Download Genetic Engineering Methods

Genetic Engineering
Methods
Outline
‹Why do it?
¾Research examples: poplar trees
‹Plant gene transfer concepts and
methods
‹Getting genes ready for transfer
(recombinant DNA/plasmids)
‹Analysis of transgenic plants
Why do it?
‹ To create new, desired trait not in
accessible gene pool
‹ To breed faster, more directly
¾ Dominant, rare alleles
‹ To amplify genetic diversity for particular
genes/traits for breeding
¾ To breed with more direction, science basis
‹ To use plants as bioproduct factories for
industry/medicine
‹ To promote biosafety for exotic/unsafe crop
(domestication, sterility)
‹ In all cases, new traits/diversity feeds into
breeding to check yield/stability of whole
organism in the field: integrated health test
Native gene alteration example:
Glutamine synthetase (GS) overexpression stimulates growth of
poplars
‹ Hyper-expression of
GS to stimulate
ammonium nitrogen
incorporation into
proteins
‹ 3-year field trial
(Spain)
‹ Increased nitrogen
storage in stem
‹ Tree height up 41%
‹ Many examples,
abiotic stress
(F. Cánovas, U. Málaga, Spain)
Gene suppression example:
Lignin modification improves
pulping
‹ Energy and
chemical costs of
pulping great
‹ Success in
changing lignin
amount and
chemistry
¾ CAD- example
Pilate et al., INRA-France and UK
Pulp yield (%)
¾ 70 million tons
pulp (USA)
¾ ~$25 billion lignin
removal (USA)
60
55
50
45
40
35 15
GE
Control
17
19
21
23
Active alkali (%)
25
27
Exotic gene function example:
Gene from bacterium makes trees
more effective at bioremediation
‹ Thousands of square
miles in U.S. mercury
contaminated
¾ Neurotoxin,
biomagnified
‹ merA transgenic
cottonwood tolerates
~400 ppm mercury
‹ Volatilizing gene
¾ Wood-sequestering
genes under
development
S. Merkle & R. Meagher, U Georgia
Biosynthetic pathway example:
Modifying color/antioxidants
Malonyl-CoA+
coumaroyl-CoA
C2
C2
P
Chalcone
Vp1
C1
R/B
Flavanone
A1
Dihydroflavonol
PI
A1
Flavan-4-ol
R/B
Flavan-3,4-diol
3-Hydroxyanthocyanidine
Bz1
Phobaphenes
Anthocyanins
(ABA)
Biosafety example
Sterile trees can dramatically
reduce gene dispersal
‹Variety of genetic mechanisms
‹A tool to reduce
risk of invasion
by new exotic
nursery/forestry
species
Invasive Douglas-firs in Argentina (B Bond)
Plant gene transfer
concepts and methods
Summary of steps in
plant genetic engineering
Antibiotic selection of transgenic cells
Transformation
Antibiotic selection
Callus formation
Shoot generation
Root generation
DNA encodes both genes and
signals for their control
External Signal
Cell Receptor
Regulatory Elements
Promoter
Gene
GO
Where
When
How
Much
STOP
TRANSCRIPTION
mRNA
TRANSLATION
Protein
Terminator
Examples of Promoter:Gene Combos
Promoter
35S-CAMV
(virus)
Gene
Bt (bacteria)
Corn Stem
Bt (bacteria)
35S-CAMV
Round-up Ready© (bacteria)
Tomato Fruit 5X
Lycopene (tomato)
Reporter genes help visualize
transgenic cells, promoter activity
Any promoter
Any easily visualized gene
Fused to
GLO-FISH
Ubiqutious
Fluorescence
(JELLY FISH)
FLORAL
(POPLAR)
GUS
(BACTERIA)
Insertion of DNA into cells via
biolistics (“gene gun”)
Transgenic cassava via biolistics
Agrobacterium tumefaciens
agent of crown gall disease
Agrobacterium is the method of
choice for plant transformation
‹A common soil pathogen that
infects an large and taxonomically
diverse range of plants
‹A natural genetic engineer--gene
transfer is essential to its
pathogenic life style
‹It transfers DNA into plant cells to
cause gall formation, which
provides a home and nutrition for it
Agrobacterium has a sophisticated
gene transfer machinery
‹Pathogenesis depends on
presence of a very large plasmid,
called the Ti (tumor inducing)
plasmid, the source of its
transferred genes (T-DNA)
‹For biotechnology, the pathogenic
genes are removed, replaced by
useful genes
Agrobacterium life cycle
Agrobacterium transfer machinery
Agrobacterium Ti plasmid genes
LB
auxin
vir genes
T - DNA
cytokinin
opine
RB
ori
opine catabolism
Ti plasmid
Right and left border (RB,LB) sequences are the only
parts of T-DNA needed to enable transfer into
plants—removal of other T-DNA genes creates a
disarmed plasmid
Disarmed and binary vector system
Binary vectors live in E. coli too, and are used to modify & shuttle genes
LB
vir
genes
Disarmed
Ti plasmid
T- DNA
RB
Binary vector
Agrobacterium
Plant Cell
A sterilized paper punch
is used to cut “disks”
from plant leaves as the
first step in genetic
engineering.
The cells on the edges of
the disk are wounded in
the process of cutting so
they can receive a new
gene from Agrobacterium
tumefaciens.
Step-by-step view of poplar transformation
Summary of steps in regenerating
transgenic plants using Agrobacterium
Getting genes ready for
transfer
Restriction
Enzymes
cut DNA at
specific
DNA
sequences
(Alcamo. 1999. DNA Technology,
2nd Ed. Harcourt Press.)
Electrophoresis separates
DNA fragments based on their length
(Alcamo. 1999. DNA Technology, 2nd Ed. Harcourt Press.)
Construction of recombinant
DNA molecules
(Alcamo. 1999. DNA Technology, 2nd Ed. Harcourt Press.)
Boyer-Cohen
Experiment,
1973 showed
how genes
could be
cloned
Plasmids are small,
Circular DNA molecules
that can replicate
independently in a
host cell.
Foreign DNA inserted
into plasmids can
generate millions of copies
of the inserted gene.
(Alcamo. 1999. DNA Technology,
2nd Ed. Harcourt Press.)
Example of a map of binary plasmid
used in plant transformation
Map of binary plasmid used in precommercial plant transformation
Analysis of transgenic
plants
Example of repeated transgenes in
a plant genome caused by
transformation process
Southern (DNA) and northern (RNA)
blots of transgenic cassava
Produced via biolistics
DNA –gene presence
RNA-gene expression
Expression analyses of Agrobacterium
transformed tobacco
Level of expression varies widely among independent
gene transfer events
Many transgenic events need to be
tested to find ones that are
agronomically suitable
‹Dozens to hundreds tested prior to
commercial use
‹Stable gene and trait expression (look
for Mendelian inheritance like native
gene)
‹Single gene insertion for stability and
simple breeding
‹Desired level and pattern of expression
(position effects)
Many transgenic events need to be
tested to find ones that are
agronomically suitable
‹No deleterious effects on plant
health/nearby genes (i.e., lack of
somaclonal variation = unintended
mutations)
‹Introgression or insertion into other
varieties for commercial use
‹Regulation considers: Plant
biochemistry, novel protein safety,
allergenic potential, environmental
impacts
The genome is a complex, messy,
mutagenized, recombinant place!
Natural transposable elements
in the maize genome