Transcript Science and GMO-relevant technology

Science and GMO-relevant
technology
• Genes and genomes – last week
– Genomes and their inheritance and variation
– Genes and their structure
– Important methods: Gene cloning, PCR and
microarrays
• Biotechnology - today
– Basic concepts of cloning/regeneration
– Transformation methods
– Transgene structure/expression
Part I:
Getting whole plants back from
cultured cells
Organogenesis
Somatic
embryogenesis
Organogenesis – sequential
differentiation of new plant
organs (shoots, roots)
First step is dedifferentiation
into callus after
treatment with
the plant
hormone auxin
Leaf-discs
Shoots usually are produced first,
then roots in organogenesis
Somatic embryogenesis – shoot-root
axis differentiated as a unit
Immature cotyledon
Somatic
embryos
Repetitive embryogenesis = cloning
Somatic embryogenesis
Embryo growth
Physiological
maturity
Dry-down
for storage propagation
Somatic embryogenesis
Germination and plant recovery
Part II:
Getting DNA into plant cells
Main methods
Agrobacterium tumefaciens
Biolistics [gene gun]
Agrobacterium is a natural plant
genetic engineer
The Ti-plasmid is
required for crown
gall disease
T-DNA = Transferred DNA
T-DNA
Ti plasmid
Ti = Tumor inducing
The Ti Plasmid
Hormones cause gall growth, opines are special nitrogen
sources
Opine Catabolism
Cytokinin Synthesis
Auxin Synthesis
T-DNA
Right Border
Left Border
200000 bp
Virulence region
independent of TDNA
Opine Metabolism
Agrobacterium transfer is complex
Borders define start and end of T-DNA
Left Border
Right Border
Nick by VirD2
Nick by VirD2
New strand synthesis
Strand displacement
Preparation of T-strand
Vir E Vir E Vir E Vir E Vir E Vir E Vir E Vir E Vir E Vir E Vir E Vir E
Vir E Vir E Vir E Vir E Vir E Vir E Vir E Vir E Vir E Vir E Vir E Vir E
Export out of the cell
Disarming the T-DNA
Border
Border
Auxin
Synthesis
Cytokinin
Synthesis
Opine
Synthesis
Cut and replace
Antibiotic
Resistance
Gene of
Interest
Reporter
Gene
A chimeric gene
Promoter
Coding sequence
Level of expression
 Constitutive
 Tissue-specific


Terminator
Polyadenylation site
 Provides stability to
mRNA

Mix and match parts
Example of a map of plasmid used
in plant transformation GUS gene
encodes
glucuronidase
(cleaves pigment
to make blue
color): GUS
reporter gene
enables easy
visualization of
successful
transformation, and
where and when
genes are
expressed
Agrobacterium engineering
Gene of interest
T-DNA
Ti Plasmid
Engineered
plant cell
Agrobacterium tumefaciens
Cocultivation of Agrobacterium with wounded
plant tissues
Agrobacterium in contact with wounded plant
tissues during cocultivation
The gene gun
Plastic bullet
DNA on gold particles
Firing pin
.22 caliber charge
Stopping plate
Gene gun bombardment of plant
tissues in Petri dish
DNA coated metal particles after “gene-gun”
insertion into tissues
Transgenic cassava via biolistics
GUS reporter gene gives blue color
Part III:
Selection of transgenic cells
Only a few cells get engineered
Challenge: Recover plants from that one cell so
new plant is not chimeric (i.e., not genetically
variable within the organism)
Hormones in plant tissue culture
stimulate division from plant cells
Antibiotics in plant tissue culture
limit growth to engineered cells
Other kinds of genes can also be used to favor transgenic cells
(e.g., sugar uptake, herbicide resistance)
Antibiotic
selection
of
transgenic
tissues in
poplar
Summary of steps in Agrobacterium
transformation
Analysis of transgenic plants
Number of gene copies can vary
Junction fragment analysis reveals
number of gene insertion sites
Restriction enzyme sites shown with
arrows
flanking DNA
DNA
inserted gene
flanking
Transgene structure and
orientation can vary
Single, simple copies much preferred for stability
Transgene expression level varies
widely between insertions (“events”)
Partly due to failure to control where gene inserts in genome
Interpreting significance of GE’s
unintended effects on genome
• Lots of unintended genetic change in
breeding
• Lots of genetic variation in gene content
and organization
• No urgency to regulate traditional breeding
Varieties derived from
induced mutations
Over 2000 crop varieties derived
from mutagenesis have been
commercialized.
Calrose 76 semi-dwarf rice
High oleic sunflower
Rio Red grapefruit
Comparing GE to other breeding methods
Expert view on chance of unintended consequences for food quality
National Research Council (2004) http://books.nap.edu/execsumm_pdf/10977.pdf
Extensive natural genetic diversity
in gene structure/content (maize)
Natural deletions of genes/chromosome sections
Summary of some GE biological
issues to consider
• Events = unique gene insertion
– They vary widely in level/pattern of expression due to
chromosomal context / modification during insertion
– The unit of regulatory consideration at present
– Mutagenic changes at insertion site highly variable (deletions,
duplications)
– Can be “read-through” (Agro DNA beyond T-DNA transferred)
• Stability of gene expression and gene silencing
– A large number of insertions are not expressed
– Some lose/change expression over time
– Must select and test events carefully – single copy preferred
Summary of some GE biological
issues to consider
• Somaclonal variation = unintended mutagenesis due to
tissue culture & regeneration system
– Can be substantial, varies widely depending on culture system
– Must weed out via crossing, intense selection of events
• Increasing use of RNAi (RNA interference), as a general
means of gene suppression in research and commerce
– A way to knock out specific genes, inhibit viruses
– Genes with inverted repeat DNA create double-stranded RNA,
which induces sequence-specific RNA degradation or inhibition
of translation – very active area of basic and applied research
LAG
LSAG
Intron
LSAG
LAG
Discussion questions
• What aspects of gene transfer are most unclear?
– What are most important to understand for
interpreting biotechnologies?
• Should individual gene transfer events be the
focus of safety evaluations?
– Or should the type of gene in a specific crop be
regulated instead?
• Should GE crops that modify the expression of
native kinds of genes (ie, not introduce novel
kinds of genes) be regulated at all?