR TO T RANSGENIC A NIMAL O RDER TRANSGENIC LINE E XPRESSING DNA V ECTOR 3. M INIPREP 2. T RANSFORM & A MPLIFY B ACTERIA 1. V ERIFY S EQUENCE

MICRO RNA S OF I NTEREST miR-315 miR-124 miR-8 miR-13b-2 A. Aboobaker, P. Tomancak, N. Patel, G. Rubin, & E. Lai, 2005

SPECIAL THANKS TO PROF. DAVID VAN VACTOR CARLOS LOYA