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Reverse Genetics

Reverse Genetics. Mouse Embryos. J. Lieb April 19, 2006. Wild-type. Bmp7 -/-. Remember Forward Genetics? : Phenotype  Gene or Mutations First  Molecular Analysis Second. Reverse Genetics Gene  Phenotype or Molecular Analysis First  Mutations Second. Example Uses:

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Reverse Genetics

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  1. Reverse Genetics Mouse Embryos J. Lieb April 19, 2006 Wild-type Bmp7 -/-

  2. Remember Forward Genetics? : Phenotype  Gene or Mutations First  Molecular Analysis Second

  3. Reverse Genetics Gene  Phenotype or Molecular Analysis First  Mutations Second • Example Uses: • Understand the function of a gene homolog characterized in another organism • Understand the function of individual amino acids or protein domains. • Create “conditional alleles” of a gene.

  4. Review of Last Lecture • "Model" Organisms in Biology • What allows us to use them? • 1. All organisms share similar cellular machinery • All animals use this machinery in similar ways to direct embryonic development • Why use them? • Perform controlled experiments on a large number of samples to learn about: • Basic Molecular Mechanisms (Yeast Cell Cycle: Cancer) • Mechanisms of Genetics (Mendel’s Peas; Fruit Flies) • Embryonic Development (C. elegans, Sea Urchins; Mice) • What does one look for when choosing a model? • Fast, cheap, easy to observe and manipulate, and has the feature you want to study

  5. Review of Last Lecture Transgenics Using the power of molecular biology to isolate and clone the DNA of our choice, and then to express it in a controlled manner in the organism of choice. • Why? • To study the role of a gene in development • To see where a gene is expressed • To understand what happens • when a gene is misregulated • To “cover” a genetic defect

  6. GENE TARGETTING • Dirigido a un gen específico. • Silenciamiento del gen a nivel del RNA. • Reemplazo o modificación del gen.

  7. SILENCIAMIENTO DEL GEN • Acción a nivel de mRNA (evitando que sea traducido) • Mecanismos: • Oligonucleótidos Antisentido : oligonucleótido con la secuencia complementaria al mRNA blanco (target ) • Ribozimas: RNAs con actividad catalítica, se une y corta al mRNA blanco (target) • iRNA : RNA de interferencia

  8. Oligonucleótidos antisentido (Antisense oligos) • DNA o RNA de hebra simple • Secuencia complementaria al mRNA target • Mecanismo de acción : formación de doble cadena y: • Bloqueo del Inicio / elongación de traducción • Alteración del procesamiento del mRNA : si está dirigido a la secuencia limite exón/intrón bloquea la función de los snRNAs que intervienen durante el splicing • Alteración de la estabilidad / vida media del mRNA: si el oligo está dirigido a una secuencia en el 3´ UTR y que impida la formación de hairpins propios del mRNA que lo estabilizen; por ejemplo: mRNAs de histonas no tienen poly A, pero sí forman hairpins en el 3´ UTR.

  9. En vertebrados existen dos tipos de histonas: • Histonas independientes de replicación : sus mRNAs son polyadenilados • Histonas dependientes de replicación, sus mRNAs: - presentan cola de polyA - presentan una estructura de tipo stem-loop en el 3´UTR - presenta una secuencia de reconocimiento para el procesamiento o maduración del extremo 3´.

  10. mRNA de histonas dependientes de replicación El oligo Antisense puede estar dirigido a: - bloquear la formación del stem-loop - bloquear la interacción del mRNA con el snRNA.

  11. El pre-mRNA de HIV integrado debe formar 2 hairpins (TAR) necesarios para la unión de la proteína TAT al RNA Antisense dirigido a bloquear la formación de los hairpins

  12. Oligonucleótidos con mayor tiempo de vida media • PNAs : Peptide-nucleic acids • Esqueleto formado por enlaces peptídicos : estabilidad, mayor tiempo de vida media • Bases nitrogenadas : confieren especificidad de acción • Principal desventaja : • - toxicidad

  13. RIBOZIMAS • RNAs con actividad catalítica, se une y corta al mRNA blanco (target) • Estructura estable : hammerhead • Pueden ser quimicamente modificados los extremos para incrementar su vida media

  14. 5’- - c g g a g u c a c u u c g - - 3’ mRNA 3’ G C C U C A U G A A G C 5’ A I A G C G G C G C U G A U AG G C C G C G U C U Mecanismo de corte por ribozima Ribozima

  15. TRANSGENIA Conceptos Diferencia entre clonación y transgenia

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

  17. 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

  18. 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?

  19. 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

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

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

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

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

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

  25. 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

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

  27. 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

  28. 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

  29. Transgenic Technology Mosaic DNA 6hrs

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

  31. 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

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

  33. Knock-out Mice Embryonic Stem cells Homologous recombination Oliver Smithties Mario Capecchi Neomycin In chromatin Episomal Neomy Neomy X X mycin mycin Approx. 500 bp

  34. Homologous recombination 1- Length of homologous sequences 2- Isogenic DNA Hom. Rec. Efficiency Base pair 25bp 2000bp

  35. Gene targeting X X NEOMYCIN NEOMYCIN Total 4 Kbp (each arm not less than 500bp) Delete coding sequences Change reading frame Transcription of Neo in antisense direction

  36. Embryonic Stem cells 3 days Blastocyst C57Bl/6 Black 129/sv (agouti) Inner Cell Mass 1-totipotent 2-tissue culture Foster mother 2 day-pregnant 3-Transfectable 4-Selection 5- Differentiation In vitro

  37. X X 1 2 1 WT Hetero Homo

  38. Site specific recombination: Cre-Lox system, Flp recombination From P1 phage Excise and integrate DNA Lox site (approx 30 bp) Cre recombinase Excise Integrate a b c e g a b c f a c e f g b

  39. Lox mouse Cre lox in the mouse X Cre mouse KO in the brain only Brain KO in the adult only Adult-P - Temporal and spatial targeting - knock-in - single point mutation - translocation

  40. What about transgenics in mammals? Day 50 (end of Week 7)

  41. A relatively new (1980s)- molecular approach Recipe for a gene "knockout“ in mice: Step 1 Problem: Find a cell line that can grow in tissue culture but also retains the potential to become part of a real embryo. Solution- Embryonic stem cells

  42. ES or Embryonic stem cells: Blastocyst-stage cells that have been coaxed and coddled into growing in culture

  43. Blastocyst stage cells can be easily incorporated into a different blastocyst stage embryo, leading to production of chimeras

  44. A mouse with “3 parents”

  45. Adding a gene: Production of Transgenic Mice

  46. Production of Transgenic Mice 2 Embryonic stem cells (ES cells) are then incorporated into blastocysts, with the hope that they “go germline”. If so, a line is created

  47. Production of Transgenic Mice 3

  48. Production of Transgenic Mice 3

  49. OK, we've added a gene (Transgenics). Now, we want to make a KO(Reverse Genetics)

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