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Gene expression systems

Gene expression systems. Recombinant gene expression systems. Direct promoter fusions GAL4/UAS system GeneSwitch system Tet regulated expression. GeneSwitch System. Invitrogen’s GeneSwitch System. Estrogen Receptor-GAL4 fusion. Tetracycline regulated expression.

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Gene expression systems

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  1. Gene expression systems

  2. Recombinant gene expression systems • Direct promoter fusions • GAL4/UAS system • GeneSwitch system • Tet regulated expression

  3. GeneSwitch System

  4. Invitrogen’s GeneSwitch System

  5. Estrogen Receptor-GAL4 fusion

  6. Tetracycline regulated expression

  7. Invitrogen’s Tet-On System (T-Rex)

  8. Tet-on in cell culture

  9. Knock-outs & Knock-ins

  10. Yeast knock-outs(S. cerevisiae) • Rationale: determine the function of all genes in the genome • Strategy: use homologous recombination to replace each open reading frame (ORF) with marker

  11. Classification of essential (inner) vs. non-essential (outer) genes

  12. Mouse Knock-outs

  13. Making a targeted mutation (“knockout”) in mouse cells Fig. 8-38 Griffiths, A., et al. (2002) Modern Genetic Analysis, 2ed.

  14. Using embryonic stem cells to make a knockout mouse Fig. 8-39 Griffiths, A., et al. (2002) Modern Genetic Analysis, 2ed.

  15. Fig. 8-38 Griffiths, A., et al. (2002) Modern Genetic Analysis, 2ed.

  16. Mouse Knock-ins

  17. Mouse Knock-ins – WHY? • Test function of a specific residue or domain • Create model for equivalent change in disease state • Tag protein • Test functional equivalence of genes • and many, many more…

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