Download Genetic engineering methods

Genetic engineering methods
Steve Strauss
Steps in
genetic
engineering
Genetic engineering vastly increases
the genetic diversity available for breeding
Difficulties of inter-species
crosses
F. Nogue – INRA France
Overview of genetic engineering
Traditional
plant breeding
x
Variety
B
Variety
A
Genetic
engineering
x
any
gene
source
What are GMOs?
• GMO =
genetically modified organism
– Same as GE or GEO = genetic engineering
= creation of recombinant DNA
modified organisms
• It’s the method: Native or “foreign” genes, modified traits
or new traits
• Genes isolated in chemical form, changed in
a test tube, and re-inserted asexually
– Vs. making crosses or random mutations in conventional breeding
• Powerful breeding tool – relatively simple traits can be
designed – but without constraints from native gene pools
– That’s why its called genetic engineering, though we are
modifying, not building, a new organism
The acronyms, evolving in
meaning
• GE (genetic engineering) = GM (genetic modification) =
asexual modification and/or insertion of DNA
GMO = genetically modified organism
GEO = genetically engineered organism
The terms “biotechnology” or “modern biotechnology” often equated with
GE or GM methods
Transgenic = GE, or transfer of genes between distant
species
Cisgenic, intragenic for transfer or modification of genes
from closely related species
How are GMO crops produced
Step 1
Getting whole plants back from cultured
cells = organismal cloning
Differentiation of new plant
organs (shoots, roots, embryos)
from single cells
First step is dedifferentiation
into “callus”
after treatment
with the plant
hormone auxin
Leaf-discs
Shoots produced first, then roots,
using specific plant hormones for
each step
Somatic embryogenesis – shoot-root
axis differentiated as a unit
Immature cotyledon
Somatic
embryos
Repetitive embryogenesis = cloning
Step 2
Getting DNA into plant cells
Main methods
• Agrobacterium tumefaciens
• Biolistics [gene gun]
Agrobacterium is a natural plant
genetic engineer
Agrobacterium engineering
Gene of interest
T-DNA
Ti Plasmid
Engineered
plant cell
Agrobacterium tumefaciens
Historically, each insertion was unique in
terms of where it landed in plant genome
Cocultivation of Agrobacterium with
plant tissues
Agrobacterium in contact with
wounded plant tissues during
cocultivation
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
Step 3
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)
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, hormone sensitivity)
Antibiotic
selection of
transgenic
tissues
Then plants are propagated
normally (seeds, cuttings) and
tested for health and new qualities
Propagation of poplars
in tissue culture
Growth in the field
Genetic engineering is defined as…
A.
B.
C.
D.
Transfer of genes from one species to another
Modifying genes to produce new traits
Asexual modification of inherited DNA
A method with risks and benefits distinct from
that of conventional breeding
E. Creation of novel-appearing life forms
Why bother with in vitro culture when doing
GE, when we can simply treat whole plants
with Agrobacterium (like happens in Nature)?
A. Resulting plants would not be sterile
B. Its just routine scientific technique
C. The resulting plants would not be genetically
homogenous
D. Plants (but not animals) have cell walls that
stop Agrobacterium gene transfer
E. Antibiotic selection is much less efficient vs.
that in Petri dishes
Poor control over
transgene insertion
• Location in genome ~random
– Chromosomal environment important to level
and pattern of expression, not just promoters
– Expression varies a great deal among gene
insertion events
• Also varying among events
– Number of copies varies from one to dozens
– Orientation of gene varies
– Genes can be turned off, or be unstable
Large consequences of poor
transgene/transformation control
for biotechnology
• Gene transfer and tissue culture process
is itself mutagenic
– Insertion into/near genes
– Increased random mutation in genome from
stress of hormones/new developmental path
• Thus, extensive selection after gene
insertion for desirable events prior to
commercial use
– Hundreds screened, over many generations
• Event = unit of regulation worldwide
Interpreting significance of GE’s
unintended effects on genome
• How does it compare to conventional
breeding?
– Lots of unintended genetic change in making
hybrids, inbreeding, random mutagenesis
• Lots of genetic variation in gene
expression and gene content in nature
– Gene presence and absence highly variable
• No urgency to regulate traditional breeding
comparable to GE in spite of this
Time to end event-specific
regulation?
No greater unintended impacts from GE vs. breeding
Coming: Gene editing technology for
diverse traits
CRISPRs
TALENs
Adapted from Pennisi, Science, 2013)
CRISPRs: Predictable, stable, certain
change of DNA sequence
~50% biallelic mutation rate for genes in poplar
Wild type`
Non-mutants
Insertion
Mutants
Data from
Estafania
Elorriaga, PhD
student, OSU
AG-2
target site
Small deletions
Large deletion
AG-1
target site
Three types of Site Directed Nuclease (SDN)
“genome editing”
Genetic engineering has a similar function
to introgression – “gene purification”
Recombinant DNA (or
GM) allows a single
gene to be introduced
into a genome. This
method can be faster
and more precise
than conventional
breeding
Elite tomato
Poor tomato but disease
resistant
Elite, disease resistant tomato
Source of gene
(disease-resistant
plant)
Gene of interest
Isolate gene of
interest using
molecular biology
methods
Once a gene is
introduced into the
plant genome it
functions like any
other gene
Recombine into
recipient plant DNA
Why are GE methods used sometimes and
molecular breeding others?
Molecular breeding
1. Desired trait must
be present in
population or a wild
relative that can
hybridize
2. Genetic resources
must be available to
breeders (compatible
relatives)
3. Plant should be
propagated
sexually, and
efficiently
Photo credits: Gramene.org
Why are GM methods used sometimes and
molecular breeding others?
Molecular breeding
1. Desired trait must be
present in population
1. Gene can come from
any source or created
anew
2. Genetic resources
must be available
GM
2. Genetic resources not
required
3. Plant should be
propagated sexually
3. Plant can be
propagated vegetatively;
sex not needed
Photo credits: Gramene.org ETH Life International
Summary
• Genetic engineering requires knowledge
of gene-trait connection, and both insertion
and regeneration methods
– Biological and physical vectors for gene
insertion
• Terms used to define genetic engineering
evolving, confusing
– Trans – vs. cis/intra-genic
• Much variation among insertions with “old”
methods
• Genome editing targets gene changes and
insertions with high efficiency