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1. Biotechnology Gene technologybased approaches Non-genetic approaches Knockout & transgenic organisms microchips Recombinant proteins Live vaccines New stem cell techniques Gene therapy, immunotherapy Industrial fermentation Monoclonal antibodies Cloned organisms Traditional stem cell technlogy Live vaccines etc. 2. Intervention Examination Recombinant gene technology Genomics 3. Gene technology Top danger 4. Moratorium on genetic manipulations - Asilomar: the conference of debate Voluntary moratorium The immediate reason: cloning of entire genome of SV40 in E. coli: contagious tumor? Fears: human health and ecosystem of Earth Asilomar Conference (1975): Something can be considered dangerous if it is proved to be dangerous Paul Berg James Watson Sidney Brenner 5. Today’s fears 1. Human-made contagious diseases - AIDS from poliomyelitis vaccine? 2. We alter human evolution 3. We interfere to the Creator’s business 4. Genetically modified foods are unhealthy 5. We change the ecosystems 6. Illegitimate recombination - Random DNA integration plasmid vector transgene chromosome transgene transgene transgene 7. Homologous recombination - targeted DNA insertion transfer vector flanking seq. foreign DNA flanking seq. chromosome target sequence chromosome foreign DNA Genetically modified sequence Knock-in Knock-out 8. 8. Cultured cell types 1. Primary cell culture 2. Tumor cell culture 3. Immortalized cell culture 10. Gene delivery techniques – to cultured cells Transfection 1. Liposome 2. Ca-phosphate 3. Electroporation 4. Gene gun Virus vector 1. Retroviruses 2. Adenoviruses 3. Adeno-associated viruses LIPOSOME DNA Protective layer crystallized drog Homing peptides Lipid Water-soluble drog bilayer 11. Gene delivery methods Liposome electroporation retrovirus 12. Foreign gene (transgene) promoter transgene T transfection 13. Foreign gene – transient gene expression 13. Foreign gene – transient gene expression 14. Foreign gene – stable gene expression 14. Foreign gene – stable gene expression 15. Genetically Modified Organisms (GMOs) Definition: addition of novel genetic material to the genome of an organism 1. Microorganisms 1. Basic research 2. Economic benefit 2. Plants 3. Animals 4. Human GMO: Genetically Modified Organism 16. Transgenic animals What are they? - Animals with one or more foreign genes in their genomes What are they good for? - (1) examination of gene function; (2) economically important GMOs, (3) gene therapy in future 17. Gene delivery techniques – to living organisms Germline gene delivery foreign gene is present in every cell 1. DNA microinjection zygote 2. Gene delivery to ES cells 3. Virus vectors (retroviruses) zygote Somatic gene delivery 1. Physico-chemical 2. Virus vectors 3. Stem cells foreign gene is NOT present in every cell Direct: in vivo Cell-mediated: ex vivo Delivery of foreign genes to zygote by pronuclear inoculation promoter foreign gene Pronuclear microinjection plasmid foreign gene with promoter + pronuclear injection reimplant injected embryos to pseudopregnant females transgenic founder transgenic offspring 18a. Foreign gene delivery 18b. to the zygote by pronuclear microinjection Pronuclei Inject foreign DNA into the male pronucleus + Fertilized mouse egg prior to fusion of male and female pronuclei Transfer injected eggs into foster mother About 10 to 30% of offspring contain injected foreign DNA. Foreign DNA is present in equal amounts in all tissues Mice expressing of foreign DNA are bred to continue DNA in germ line Delivery of foreign genes by retrovirus vector 19. Delivery of foreign genes to ES cells Internal cell mass Blastocyst 20. 21. 3. 1. 2. Cloned gene 5. 4. Gene delivery to ES cells 6. 8. 9. 22. Regulation of gene expression A. Transgene expression (gain of function 1. Constitutive (continuous) a. Ubiquitous expression UP transgene b. “Semi-ubiquitous expression PNP transgene c. Cell-specific expression TSP transgene IP transgene 2. Inducible B. Gene inactivation (loss of function) 1. Knock-out technology 2. RNA interference Cell-specific gene expression BAC - technology Regulatory elements Genomial DNA BAC 100,000- 400,000 bp RE digestion Original gene (exon + intron) Ligation Foreign gene Recombinant BAC Transformation Micro-inoculation Transgenic mouse 23. 24. Knock-out animals Investigation paradigm: the function of the knocked out gene is the reverse as the obtained phenotype is 2007 M. Evans: development of ES cell technology (ES cells are used for knock-out technology) M. Capecchi & O. Smithies: homologous recombination: - knock-out technology - replacement of defected genes with functional ones: importance in medicine 25. Generation of knock-out animals Genomic DNA Zygote blastula neo flanking seq. flanking seq. „Targeting” plasmid construction tk Inner cell mass Genomic DNA ES cells neo Genomic DNA Homologous recombination Genomis DNA neo Genomic DNA tk Illegitimate recombination Donor parents-1 Donor parents-2 x 1. neo x tk 3. 2. 5. Targeting sequence 4. Microinjection ICM Blastocyst ES cell culture Mixing of ES cells with the foreign DNA Selection of transgenecontaining cells 6. Recipient parent Transgenic ES cell integration to the embryo 8. Gene knock-out in ES cells Chimeric mice 9. x Chimera Homozygote normal Heterozygote Normal 10. x Heterozygote mice Homozygote knockout 26. 27. In vivo RNA interference 3b. lentivirus adenovirus What RNAi is good for? (1) analysis of gene function dsRNA shRNA- expressing DNS siRNA 3a. 1. cell membrane 1. 2. nuclear membrane DICER integration shRNA siRNA (2) shRNA episome DICER therapy: RISC (a) blocking the expression of mutant gene Stabile expression RISC Transient expression siRNA (b) blocking the overexpression of protooncogens in tumors Activated RISC mRNA What do we deliver? Where to deliver? (1) long dsRNA (2) siRNA LOCAL - SYSTEMIC Intracerebral Intravascular perfusion, Hypodermal injection (not applicable in mammals, since it evokes apoptosis) (systemic or local) Intraperitoneal (3) siRNA encoding DNA (systemic or local) (3a) Plasmid DNA (3b) Virus vector (lentivirus, adenovirus, adeno-associated virus) Types of siRNAs (2) In vitro transcription (1) Chemical synthesis extracellular space (3b) Virus vector (3a) Plasmid vector long dsRNA Dicer Dicer cytoplasm shRNA Nucleus shRNAcoding DNA 28. Dicer 29. RNA interference nucleus cytoplasm shRNA exportin DICER Degraded mRNA P body target mRNA DICER Acivated RISC Single-stranded siRNA shRNA: small hairpin RNA 30. The CRISPR system CRISP: clustered regularly interspaced short palindromic repeats