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Gene Technology

A New Tool Box for Recombinant DNA. Gene Technology. Expression systems Monoclonal Antibodies Transformation systems Expression in Cell lines versus Transgenic plants and transgenic animals Knock-out system to study diseases Gene therapy. Lecture 7 – Chapter 6 –part 2. Gene Technology.

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Gene Technology

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  1. A New Tool Box for Recombinant DNA Gene Technology • Expression systems • Monoclonal Antibodies • Transformation systems • Expression in Cell lines versus Transgenic plants and transgenic animals • Knock-out system to study diseases • Gene therapy Lecture 7 – Chapter 6 –part 2

  2. Gene Technology Expression systems Prokaryotic Expression vector Eukaryotic Expression vector

  3. Gene Technology Expression systems

  4. Gene Technology Expression systems -> Mainly in Prokaryotic systems -> Mainly in Eukaryotic systems

  5. Gene Technology Expression systems Most Expression systems are inducible !!!! In Prokaryotes (E. coli) -> many of them are based on the lac regulation (LacI /IPTG) -> lac system, tac system, T7 system,… Selection markers: -> antibiotic resistance genes (AmpR, TetR, CmlR) In Eukaryotes: -> more complex -> expression in yeast, insect cells, mammalian cells (cancer cell lines) (-> advanced gene technology) -> transgenic plants and animals Selection markers: -> genes coding for metabolic enzymes (complementation) -> antibiotic resistance genes -> genes coding for enzymes degrading toxic substances

  6. Gene Technology The Tet-off system: Originally from E. coli Tetracycline resistance protein (tetR) coded on TN10 in E. coli genome -> in absence of tetracycline -> repressor (tetR) binds -> no transcription -> tetracycline present -> no binding of repressor -> transcription Adaption for mamalien cells -> novel transcription factor (DNA-binding from TetR and Activator domain from herpes virus protein VP16) -> activator Binds to tet operator when no tetratcycline -> transcription Tetracycline present-> transcrition factor dissociates -> No transcription Turn off a gene by adding tetracycline Expression systems

  7. Gene Technology Monoclonal Antibodies Antigen stimulates production of lymphocytes (spleen cells) -> express antibodies Fusion of lymphocytes with cancer cells (myeloma cells) -> hybridoma cells Selection for hybridoma cells: Myeloma cells do not produce hypoxanthine phosphoribosyltransferase (HPRT) Lymphocytes are not alive over many generations in culture -> Selection for growth on hypoxanthine (HAT) -> just hybridoma cells can survive

  8. Gene Technology Transformation systems Gene transfer into the host organism -> depended on type of cells • In Prokaryotes: • - Transformation -> uptake of naked DNA (chemical transformation, electroporation) • - Conjugation -> DNA transfer by cell – cell contact • Transduction -> DNA transfer by bacteriopage infection • In Eukaryotes:->used with fungi, animal and plant cells: • Electroporation • Microinjection • protoplasts • Lipid treatment

  9. Gene Technology Transformation systems - Electroporation Used for: -> Bacteria -> Fungi -> mammalian cells -> some plant cells

  10. Gene Technology Transformation systems - Lipofection Used mainly for animal cells

  11. Gene Technology Transformation systems – Protoplast transfer Plants have a cell wall (cellulose) -> degradation of wall -> protoplasts Fungi have also a cell wall (chitin or cellulose) Protoplasts can take up DNA -> Regenerate to plant or fungus Regeneration of some protoplast difficult !!!

  12. Gene Technology Transformation systems – Shutgun Used for animal and plant cells

  13. Gene Technology Transformation systems – Transfection Used for: -> bacteria -> animal cells -> some plant cells

  14. Gene Technology Special Transformation systems for plants Ti plasmid of Agrobacterium tumefaciens

  15. Gene Technology Special Transformation systems for plants Ti plasmid of Agrobacterium tumefaciens

  16. Gene Technology Definition of Transgenic Transgenic -> stable introduction of a gene into another organism -> For Unicellular organisms (such as bacteria or yeast) all transformed cells are -> transgenic -> For multicellular organisms (such as animals, plants,..) difference between: - manipulation of single cells -> cell line (expression in insect cells or mammalian cells) - manipulation of a whole plant or animal -> transgenic (can have a transgenic offspring!!!) -> more difficult and expensive to create whole modified organism (transgenic) than just cell line!!!

  17. Gene Technology Transgenic versus Cloning Transgenic -> creation of transgenic animal or plant (introduction of foreign gene into organism) -> transgenic organisms produced by introduction of foreign gene into germ line (-> transgenic offspring!!!) -> introduction of gene into somatic cells -> gene therapy Cloning -> obtaining an organism that is genetically identical to the original organism -> such as Dolly the sheep -> asexual propagation of plants (taking cuttings) -> natural clones -> twins

  18. Gene Technology Transgenic plant PG (polygalacturonase) -> Sensitivity of tomatoes to bruising Reduced level-> should give harder fruit during shipping Result: lower level -> did not give harder fruit (more factors responsible for process) Expression of Antisense RNA of transcript of PG -> reduces level of protein produced

  19. Gene Technology Transgenic animal Transformation by Microinjection

  20. Gene Technology Transgenic animal Embryonic stem cells used to create mice with altered genome Changes in the germ line -> transgenic offspring!!! Embryonic stem cells

  21. Gene Technology Transgenic Cattle, Sheep, Goat, Pigs Production of pharmaceutical proteins -> drugs Problems: Highly inefficient Only 20% of the eggs survive and only 5% of them produce product

  22. Gene Transfer - what happens on DNA level • Integration into chromosome -> Recombinantion • Recombinantion can be -> homologous – non-homologous • non-homologous event -> more frequently • homologous event -> less frequent but desired • Knock-out mutants -> disrupt functional gene by integration of another gene into target gene • Used for: • -> study human diseases by creating model organisms • -> make minus mutant

  23. Gene Technology Knock out systems Homologous recombinantion used to make knock out systems Used: -> study diseases -> generate specific model organism neo: resistance to antibiotic G418 tk: thymidine kinase (if tk produced cells are killed if exposed to gancylovir) Yellow areas -> gene of interest that should be knocked out

  24. Gene Technology How do determine function of a gene?

  25. Gene Technology Gene Therapy – gene transfer into somatic stem cells Viral gene transfer into somatic stem cells -> gene therapy

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