University of Indonesia / Jakarta: Genetic Engineering: State of The Art and Trends

University of Indonesia / Jakarta
February 25th 2019
Genetic Engineering: State of the Art and Trends
Hans-Jürgen Mägert, Anhalt University of Applied Sciences, Köthen, Germany

Genetic Engineering: State of the Art and Trends
1. INTRODUCTION
Definition of genetic engineering, milestones in history
2. MAIN PART
2.1. Classical genetic engineering

– Recombination by restriction and ligation
– Generation of libraries
– DNA/DNA hybridization
2.2. Advanced classical genetic engineering

– Polymerase chain reaction (PCR)
– T-vector cloning of amplicons
– Recombination by use of recombinases
– Recombination by PCR
2.3. New era in genetic engineering / trends

– Next generation sequencing
– In silico screening for functional genes / proteins
– Synthetic biology
– Using DNA as memory
– CRISPR/Cas9 system for genome editing
Literature
Cornel Mülhardt: Molekularbiologie / Genomics

Spektrum Akademischer Verlag, Heidelberg Berlin Oxford 2009
Tom Strachan, Andrew P. Read: Human Molecular Genetics

Taylor & Francis, 2010
Rolf D. Schmid: Pocket Guide to Biotechnology and Genetic Engineering

Wiley-VCH, Weinheim 2003
Michael L. Metzker: Sequencing technologies – the next generation

Nat. Rev. Genet. 11: 31-46, 2010
Jay Shendure and Hanlee Ji: Next-generation DNA sequencing

Nat. Biotechnol. 26: 1135-1145, 2008
Eric Young and Hal Alper: Synthetic biology: Tools to design, build, and optimize
cellular processes
J. Biomed. Biotechnol. 2010, Article ID 130781
Jeffery D. Sander and J. Keith Joung: CRISPR-Cas systems for genome editing,
regulation and targeting
Nat. Biotechnol. 32: 347-355, 2014
Many Websites
Genetic Engineering - Definition
Genetic engineering includes the entirety of

methods.......for generation of new genetic combi-
nations and the reintroduction and replication of
the recombinant genetic material in a different
biological environment.
Clone - Definition
clone
1. A group of organisms or cells that have arisen from a
single individual by asexual reproduction and are
therefore all genetically identical, e.g. a group of plants
propagated by bulbs or cuttings.
2. (gene clone) An exact replica of a gene.
A Dictionary of Science, Oxford University Press, ©
Market House Books Ltd 1999
Red genetic engineering
Gray genetic engineering
White genetic engineering
Green genetic engineering

Red genetic engineering
Genetic engineering in the field of
medicine:
– Basic research
– Establishment of cell lines suitable for the

screening for drug candidates
– Recombinant production of human agents
– Gene therapy
Gray genetic engineering
Applications of genetic engineering in the field
of environmental management:
– Cleaning of environment by phytoremediation
– Production of naturally degradable polymers (“natural

plastic")
– Establishment of cellular systems capable of detecting

pollutants
White genetic engineering
– „Smooth“ chemistry by optimization and

production of (industrial) enzymes
Green genetic engineering
Use of genetic engineering in agriculture
– Plants: herbicide tolerance, insect resis-

tance, virus resistance, change of the
composition of ingredients of plants
– Animals: increase of muscle meat

Also: Blue genetic engineering
(with marine organisms)
Black genetic engineering

(military applications?)
Milestones in the History of Genetic Engineering
The Early Beginning.....
Nobel Prize
1869 Friedrich Miescher (CH) – Isolation of nucleic acids
1933 (Ph. Med.) Thomas H. Morgen (USA) – Relevance of chromosomes
1940s Erwin Chargaff (A/USA) – Chemical composition of DNA
1962 (Ph. Med.) James Watson (USA) – Molecular structure of DNA

Francis Crick (GB)
Maurice Wilkins (GB)
DNA - Double Helix
Rosalind Franklin
DNA - X-Ray Diffraction Pattern
James Watson Francis Crick
Nobel Prize

Francis Crick (GB)
1968 (Ph. Med.) Robert W. Holley (USA) – Genetic Code

Har G. Khorana (USA)
Marshall W. Nierenberg
Genetic Code
The Beginning of Genetic Engineering
Nobel Prize

Francis Crick (GB)
1968 (Ph. Med.) Robert W. Holley (USA) – Genetic code

1978 (Ph. Med.) Werner Arber (CH) – Restriction endonucleases

Daniel Nathans (USA)
Hamilton O. Smith
Restriction Endonucleases
Class I Class II
recognition sequence specific, recognition sequence specific,
cutting site unspecific and cutting site specific and
distant from the recognition within the recognition sequence
sequence
Class III
recognition sequence specific,
cutting site specific but distant from the
recognition sequence
DNA Restriction Analysis
Insertion of Foreign DNA into a Plasmid
Bacterial Lawn with Phage Plaques (Clones)
Impact
– Cloning of genes for precise analysis
– Production of proteins in host organisms

Nobel Prize
1940s Erwin Chargaff (A/USA) – Chemical composition of DN

Francis Crick (GB)
1968 (Ph. Med.) Robert W. Holley (USA) – Genetic code

1978 (Ph. Med.) Werner Arber (CH) – Restriction endonucleases

Daniel Nathans (USA)
Hamilton O. Smith
1980 (Chem.) Frederick Sanger – DNA sequencing

DNA Fluorescence Sequencing Interferogram
Impact
– Conclusions from gene sequences on the encoded proteins
– Diagnostics – detection of mutations
– Check of constructs generated by genetic engineering

„Modern Age“ of Genetic Engineering
Nobel Prize
1993 (Chem.) Kary Mullis – Polymerase chain reaction

Polymerase Chain Reaction (PCR)
Impact
– Fast cloning of genes and cDNAs
– Generation of various DNA constructs
– Molecular Diagnostics
Kary Mullis
Nobel Prize
2007 (Ph. Med.) Mario Capecchi (USA) – Stem cells / DNA recombination,
Martin Evans (GB) transgenic animals
Oliver Smithies (USA)
Impact
– Generation of transgenic animals e.g. for analysis of

gene functions („knock out mice“) or as animal models
for human diseases
– Generation of transgenic animals for the production of

(human) proteins (drugs)
Leptin „Knock Out“ Mouse
Nobel Prize in Medicine 2007 for Oliver Smithies,
Mario Capecchi and Martin Evans
Transgenic fluorescent mice
Also generation of transgenic plants with.....
– Herbicide tolerance
– Insect resistance
– Virus resistance
– Change in metabolite pattern

„Golden Rice“ contains ß-carotene (provitamin A) (Ingo Potrykus, Peter Beyer)
Nobel Prize
1990 Start of the human genome project

Nobel Prize
2006 (Ph. Med.) Andrew Z. Fire (USA) – RNA interference (RNAi)

Craig Mello (USA)
RNA - Interferece
Impact
– Analysis of gene functions at the cellular level
– Tool for suppression of gene expression for analytical

purposes or for changing the pattern of metabolites in
plants
Craig C. Mello Andrew Z. Fire
Nobel Prize

Craig Mello (USA)
2003 Announcement of the decryption of the human genome

Impact
– Molecular diagnostics
– Discovery of novel therapeutic targets

Nobel Prize

Craig Mello (USA)
2008 (Chem.) Osamu Shimomura (USA) – Green fluorescent protein

Martin Chalfie (USA)
Roger Tsien (USA)
„Glow Cat“
Impact
– „Reporter“ for analysis of cellular processes
– Application also in screening assays for identification

of medically relevant factors
Nobel Prize

Craig Mello (USA)

Roger Tsien (USA)
2000 Beginning of „synthetic biology“*
2004 Beginning of the development of „next generation“ DNA sequencers

„Next Generation Sequencer“
Impact
– Sequencing of entire genomes in a few days (or soon in a

few hours)
– Bioinformatic analysis
– Molecular diagnostics
– Discovery of novel therapeutic targets
– Patient-adjusted therapies (pharmacogenetics)
– Synthetic biology
– Much more.....
Nobel Prize

Craig Mello (USA)

Roger Tsien (USA)
2000 Beginning of „synthetic biology“*
2004 Beginning of the development of „next generation“ DNA sequencers
2007 Group of Greg Venter generates artificial viable „minimal bacterium“
2011 Similar experiment by Japanese scientists with artificial cell body

Synthetic Biology
– Availability of libraries with well-defined genetic building

blocks
– Use of these building blocks for generation of new organisms

producing or doing whatever you want
DNA Synthesizer
Synthesized
genetic
building blocks
Nucleus
Cell Mitochondria
Products
http://www.synbioproject.org
Impact
– Metabolic engineering - production of desired chemical

compounds
– Detection of chemical pollutants and weapons
– Disease diagnosis and treatment
– Generation of biofuels
– Much more.....
2012 - CRISPR/Cas9
By Emmanuelle Charpentier and Jennifer Doudna
The novel CRISPR/Cas9 technology enables highly

specific (targeted) manipulations of the genome
CRISPR = Clustered Regularly Interspaced Short

Palindromic Repeats
Emmanuelle Charpentier
2012 - CRISPR/Cas9
By Emmanuelle Charpentier and Jennifer Doudna
The novel CRISPR/Cas9 technology enables highly

specific (targeted) manipulations of the genome
CRISPR = Clustered Regularly Interspaced Short

Palindromic Repeats
Thank you very much for your attention!

University of Indonesia / Jakarta: Genetic Engineering: State of The Art and Trends

Загружено:

Сведения о документе

Оригинальное название

Авторское право

Доступные форматы

Поделиться этим документом

Поделиться или встроить документ

Параметры публикации

Этот документ был вам полезен?

Это неприемлемый материал?

Авторское право:

Доступные форматы

University of Indonesia / Jakarta: Genetic Engineering: State of The Art and Trends

Загружено:

Авторское право:

Доступные форматы

University of Indonesia / Jakarta

February 25th 2019

Genetic Engineering: State of the Art and Trends

Hans-Jürgen Mägert, Anhalt University of Applied Sciences, Köthen, Germany

Definition of genetic engineering, milestones in history

2.1. Classical genetic engineering

2.2. Advanced classical genetic engineering

2.3. New era in genetic engineering / trends

Cornel Mülhardt: Molekularbiologie / Genomics

Tom Strachan, Andrew P. Read: Human Molecular Genetics

Rolf D. Schmid: Pocket Guide to Biotechnology and Genetic Engineering

Michael L. Metzker: Sequencing technologies – the next generation

Jay Shendure and Hanlee Ji: Next-generation DNA sequencing

Genetic engineering includes the entirety of

Gray genetic engineering

White genetic engineering

Green genetic engineering

– Establishment of cell lines suitable for the

– Recombinant production of human agents

– Cleaning of environment by phytoremediation

– Production of naturally degradable polymers (“natural

– Establishment of cellular systems capable of detecting

– „Smooth“ chemistry by optimization and

Use of genetic engineering in agriculture

– Plants: herbicide tolerance, insect resis-

– Animals: increase of muscle meat

Black genetic engineering

1869 Friedrich Miescher (CH) – Isolation of nucleic acids

1933 (Ph. Med.) Thomas H. Morgen (USA) – Relevance of chromosomes

1940s Erwin Chargaff (A/USA) – Chemical composition of DNA

1962 (Ph. Med.) James Watson (USA) – Molecular structure of DNA

1869 Friedrich Miescher (CH) – Isolation of nucleic acids

1933 (Ph. Med.) Thomas H. Morgen (USA) – Relevance of chromosomes

1940s Erwin Chargaff (A/USA) – Chemical composition of DNA

1962 (Ph. Med.) James Watson (USA) – Molecular structure of DNA

1968 (Ph. Med.) Robert W. Holley (USA) – Genetic Code

1869 Friedrich Miescher (CH) – Isolation of nucleic acids

1933 (Ph. Med.) Thomas H. Morgen (USA) – Relevance of chromosomes

1940s Erwin Chargaff (A/USA) – Chemical composition of DNA

1962 (Ph. Med.) James Watson (USA) – Molecular structure of DNA

1968 (Ph. Med.) Robert W. Holley (USA) – Genetic code

1978 (Ph. Med.) Werner Arber (CH) – Restriction endonucleases

– Cloning of genes for precise analysis

– Production of proteins in host organisms

1869 Friedrich Miescher (CH) – Isolation of nucleic acids

1933 (Ph. Med.) Thomas H. Morgen (USA) – Relevance of chromosomes

1940s Erwin Chargaff (A/USA) – Chemical composition of DN

1962 (Ph. Med.) James Watson (USA) – Molecular structure of DNA

1968 (Ph. Med.) Robert W. Holley (USA) – Genetic code

1978 (Ph. Med.) Werner Arber (CH) – Restriction endonucleases

1980 (Chem.) Frederick Sanger – DNA sequencing

– Conclusions from gene sequences on the encoded proteins

– Diagnostics – detection of mutations

– Check of constructs generated by genetic engineering

1993 (Chem.) Kary Mullis – Polymerase chain reaction

– Fast cloning of genes and cDNAs

– Generation of various DNA constructs

1993 (Chem.) Kary Mullis – Polymerase chain reaction

– Generation of transgenic animals e.g. for analysis of

– Generation of transgenic animals for the production of

– Change in metabolite pattern

1993 (Chem.) Kary Mullis – Polymerase chain reaction

1990 Start of the human genome project