Philipp Christen  .  Rolf Jaussi .  Roger Benoit

Biochemie und Molekularbiologie

Eine Einführung mit 40 Lerneinheiten




39   Gentechnik


Inhaltsverzeichnis

Einführung Website

Merksätze

Kontrollfragen

39.1 Werkzeuge der Gentechnik: Restriktionsenzyme und andere Nucleasen, Ligasen, DNA-Polymerasen und Rekombinationsenzyme

39.2 Plasmide als Vektoren (Genfähren)

39.3 Viren als Vektoren; Gentherapieversuche

39.4 Künstliche Chromosomen als Vektoren

39.5 PCR (Polymerase chain reaction)

39.6 Genbanken: cDNA und genomische DNA

39.7 Bestimmung der Nucleotidsequenz von DNA

39.8 Southern, Northern und Western blotting

39.9 Expression rekombinanter Proteine und RNAs

39.10 Gezielte und zufällige Mutagenese

39.11 Präsentation von Genprodukten auf Bakteriophagen (Phage display) oder Ribosomen (Ribosome display); gerichtete molekulare Evolution

39.12 Klonierung von Zellen und Organismen; transgene Organismen

Weiterführende Literatur

Links

39.1 Werkzeuge der Gentechnik: Restriktionsenzyme und andere Nucleasen, Ligasen, DNA-Polymerasen und Rekombinationsenzyme

Restriction enzymes (David S. Goodsell, RCSB PDB): Struktur und Funktion der Restriktionsenzyme (mit Links zu 3D-Strukturen von EcoRI and EcoRV im Komplex mit DNA)

Molecular biology in medicine (M.W. King, themedicalbiochemistrypage.org): Molekulargenetische Methoden in der Medizin

REBASE (New England Biolabs, rebase.neb.com): Datenbank der Restriktionsenzyme

New England BioLabs (www.neb.com): Wichtiger Anbieter von Enzymen für die Molekularbiologie; Datenbank mit vielen guten Erklärungen und Beschreibung von Methoden

DNA Ligase (David S. Goodsell, RCSB PDB): Struktur und Funktion

Cre recombinase / DNA complex intermediate (Gopaul et al. 1998, EMBO J. 17, 4175-4187; RCSB PDB ): Struktur einer "Holliday Junction"

39.2 Plasmide als Vektoren (Genfähren)

Vector database (Addgene; www.addgene.org): Vektoren-Datenbank

DNASU plasmid repository (dnasu.org): Plasmid-Datenbank

39.3 Viren als Vektoren; Gentherapieversuche

M13 bacteriophage (Wikipedia)

Bacteriophage lambda vectors (P. McClean, www.ndsu.edu)

Gentherapie (DocCheck Flexikon; flexikon.doccheck.com)

Gentherapie (Y. Arsenijevic; Forum Genforschung der Akademie der Naturwissenschaften Schweiz (SCNAT); www.naturwissenschaften.ch)

39.4 Künstliche Chromosomen als Vektoren

DNA sequencing technologies key to the human genome project (H. Chial 2008, Nature Education 1(1):219): Technologien zur DNA-Sequenzierung, inclusive BACs

Comparative genome hybridization (B.J. Trask 2002, Nature Reviews Genetics 3, 769-778/ Nature Education): Beispiel einer Anwendung von BACs

Yeast artificial chromosome (Wikipedia)

39.5 PCR (Polymerase chain reaction)

Polymerase chain reaction PCR (DNA Learning Center, YouTube): PCR Animation

Press release, Kary B. Mullis, Michael Smith (Nobelprize.org): Pressemitteilung zum 1993 Nobelpreis zur Entwicklung von Methoden der DNA-basierten Chemie

PCR (www.ncbi.nlm.nih.gov): Erklärung der PCR

SYBR green I-based qPCR technical animation (www.sigmaaldrich.com): Animation zur qPCR mit SYBR green

Real-time PCR probe animation video (Biosearch technologies, www.biosearchtech.com): Animation zu den detaillierten Mechanismen der qPCR

Gibson Assembly (www.neb.com): Animation

39.6 Genbanken: cDNA und genomische DNA

GenBank (www.ncbi.nlm.nih.gov/genbank)

ORIGENE (www.origene.com): cDNA-Datenbank

39.7 Bestimmung der Nucleotidsequenz von DNA

The Nobel Prize in chemistry 1958 (Nobelprize.org): Frederick Sangers erster Nobelpreis

The Nobel Prize in chemistry 1980 (Nobelprize.org): Frederick Sangers zweiter Nobelpreis

J. Craig Venter Institute (www.jcvi.org): Institut von Craig Venter

Sequencing human genome: The contributions of Francis Collins and Craig Venter (Adams 2008, Nature Education 1(1):133): Zu den Beiträgen von Francis Collins und Craig Venter zur Sequenzierung des menschlichen Genoms

39.8 Southern, Northern und Western blotting

Southern blotting (Biology with Animations, YouTube)

Western blot transfer methods (www.piercenet.com)

Northern blot (Wikipedia)

39.9 Expression rekombinanter Proteine und RNAs

Cell-free protein expression (Promega Corporation): Übersicht zur zellfreien Proteinexpression

Gene silencing (www.ncbi.nlm.nih.gov): Kurzübersicht zum Gen-Silencing

Small interfering RNA (David S. Goodsell, RCSB PDB): siRNA Struktur und Funktion

Video animation: RNA interference (YouTube, Nature Video)

39.10 Gezielte und zufällige Mutagenese

Site-directed mutagenesis (New England Biolabs, www.neb.com): Übersicht zu Methoden der gezielten Plasmid-Mutagenese

Methods for site-directed mutagenesis (Integrated DNA technologies, www.idtdna.com): Methoden zur gezielten Plasmid-Mutagenese

Methoden zur Klonierung und Mutagenese von Plasmiden

CRISPR/Cas9 and targeted genome editing: A new era in molecular biology (www.neb.com)

Quick learning of CRISPR/Cas9 (OriGene Technologies Inc, YouTube)

Genome editing with CRISPR-Cas9 (McGovern Institute for Brain Research at MIT, YouTube): Einführende Animation

Genome engineering with CRISPR-CAS9 (iBiology, www.ibiology.org): Einführung durch Jennifer Doudna, kurzer Film

Davos 2015 - Rewriting human genes (World Economic Forum, YouTube): Discussion zur CRISPR-CAS9 Technologie

Ribosome display (Wikipedia)

Phagen-Display (Wikipedia)

Science explains: CRISPR diagnostics (Science Magazine, YouTube)

39.11 Präsentation von Genprodukten auf Bakteriophagen (Phage display) oder Ribosomen (Ribosome display); gerichtete molekulare Evolution

Directed evolution (Wikipedia): Gerichtete Evolution

Engineering and directed evolution of proteins (Plückthun lab, www.bioc.uzh.ch): Konstruktion und gerichtete Evolution von Proteinen

What is directed evolution? (www.kapabiosystems.com): Animation zur gezielten molekularen Evolution

Phage display & antibody library services (www.creative-biolabs.com): Übersicht zu einem Phage-Display Service

39.12 Klonierung von Zellen und Organismen; transgene Organismen

Genetically modified organisms (Philips 2008, Nature Education 1;1:213): Artikel zu genetisch modifizierten Organismen

Transgenic animals in agriculture (Wheeler 2013, Nature Education Knowledge 4;11:1): Transgene Tiere in der Agrikultur

Cloning (www.genome.gov): Übersicht zum Klonieren

Cloned animals (www.theguardian.com): Liste erfolgreich klonierter Tiere

Cloning (learn.genetics.utah.edu)

Transgenic crops (extension.colostate.edu): Transgene Pflanzen

Knockout mice (www.genome.gov): Übersicht zu Knockout-Mäusen

Transgenic animals (users.rcn.com): Transgene Tiere

Custom knockin generation (www.genoway.com): Website eines Anbieters von Knock-in-Mäusen, mit Informationen zum Prinzip

Weiterführende Literatur

Pingoud et al. 2014, Nucleic Acids Res - "Type II restriction endonucleases--a historical perspective and more"

Loenen et al. 2014, Nucleic Acids Res - "Type I restriction enzymes and their relatives"

Cohen et al. 1973, Proc Natl Acad Sci USA - "Construction of biologically functional bacterial plasmids in vitro"

Novick 1987 Microbiol Rev - "Plasmid incompatibility"

Dimitriu et al 2014, Proc Natl Acad Sci USA - "Genetic information transfer promotes cooperation in bacteria"

Rakowski and Filutowicz 2013, Plasmid - "Plasmid R6K replication control"

Camps 2010, Recent Pat DNA Gene Seq - "Modulation of ColE1-like plasmid replication for recombinant gene expression"

Alekshun and Levy 2007, Cell - "Molecular mechanisms of antibacterial multidrug resistance"

Nakaido 2009, Annu Rev Biochem - "Multidrug resistance in bacteria"

Christensen 2001, Mol Biotechnol - "Bacteriophage lambda-based expression vectors"

Nature January 2015, Gene Therapy

Macpherson and Rasko 2014, Intern Med J - "Clinical potential of gene therapy: towards meeting the demand"

Duncan and Hadlaczky 2007, Curr Opin Biotechnol - "Chromosomal engineering"

Mullis and Faloona 1987, Methods Enzymol - "Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction."

Ishino and Ishino 2014, Front Microbiol - "DNA polymerases as useful reagents for biotechnology - the history of developmental research in the field"

Heckman and Pease 2007, Nat Protoc - "Gene splicing and mutagenesis by PCR-driven overlap extension"

Gibson et al 2009, Nat Methods - "Enzymatic assembly of DNA molecules up to several hundred kilobases"

Lu 2005, Trends Biotechnol - "Seamless cloning and gene fusion"

Olieric et al. 2010, BMC Biotechnol - "Automated seamless DNA co-transformation cloning with direct expression vectors applying positive or negative insert selection"

Benson et al. 2007, Nucleic Acids Res - "GenBank"

Venter et al. 2001, Science - "The sequence of the human genome"

Venter 2011, Science - "Genome-sequencing anniversary. The human genome at 10: successes and challenges"

Nicholas and Nelson 2013, J Invest Dermatol - "North, south, or east? Blotting techniques"

Rosano and Ceccarelli 2014, Front Microbiol - "Recombinant protein expression in Escherichia coli: advances and challenges"

Studier 2014, Methods Mol Biol - "Stable expression clones and auto-induction for protein production in E. coli"

Goeddel et al. 1979, Proc Natl Acad Sci USA - "Expression in Escherichia coli of chemically synthesized genes for human insulin"

Kimple et al. 2013, Curr Protoc Protein Sci - "Overview of affinity tags for protein purification"

Bandaranayake and Almo 2014, FEBS Lett - "Recent advances in mammalian protein production"

Jarvis 2009, Methods Enzymol - "Baculovirus-insect cell expression systems"

Palmberger et al. 2013, Bioengineered - "MultiBac turns sweet"

Chaudhary et al. 2012, Nat Protoc - "Overexpressing human membrane proteins in stably transfected and clonal human embryonic kidney 293S cells"

Einstein and Yeo 2016, Science - "Making the cut in the dark genome"

Hoffmann and Bode 2016, Science - "A radical approach to posttranslational mutagenesis"

Ledford 2016, Nature - "CRISPR: gene editing is just the beginning"

Saha et al. 2021, Nature - "The NIH somatic cell genome editing program"

Doudna 2020, Nature - "The promise and challenge of therapeutic genome editing"

Wetzel et al. 2021, BIOspektrum - "Von Genen zu Chromosomen: Pflanzenzüchtung mit CRISPR-CAS"

Pacesa et al. 2022, Nature - "R-loop formation and conformational activation mechanisms of Cas9"

Dolgin 2020, Nature - "The kill-switch for CRISPR that could make gene-editing safer"

Dimova and Kietzmann 2018, BIOspektrum - "Genomveränderungen-CRISPR/Cas9 als Methode der Wahl oder Qual?"

Jayaraman 2009, J Genet - "Mutators and hypermutability in bacteria: the Escherichia coli paradigm"

Muteeb and Sen 2010, Methods Mol Biol - "Random mutagenesis using a mutator strain"

Kushwaha et al. 2014, J Vis Exp - "A protocol for phage display and affinity selection using recombinant protein baits"

Joyce 2007, Angew Chem Int Ed Engl - "Forty years of in vitro evolution"

Pande et al. 2010, Biotechnol Adv - "Phage display: concept, innovations, applications and future"

Tamerler et al. 2010, Biopolymers - "Molecular biomimetics: GEPI-based biological routes to technology"

Fujii et al. 2014, Nat Protoc - "Liposome display for in vitro selection and evolution of membrane proteins"

Doyle et al. 2012, Transgenic Res - "The construction of transgenic and gene knockout/knockin mouse models of human disease"

Klümper and Qaim 2014, PLoS One - "A meta-analysis of the impacts of genetically modified crops"

Hallowell et al. 2014, J Med Ethics - "Revealing the results of whole-genome sequencing and whole-exome sequencing in research and clinical investigations: some ethical issues"

Turner 2003, Nat Biotechnol - "The tyranny of 'genethics'"

Ye 2022, Nature - "Embryos with DNA from three people develop normally in first safety study'"

Weinstock et al. 2016, Nat Methods - "Vibrio natriegens as a fast-growing host for molecular biology"

Dunbar et al. 2018, Science - "Gene therapy comes of age"


Revision Juli 2024