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Titre de la présentation

This presentation discusses the concept of transgenic animals and the manipulation of mammalian embryos in research, including gene knockout, gene expression profiling, and the use of embryonic stem cells. The benefits and applications of these technologies in various fields are also explored.

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  1. Titre de la présentation

  2. Figure 2.1 The hierarchy of biological order from atom to organism

  3. Figure 1.5 The genetic material: DNA

  4. Figure 1.5x DNA

  5. Procaryotes: un seul compartiment

  6. L’ADN dans le Chromosome

  7. L’ADN dans le Chromosome

  8. Figure 5.x3 James Watson and Francis Crick

  9. Figure 16.4 Rosalind Franklin and her X-ray diffraction photo of DNA

  10. Figure 20.1 An overview of how bacterial plasmids are used to clone genes

  11. Figure 20.2 Using a restriction enzyme and DNA ligase to make recombinant DNA

  12. Figure 20.3 Cloning a human gene in a bacterial plasmid: a closer look (Layer 1)

  13. Figure 20.3 Cloning a human gene in a bacterial plasmid: a closer look (Layer 2)

  14. Figure 20.3 Cloning a human gene in a bacterial plasmid: a closer look (Layer 3)

  15. Figure 20.4 Using a nucleic acid probe to identify a cloned gene

  16. Figure 20.16 One type of gene therapy procedure

  17. Figure 20.18 “Pharm” animals

  18. Figure 20.19 Using the Ti plasmid as a vector for genetic engineering in plants

  19. Figure 20.20 “Golden” rice contrasted with ordinary rice

  20. Figure 20.x1a Laboratory worker reviewing DNA band pattern

  21. Figure 20.x2 Injecting DNA

  22. TRANSGENIC TECHNOLOGIES in Biomedical research Prof. Michel L. Tremblay michel.tremblay@mcgill.ca

  23. Definition: Transgenic Animal • Animal in which a segment of DNA has been physically inserted into the genome. • The genome of all cells of the organism contains the DNA segment. • The DNA segment is present in the germ line so it can be transmitted to progeny as a Mendelian trait.

  24. Retroviral Mediated Gene Transfer Mouse embryos MoMuLV Producing Cells Insertion of viral DNA containing transgene into embryos Transgenic Mouse

  25. PMS + HCG to superovulate 1-cell pronuclear stage mouse embryo. X 2-cell stage embryo Injection Pipette Holding Pipette Oviduct Transfer + +  Pregnant Foster Mother

  26. F e r t i l i z a t i o n R e i m p l a n t a t i o n M a t i n g M i c r o i n j e c t i o n C e l l D i v i s i o n G e s t a t i o n Events During Transgenesis E 1 E 2 E 2 1 D A Y 1 2 3 4 5 B i r t h E m b r y o R e c o v e r y PMS hCG 1 2 A M 1 2 A M 1 2 A M 1 2 A M 1 2 A M 1 2 A M 1 2 A M

  27. Embryonic Stem Cells • Pluripotent stem cells isolated form the inner cell mass of a blastocyst stage mouse embryo. • Cells retain ability to differentiate into all organs and cells. • Cells can be propagated under conditions in vitro that allow them to retain their totipotency- prevent differentiation in vitro. • Can be induced to differentiate in vitro into a number of cell types. ES cells can mimic early mammalian development in vitro. • Can be manipulated in vitro to alter the genome through homologous recombination.

  28. Pure Population of Targeted ES Cells ES cell Culture Targeted ES Cells Targeted ES Cells Injected into Blastocyst Uterine Transfer Chimera  Pregnant Foster Mother Blastocyst derived from mouse with white coat. X Germline transmission of ES cell-derived genome. Breed to WT white mouse

  29. B C D E F G E F G B C D Gene Knockout by Homologous RecombinationUsing an “O” or “Insertion” Type Construct A B C D E F G H I X neo A B C D E F G H I neo

  30. B C F G C F Gene Knockout by Homologous RecombinationUsing an “W” or “Replacement” Type Construct A B C D E F G H I X X TK neo A B G H I neo

  31. WHY? • Identification of gene function • Generation of animal models of diseases • Drug validation • Cell and organ research • Others…

  32. How many genes are there in mammalian cells? E. coli 4.6 Mb 4,288 genes S. cerevisiae 13.5 Mb 6,034 genes D. melanogaster 165 Mb 12,000 genes C. elegans 97 Mb 19,099 genes H. Sapiens 3,300 Mb 40,000 genes Genome project was completed in 2002 (still regions that are unclear) Genomics and proteomics Gene expression profiling (Exon profiling) Studied on the mechanism of gene expression. Phenotypes Transgenic technologies

  33. MAMMALIAN EMBRYO MANIPULATIONS Animal models? In theory, all mammalian embryos can be used. Research Mouse and rats Farm animals Commercial uses: Human Improve health, Cure diseases , others?

  34. Life span: approx. 2.5 years The MOUSE Gestation : 21 days Litter size: 8 to 12 Generation time: three months Several inbred and outbred strains Genomic database Most advance genetic technologies Cost per mouse/$2 to 24$ Housing cost Over 90% identical to human genome Large enough for physiological studies

  35. MAMMALIAN EMBRYO MANIPULATIONS NON-TRANSGENIC No modifications to the genome. TRANSGENIC Modifications to the genome. Germline mutations Somatic cell mutations

  36. Morula Fertilized egg Oocyte Zygote 18hrs 6hrs 36hrs 48hrs Blastocyst Implantation 3 days 4 days

  37. Fertilized egg Oocyte Blastocyst Morula -Nuclear Transfer -Cloning Freezing Splitting Genotyping Infection -Embryonic stem Cells -Gene targeting Transgenic By Microinjection

  38. Nuclear Transfer Technology For cloning Oocyte Nucleus of stem cells or others

  39. Nuclear Transfer Technology Usage? Cloning of: - Valuable cheptel (farm animals) - Endangered species - Basic research in stem cell tech. - Others?

  40. Gene Locus: Includes both the promoter and the transcription unit! Distal >100kb Proximal Approx. 1kb Coding and uncoding sequences from 1kb to >200kb Promoter Transcription Unit

  41. AAAA Transgenic Technology Intron Promoter cDNA AAAA Gene of interest Temporal and spatial expression Transgene <1kb to >200kb

  42. Transgenic Technology 1- Tissue specific (brain, liver, muscle …) Promoter 2- Ubiquitous 3- Inducible (tetracyclin, interferon... cDNA Gain of Function Loss of Function - wild-type gene - mutant - dominant negative - antisense - ribozyme

  43. Identification of the important features of a promoter. A: Comparison of sequences: Increasing uses Databases: Genebank and others. B: Use of reporter genes: Gene x Promoter of gene x - B galactosidase - Luciferase - GFP - others…. Reporter gene Promoter of gene x

  44. Transgenic Technology Mosaic DNA 6hrs

  45. Fos Fos Fos Transgenic Technology X Ste. X CD1 FVB/N 6hrs Reimplantation in the oviduct Pregnancy

  46. Transgenic Technology Disadvantages Advantages - Random integration - Multiple integration sites - Each animal has different genotype - Short time to produce (21 days) - High level of expression - Cheaper cost - Simple vectors

  47. Embryonic stem Cells and Gene targeting Gene knock-out and Gene Knock-in

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