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Negative Gene Sensor Systems for microRNA Detection using Cherry

Negative Gene Sensor Systems for microRNA Detection using Cherry. Petar V. Todorov. Van Vactor Laboratory. Carlos Loya. What is microRNA?. Non-protein coding ribonucleic acid Single-stranded ~21 nucleotides Important regulator of gene expression Acts by binding mRNA

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Negative Gene Sensor Systems for microRNA Detection using Cherry

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  1. Negative Gene Sensor Systems for microRNA Detection using Cherry Petar V. Todorov Van Vactor Laboratory Carlos Loya

  2. What is microRNA? • 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 http://en.wikipedia.org/wiki/File:Microrna_secondary_structure.png V. Narry Kim, Jinju Han & Mikiko C. Siomi, 2009

  3. CAP eIF4E eIF4G AGO2 PABP AAAA GW182 Inhibition at Initiation of Translation 1 AGO2 2 AAAA GW182 3 Inhibition of Translation Elongation AGO1/2 AAAA Endonucleolytic cleavage AND Degradation Carlos Loya

  4. Current Methods for miR Detection: In situ hybridization (Drosophila Embryo) miR-315 miR-8 miR-124 miR-13b-2 A. Aboobaker, P. Tomancak, N. Patel, G. Rubin, & E. Lai, 2005

  5. Negative Sensor System Tubulin promoter miR-x Binding Site x3 EGFP FLUORESCENCE!

  6. Current Methods for miR Detection: Negative Fluorescence Sensor Western Blots Brain Brain (Extracts) miR-8 Sensor (EGFP) Control ∆miR-8 α-GFP α- Tub Muscle (Extracts) ∆miR-8 W1118 Motor neuron Control ∆miR-8 α-GFP MUSCLE (PHALLOIDIN) α- Tub Carlos Loya et al. 2009

  7. Cloning Map pAc-Cherry KpnI-Cherry-NotI ptub- -SV40 -Cherry- ptub-Cherry- SV40 ptub- -SV40 -EGFP- ptub-KpnI-EGFP-NotI-SV40 Restriction Digest PCR & Purify Restriction Digest & Purify Run Gel & Extract Ligate

  8. ptub-EGFP-SV40 Vector EGFP NotI KpnI SV40 ptub-EGFP-SV40 Tubulin promoter

  9. Restriction Digest: KpnI & NotI EGFP -EGFP- NotI KpnI SV40 Tubulin promoter ptub- -SV40

  10. 10kb Ladder ptub- -SV40 & EGFP

  11. Cloning Map pAc-Cherry KpnI-Cherry-NotI ptub- -SV40 -Cherry- ptub-Cherry- SV40 ptub- -SV40 -EGFP- ptub-KpnI-EGFP-NotI-SV40 Restriction Digest PCR & Purify Restriction Digest & Purify Run Gel & Extract Ligate

  12. 95°C denature 60°C anneal primers 72°C replicate DNA Cherry

  13. 95°C denature 60°C anneal primers 72°C replicate DNA

  14. 95°C denature 60°C anneal primers 72°C replicate DNA

  15. Cherry NotI KpnI

  16. Cherry NotI KpnI SV40 ptub-Cherry-SV40 Tubulin promoter ptub- -SV40

  17. From Vector to Transgenic Animal 1. Transform & Amplify Bacteria 2. Miniprep 3. Order transgenic line Expressing DNA Vector Verify Sequence

  18. microRNAs of Interest miR-315 miR-8 miR-124 miR-13b-2 A. Aboobaker, P. Tomancak, N. Patel, G. Rubin, & E. Lai, 2005

  19. Special Thanks TO Prof. David Van Vactor Carlos Loya

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