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Reverse Genetics J. Lieb Mouse Embryos 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: • 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. Review of Last Lecture "Model" Organisms in Biology What allows us to use them? 1. All organisms share similar cellular machinery 2. 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 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 GENE TARGETTING • Dirigido a un gen específico. • Silenciamiento del gen a nivel del RNA. • Reemplazo o modificación del gen. 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 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. 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´. 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. 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 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 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 Mecanismo de corte por ribozima 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 C U I G A A U G AG CG GC GC CG GC G U CU Ribozima TRANSGENIA Conceptos Diferencia entre clonación y transgenia WHY? •Identification of gene function •Generation of animal models of diseases •Drug validation •Cell and organ research Others… How many genes are there in mammalian cells? E. coli S. cerevisiae D. melanogaster C. elegans H. Sapiens 4.6 Mb 13.5 Mb 165 Mb 97 Mb 3,300 Mb 4,288 genes 6,034 genes 12,000 genes 19,099 genes 40,000 genes Genome project was completed in 2002 (still regions that are unclear) Genomics and proteomics Gene expression profiling Phenotypes (Exon profiling) Studied on the mechanism of gene expression. Transgenic technologies MAMMALIAN EMBRYO MANIPULATIONS Animal models? In theory, all mammalian embryos can be used. Mouse and rats Research Farm animals Commercial uses: Human Improve health, Cure diseases , others? The MOUSE Life span: approx. 2.5 years 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 MAMMALIAN EMBRYO MANIPULATIONS NON-TRANSGENIC No modifications to the genome. TRANSGENIC Modifications to the genome. Germline mutations Somatic cell mutations Transgenic and embryo manipulation in mammals 2 1 Transgenic Animals by pronucleus injection 3 -Genotyping of genetic diseases -Freezing and storing -Embryo cleavage and cloning Spermatozoide Natural or in vitro fertilization Morula Oocyte Enucleation Nuclear transfer and cloning 8 4 Stem cells Cell therapy 7 5 Neuronal cells - Isolaton of embryonic stem cells - Gene targeting in vitro Différentiation Muscle cells Blastocyst Epithelial cells 6 Primary cells culture Oocyte Fertilized egg Morula Zygote 6hrs 18hrs 36hrs Blastocyst Implantation 4 days 3 days 48hrs Oocyte -Nuclear Transfer -Cloning Fertilized egg Blastocyst Morula Freezing Splitting Genotyping Infection Transgenic By Microinjection -Embryonic stem Cells -Gene targeting Nuclear Transfer Technology For cloning Oocyte Nucleus of stem cells or others Nuclear Transfer Technology Usage? Cloning of: - Valuable cheptel (farm animals) - Endangered species - Basic research in stem cell tech. - Others? Gene Locus: Includes both the promoter and the transcription unit! Distal Proximal >100kb Approx. 1kb Promoter Coding and uncoding sequences from 1kb to >200kb Transcription Unit Transgenic Technology Promoter Intron cDNA AAAA Temporal and spatial expression Gene of interest AAAA Transgene <1kb to >200kb Transgenic Technology Promoter cDNA 1- Tissue specific (brain, liver, muscle …) 2- Ubiquitous 3- Inducible (tetracyclin, interferon... Gain of Function - wild-type gene - mutant Loss of Function - dominant negative - antisense - ribozyme Identification of the important features of a promoter. A: Comparison of sequences: Increasing uses Databases: Genebank and others. B: Use of reporter genes: Promoter of gene x Promoter of gene x Gene x Reporter gene - B galactosidase - Luciferase - GFP - others…. Transgenic Technology DNA 6hrs Mosaic Transgenic Technology Fos X X Ste. CD1 FVB/N Fos 6hrs Reimplantation in the oviduct Fos Pregnancy Transgenic Technology Advantages Disadvantages - Short time to produce (21 days) - High level of expression - Cheaper cost - Simple vectors - Random integration - Multiple integration sites - Each animal has different genotype Embryonic stem Cells and Gene targeting Gene knock-out and Gene Knock-in Knock-out Mice Embryonic Stem cells Homologous recombination Oliver Smithties Mario Capecchi Neomycin Episomal In chromatin Neomy X mycin Neomy X mycin Approx. 500 bp Homologous recombination 1- Length of homologous sequences Hom. Rec. Efficiency Base pair 25bp 2000bp 2- Isogenic DNA 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 Embryonic Stem cells Blastocyst 3 days C57Bl/6 Black 129/sv (agouti) Inner Cell Mass 1-totipotent Foster mother 2 day-pregnant 2-tissue culture 3-Transfectable 4-Selection 5- Differentiation In vitro X X 1 WT 2 Hetero 1 Homo Site specific recombination: Cre-Lox system, Flp recombination From P1 phage Excise and integrate DNA Cre recombinase Excise a b a c b a Integrate e c c b Lox site (approx 30 bp) g f e f g Lox mouse Cre lox in the mouse X Cre mouse KO in the brain only KO in the adult only Brain Adult-P - Temporal and spatial targeting - knock-in - single point mutation - translocation What about transgenics in mammals? Day 50 (end of Week 7) 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 ES or Embryonic stem cells: Blastocyst-stage cells that have been coaxed and coddled into growing in culture Blastocyst stage cells can be easily incorporated into a different blastocyst stage embryo, leading to production of chimeras A mouse with “3 parents” Adding a gene: Production of Transgenic Mice 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 Production of Transgenic Mice 3 Production of Transgenic Mice 3 OK, we've added a gene (Transgenics). Now, we want to make a KO (Reverse Genetics) Recipe to "knockout" a gene: Step 2 A normal cell has two copies of gene X: mRNA Gene X mRNA Mario Capecchi Gene X Scientists use homologous recombination to insert gene for resistance to the drug neomycin into the middle of one of the copies of gene X, destroying its function. Neo resistance gene No mRNA Gene X mRNA Gene X Recombinant ES cells can then be selected in culture. Oliver Smithies (UNC) Technique for Gene Targeting (1 of 3) Technique for Gene Targeting (2 of 3) Technique for Gene Targeting (3 of 3) Morphological Analysis of Bmp7 Knockout Mice Morphological Analysis of Bmp7 Knockout Mice Mouse models of human disease help us to design and test new treatments www.hgu.mrc.ac.uk/Research/Devgen/Cysfib/julia.htm www.cf.ac.uk/biosi/staff/jacob/teaching/ionchan/cftr.jpg A transgenic human Treated for SCID Other Reverse Genetic Approaches • Site-directed mutagenesis • RNAi • Chemicals (Chemical Genetics) Site-directed mutagenesis Gene Replacement RNA Interference Method 1 Method 2 Method 3 Mechanism of RNAi iRNA RNA de interferencia RISC: RNA induced silencing complex RNAi en la expresión de GFP Fig 4. Small interfering RNAs vs Small temporal RNAs Forward and Reverse "Chemical Genetics" REEMPLAZO DE GENES • Integración del fragmento de DNA en el genoma del hospedero- en el sitio del gen homólogo o en sitios al azar. La integración en sitios al azar es más frecuente. • Si existen secuencias homólogas en el genoma puede haber recombinación homóloga. Se da el reemplazo del gen específico del hospedero (Knock-out). • En la integración al azar, la expresión del gen puede verse afectada según el lugar de inserción, puede interrumpir o afectar a otros genes. • Células somáticas vs. Células germinales - transgénico. GENERACION DE ANIMALES TRANSGENICOS 1. Cultivo in vitro de células troncales de embrión (Embryonic stem- ES). 2. Preparación del gen a insertar. (Ej: BMP7 clonado, es interrumpido por el gen de resistencia a Neomicina como marcador de selección). 3. Transferencia del DNA exógeno a las células. 4. Selección de las células donde ha ocurrido el reemplazo del gen utilizando el marcador de selección (resistencia a Neomicina) 5. Las células seleccionadas se insertan en un embrión nuevo, el cual es colocado al útero. 6. Progenie resultante: quimeras con algunos tejidos heterocigotes y otros tejidos silvestres. 7. El cruce de una quimera con un animal silvestre dará una progenie heterocigote (BMP7+/BMP7-) si las cells ES modificadas han contribuido a la linea germinal. 8. Del cruce entre los heterocigotes, aprox. 25% de la progenie será homocigote trangénico BMP7- /BMP7- Ejemplo: knock-out del gen BMP-7 MARCADORES DE SELECCIÓN • Selección positiva : célula + marcador insertado la célula vive • Selección negativa: célula + marcador insertado la célula muere Por ej: • Gen de resistencia a Neomicina (marcador de selección positiva) Cell + Neor en presencia de Neomicina sobrevive • Gen Timidina Kinasa del HSV (marcador de selección negativa) Cell + TK HSV en presencia de ganciclovir muere