4 Eukaryotic Vectors

4.1 Saccharomyces cerevisiae Vectors

4.2 Mammalian Cell Vectors

4.3 Plant Vectors

4.1 Sacchomyces cerevisiae Vectors

4.1.1 YIp = Yeast Integrating plasmid4.1.2 YRp = Yeast Replicating plasmid4.1.3 YCp = Yeast Centromeric plasmid4.1.4 YEp = Yeast Episomal plasmid4.1.5 YAC = Yeast Artificial Chromosome

4.1.1 Integrating Plasmid (YIp)

Integrates into the yeast genome to become passively replicated

Integration occurs via homologous recom-bination

Unstable in the absence of selective pressure= integration reversible

The Yeast Integrating Vector YIp5

4.1.2 Replicating Plasmid (YRp)

Plasmids contain an ARS (Autonomously Replicating Sequence)

ARS: 25 – 65 bp in length

Plasmids unstable due to the lack of a centromer

The Yeast Replicating Vector YRp7

4.1.3 Centromeric Plasmid (YCp)

Plasmid with an ARS and a CEN Are stably replicated and segregated Play an important role in the Y2H (Yeast Two (2) Hybrid system

The Yeast Centromeric Vector YCp50

4.1.4 Episomal Plasmid (YEp)

The Yeast 2 μm Plasmid

The Yeast Episomal Vector YEp24

4.1.5 Artificial Chromosome (YAC)

A Yeast Artificial Chromosome Vector

The YAC Cloning System

4.2 Vectors for Mammalian Cell

Cultures

1. SV40-Based vectors

2. Vectors based on retroviruses

4.2.1 Vectors Based on SV40

SV40 = Simian Virus #40

Genetic Map of the Mammalian Cell Virus SV40

Permissive cells

Virus replication in African Green Monkey cells

Non-permissive cells

Integration in mouse or hamster cell lines

Phases of lytic infection:

1. First 8 h: Virus is uncoated, DNA moves to the nucleus

2. Next 4 h = early phase: Synthesis of early mRNA and early proteins

3. Next 36 h = late phase: Synthesis of viral DNA, late mRNA, late proteins, production of virusparticles

Regulatory Elements Within the ori Region of SV40

G/C-rich 21-bp direct repeats: recognized by cellular transcription factors

T-antigenMultifunctional phosphoprotein involved in1. Initiation of viral DNA synthesis2. Stimulates its own synthesis3. Stimulates host DNA replication4. Stimulates transcription of host genes

including DNA ligase, DNA polymerase α

t-antigenEssential for integration of SV40 DNA in non-permissive cells

Three types pf SV40-based vectors:

1. Replacement vectors leading to the production of virus particles (need a helper virus): size restricted

2. Autonomously replicating vectors (need T-antigen): not size restricted

3. Transient expression vectors

SV40-Based Transient Expression Vector pMSG

Selection marker

4.2.2 Vectors Based on Retroviruses

The Life Cycle of a Retrovirus(two identical RNA molecules + reverse transcriptase)

Provirus

Structure and Products of a Provirus

enhancer + promoter

LTR = Long Terminal Repeat

The Retrovirus Cloning System

ψ = packaging sequence

Formation of

Recombinant

Retrovirus Particles

Transgenic Mouse With the Human Growth Hormone Gene

4.3 Vectors for Plants

There are four important methods:

1. The Ti plasmid

2. The biolistic method

3. Protoplast transformation

4. Homologous recombination

4.3.1 The Ti Plasmids

SB Gelvin (2003) Microbiol Mol Biol Rev 67: 16

Jeff Schell

1935 - 2003

Crown Gall Disease

Transfer of the T

DNA Into Plant

Cells

border sequences

Schematic Drawing of a Ti Plasmid

~200 kb

Opines: Series of unusual amino acid or sugar derivatives specifically synthesized by the crown gall tumor cells

agrocinopine A and B

agropine nopaline octopine

Model of the Biogenesis of the T-PilusRod Mediated by the Transmembrane

Apparatus

E-M. Lai (2000) Trends Microbiol. 8: 361-369.

Some of the Vir Proteins Involved in the Transfer of T-DNA

There Are Two Strategies How to Transfer Recombinant Genes Into

Plant Cells

1. The binary strategy

2. The cointegration strategy

The Binary Vector Strategy

The Cointegration Strategy

Construction of

Recombinant

Plants

Part I

Construction of

Recombinant

Plants

Part II

The Ti Plasmid Can Also Transfer Chromosomal DNA

Analysis of Arabidopsis thaliana insertions:~0.4% of the insertion sites contain bacterial chromosomal DNA (up to ~18 kb) Happens also in rice

B Ülker (2008) Nature Biotechnol. 26: 1015

Host Range of Agrobacterium

The host range of A. tumefaciens has been extended to

yeast filamentous fungi cultivated mushrooms human cultured cells

B Lacroix (2006) Trends Genetics 22: 29

4.3.2 The Biolistic Method

Gene Delivery Microprojectiles Coated

with DNA

wolfram or gold particles

Gene Gun

Transient Expression using the Biolistic Gun

4.3.3 Protoplast Transformation

Protoplasts are prepared and treated with DNA

Definition:Protoplasts (= spheroplasts) are cells without their cell wall

4.3.4 Homologous Recombination

Aim:Sequence-specific insertion

Problem:Retrovirus DNA and T-DNA insert at random into one of the chromosomesThis can cause a mutation

Components of an Artificial Sequence-Specific Recombination

System Causing Double-Strand Breaks at a Predetermined Site

1. Zinc-finger domain which recognizes specifically three base-pairs

2. DNase domain which carries out a double-strand break

Catalysis of a Site-Specific Double-Strand Break

KA High (2005) Nature 435: 577-579.

Site-Specific Double-Strand Break

1. Zinc-finger nuclease (ZFN): Are composed of a DNA-binding domain and a DNA-cleaving domain

2. Each zinc finger binds to a particular three-base DNA sequence

3. Linking four zinc fingers allows recognition of 12 bases

4. Two ZFNs are needed to obtain a double-strand break

Sequence-Specific Endogenous Gene Modification Using ZFN

MH Porteus (2009) Nature 459: 337

First Transgenic Plants

Maize: IPH1 locus: Integration of a herbicide-resistance gene (3-20% correct integrations)

VK Sukla (2009) Nature 459: 437

Tobaco: SuRB gene: small changes

JA Townsend (2009) Nature 459: 442

4.3.5 Chloroplast Transformation

Aim:To insert transgenes into chloroplast DNA

Insertion of Genes Into Chloroplast DNA = Transplastomic Plants

Benefits:1. High level of transgene expression 2. Delivery of multiple genes in a single

transformation event 3. Absence of gene silencing 4. Absence of position effect due to site-

specific transgene integration 5. Transgene product localized within the

chloroplast

Characteristics of Chloroplasts, I

1. Found in plant cells and eukaryotic algae 2. Are considered as derivatives of a

cyanobacterial anchestor3. Site of photosynthesis 4. Sequestration of carbon, production of

starch, synthesis of amino acids and fatty acids

5. Land plant chloroplasts typically contain 110 to 120 unique genes

Characteristics of Chloroplasts, II

6. In most angiosperm plant species, plastid genes are inherited maternally (not disseminated by pollen)

7. Up to 100,000 copies of the chloroplast genome in each plant cell: Foreign protein can accumulate up to 46% of total leaf protein

D Verma (2007) Plant Physiol 145: 1129

Selectable Marker for Chloroplasts

1. aadA: spectinomycin resistance 2. neo: kanamycin resistance 3. badh: betaine aldehyde dehydrogenase;

converts the toxic compound betaine aldehyde to glycine betaine

Chloroplast Transformation

Biolistic method: Gold particles coated with the recombinant plasmid DNA

Which transgenes are introduced into plants ?

Transgenic plants of the

First generation

Second generation

Transgenic plants of the first

generation contain the following

genes:

1. Herbicide tolerance2. Virus resistance3. Fungus resistance4. Insect resistance5. Dessication resistance6. Salt tolerance

Insect Resistance

Bt toxin (protein); encoded by Bacillusthuringiensis

The Bt toxin is eaten by the insects andperforates the gut

Bt toxin is used since about 1920- Bacteria are sprayed on the plants- Protein produced and sprayed on the plants- Toxin is produced by transgenic plants

There are more than 100 different Bt toxins

Transgener Mais

Problem: Maiszünsler

Pilze produzieren Gifte = Mykotoxine

Der Maiszünsler und mögliche Folgen

Mehr als 300 verschiedene Mykotoxine bekannt Jedes Mykotoxin übt spezifische Wirkung aus:- Zearalenon: Fruchtbarkeitsstörungen - Fumonisin: verhindert die Aufnahme von Folsäure (Vitamin B9)

Mykotoxingehalt in BT-Mais stark reduziert (50%)

Mykotoxine

Mutterkorn: Claviceps purpurea

DarmkrämpfeDurchblutungsstörungen5-10 g tödlich

Transgenic plants of the second generation:

Golden riceCanola producing omega-3 fatty acids (unsaturated fatty acids)

Golden Rice = Provitamin-A-Reis

Geschälter Reis enthält kein ß-Carotin

Über 200 Millionen Kinder und Frauenleiden an Vitamin-A Mangel

Über 500 000 Kinder erblinden pro Jahr(60 pro Stunde)

2 Millionen Kinder sterben pro Jahr anden Folgen des Vitamin-A Mangels

Ingo Potrykus (ETH-Zürich, Schweiz)

Peter Beyer (Uni-Freiburg, Deutschland)

Gentechnisch veränderter Reis mit Provitamin A

Konventionelle Züchtung und

Gentechnik führen zum gleichen Ziel

Beispiel: Amylopectin-Kartoffel

Kartoffeln produzieren zwei Stärke-Varianten:Amylose (20%) und Amylopectin (80%)

Ziel: Kartoffeln, die nur Amylopectin produzieren

(Bindemittel, z.B. in Kleister)

Transgenic Plants for Phytoremediation

Phytoremediation: Use of plants to clean up environmental pollution

Plants can absorb a diversity of natural and man-made toxic compounds: Detoxification mechanisms

E Pilon-Smits (2005) Annu Rev Plant PhysiolPlant Mol Biol 49: 643

PhytoremediationRhizofiltration: Adsorption to the rootsPhytoextraction: Uptake inside plant tissuesPhytotransformation:Transformation by plant enzymes

Rhizosphere bioremediation: Degradation by microbesPhytostabilization: Incorporation in soil material

B van Aken (2008) Trends Biotechnol 26: 225

Success in Phytoremediation

Transgenic plants have been developed able to memove

nitroaromatic explosives TNT and RDX herbicidesmetals: mercury, lead, cadmium, selenium

CL Rugh (1998) Nat Biotechnol 16: 925N Hannink (2001) Nat Biotechnol 19: 1168

WY Song (2003) Nat Biotechnol 21: 914EL Rylott (2006) Nat Biotecnol 24: 216

EAH Pilon-Smits (2006) Front Ecol Environ 4: 203

Characteristics of Poplar Trees (Genus Populus)

SL Doty (2007) PNAS 104: 16816

1. Contains fast-growing species 2. Extensive root system 3. High water uptake 4. Can be transformed using Ti plasmid5. Can remove trichlorethylene and carbon

tetrachlorids from the air

Phytoremediation of Volatile Environmental Pollutant with Poplar

Trees

SL Doty (2007) PNAS 104: 16816

Introduction of the mammalian gene coding for cytochrome P450: Transgenic trees were able to remove volatile hydrocarbons, including trichloroethylene, vinylchloride, carbon tetrachloride, benzene and chloroform

Verletzte Pflanzen rufen die Feinde ihrer Feinde

Arabidopsis thaliana:Transgene Pflanzen produzieren zwei neue IsoprenoideWerden bei InsektenfraßfreigesetztLocken räuberische Milben an

Weltweite Anbauflächen von GVO Pflanzen

2000: 40 Mill. Hektar

2008: 125 Mill. Hektar

Weltweite Anbauflächen von GVO Pflanzen

http://www.transgen.de/anbau/eu_international/201.doku.html

Anbauflächen in Mill. Hektar 2008USA 62,5 S,M,B,R, Squash,PapayaArgentinien 21,0 S,M,BBrasilien 15,8 S,M,BIndien 7,6 BKanada 7,6 R,M,S, ZuckerrübeChina 3,8 B, Pappeln, Papaya, TomatenParaguay 2,7 SSüdafrika 1,8 M,S,BUruguay 0,7 S,MEU 0,1 M http://www.transgen.de/anbau/eu_international/

531.doku.html