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What causes LCA2 blindness?

What causes LCA2 blindness?. light. change [Na + ] send signal on optic nerve. trans-retinal. cis-retinal. RPE65. LCA2 blindness:. light. change [Na + ] send signal on optic nerve. trans-retinal. cis-retinal. trans-retinal. normal allele. LCA2 allele.

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What causes LCA2 blindness?

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  1. What causes LCA2 blindness? light change [Na+] send signal on optic nerve trans-retinal cis-retinal RPE65

  2. LCA2 blindness: light change [Na+] send signal on optic nerve trans-retinal cis-retinal trans-retinal

  3. normal allele LCA2 allele Is LCA2 allele dominant or recessive? chromosome 1 transcription translation RPE65 gene no functional protein RPE65

  4. What are the genotypes? What is the probability of another LCA2 child? Rr Rr R = normal RPE65 r = LCA2 rr 1 in 4 chance next child will be blind by age 20

  5. How could we prevent or cure this disease? Rr Rr R = normal RPE65 r = LCA2 rr 1 in 4 chance next child will be blind by age 20 What would we need to have in order to do gene therapy?

  6. Where can we find the RPE65 gene? RPE65 gene Joe human cell human DNA OK, but now what?

  7. Gene cloning human RPE65 gene • Isolate a specific gene of interest • Insert into a plasmid • Transfer to bacteria • Grow bacteria to get many copies • Express the protein product • Why? • Sequence the gene • Study the enzyme • Understand regulation • Genetic screening • Gene therapy …etc. plasmid recombinant DNA human RPE65 enzyme E. coli

  8. Steps in gene cloning human RPE65 gene • Isolate DNA including YFG • Join to plasmid vector (ligation) • Introduce into host (transformation) • Find correct clone • Express the protein product ligation plasmid recombinant DNA transformation human RPE65 enzyme E. coli

  9. 1. Isolate DNA including YFG • Extract from cells • Cut into manageable fragments RPE65 gene RPE65 gene human DNA

  10. Restriction digest human DNA GAATTC CTTAAG GAATTC CTTAAG GAATTC CTTAAG cloning vector (plasmid)

  11. AATTC G G CTTAA “sticky” ends G CTTAA AATTC G 2. Join to plasmid vector (ligation) restriction fragment cloning vector (plasmid)

  12. DNA ligase 2. Join to plasmid vector (ligation) GAATTC CTTAAG GAATTC CTTAAG recombinant plasmid what’s missing? what enzyme should we use?

  13. plasmid library 2. Join to plasmid vector (ligation) plasmid vector + human DNA fragments

  14. 3. Introduce into host (transformation) + recombinant DNA E. coli CaCl2 or electric shock recombinant E. coli

  15. 3. Introduce into host (transformation)

  16. agar plate with ampicillin 3. Introduce into host (transformation) • Select cells that have plasmid by antibiotic resistance

  17. 4. Find the correct clone How do we know which of all these colonies came from a cell that took up a plasmid carrying RPE65?

  18. 4. Find the correct clone • Enzyme assay for RPE65 trans- retinal HPLC This won’t work. Why not? proteins from lysed bacteria

  19. Why my clones can’t make RPE65 protein: • RPE65 gene has introns; bacteria can’t splice • Expression signals: • Transcription: bacteria need -10 and -35 human gene has TATA, enhancers, etc. • Translation: bacteria need Shine-Dalgarno human gene won’t have it ? ? TAG ATG enhancers TATA

  20. cDNA cloning: DNA copy of RNA • Spliced mRNA → coding sequence with no introns DNA nucleus mRNA cytoplasm mature RNA AAAAAAAAAAAAAAA reverse transcriptase Why does it have to be DNA? DNA

  21. AAAAAAAAAA mRNA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA cDNA cloning • Purify mRNA: from what kind of cells? from where in the cell?

  22. AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA TTTTTTT TTTTTTT TTTTTTT TTTTTTT cDNA cloning • Add reverse transcriptase to make cDNA TTTTTTT

  23. cDNA cloning • Add reverse transcriptase to make cDNA

  24. cDNA cloning • Ligate to a plasmid vector +

  25. cDNA cloning • Transform into E. coli • Find correct clone cDNA library Now could we express the protein product??

  26. -10 -35 EcoRI S-D RPE65 cDNA EcoRI Expression vector • Plasmid with transcription and translation signals -10 -35 EcoRI S-D expression vector

  27. 4. Find the correct clone • Enzyme assay for RPE65 trans- retinal HPLC proteins from lysed bacteria

  28. purify plasmid DNA sequencing express protein etc. Cloned gene is ready for use!

  29. PCR Cloning by PCR • Polymerase chain reaction • If DNA sequence is known, amplify specific gene directly RPE65 gene human DNA

  30. Cloning by PCR • Human DNA • RPE65-specific 20-nt primers • Taq DNA polymerase • dNTPs part of RPE65 5′ ATGTCTATCCAGGTTGAGCATCCTGCTGGTGGTTACAAGAACTGTTTGAAACTGTGGAGG 3′ TACAGATAGGTCCAACTCGTAGGACGACCACCAATGTTCTTGACAAACTTTGACACCTCC heat heat primer 5′ ATGTCTATCCAGGTTGAGCATCCTGCTGGTGGTTACAAGAACTGTTTGAAACTGTGGAGG TTTGACACCT 5′ 5′CCAGGTTGAG TACAGATAGGTCCAACTCGTAGGACGACCACCAATGTTCTTGACAAACTTTGACACCT 5′ TACAGATAGGTCCAACTCGTAGGACGACCACCAATGTTCTTGACAAAC primer CATCCTGCTGGTGGTTACAAGAACTGTTTGAAACTGTGGA

  31. Cloning by PCR

  32. Cloning by PCR • Once amplified, ligate and transform as before + plasmid vector amplified copies of RPE65 gene

  33. Genetic engineering

  34. Genetic engineering • Modified microorganisms: • Insulin, growth hormone, clotting factors, EPO… • HPV vaccine • Ethanol from cellulose • Oil-eating bacteria • Modified plants and animals • BT corn • Roundup-ready soybeans • Golden rice • Modified humans • Gene therapy

  35. Recombinant DNA technology: Unlimited possibilities Many questions

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