Genetic engineering methods
Steve Strauss
Steps in genetic engineering
Genetic engineering vastly increases the genetic diversity available for breeding
F. Nogue – INRA France
Difficulties of inter-species crosses
Overview of genetic engineering
Traditionalplant breeding
x
Variety A
Variety B
Geneticengineering
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 organismGEO = 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 1Getting whole plants back from cultured
cells = organismal cloning
Differentiation of new plant organs (shoots, roots, embryos)
from single cells
Leaf-discs
First step is de-differentiation into “callus” after treatment with the plant hormone auxin
Shoots produced first, then roots, using specific plant hormones for
each step
Somatic embryogenesis – shoot-root axis differentiated as a unit
Immature cotyledon Somaticembryos
Repetitive embryogenesis = cloning
Step 2
Getting DNA into plant cellsMain methods• Agrobacterium tumefaciens• Biolistics [gene gun]
Agrobacterium is a natural plant genetic engineer
Agrobacterium engineering
Gene of interest
Agrobacterium tumefaciens
Engineeredplant cell
T-DNA
Ti Plasmid
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 3Selection 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 culturelimit 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
Growth in the fieldPropagation of poplars in tissue culture
Then plants are propagated normally (seeds, cuttings) and
tested for health and new qualities
Genetic engineering is defined as…
A. Transfer of genes from one species to anotherB. Modifying genes to produce new traitsC. Asexual modification of inherited DNAD. A method with risks and benefits distinct from
that of conventional breedingE. 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 sterileB. Its just routine scientific techniqueC. The resulting plants would not be genetically
homogenousD. Plants (but not animals) have cell walls that
stop Agrobacterium gene transferE. 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
TALENs
CRISPRs
Adapted from Pennisi, Science, 2013)
CRISPRs: Predictable, stable, certain change of DNA sequence~50% biallelic mutation rate for genes in poplar
AG-1target site
AG-2 target site
Wild type`
Non-mutants
Mutants
Insertion
Large deletionSmall deletions
Data from EstafaniaElorriaga, PhD student, OSU
Three types of Site Directed Nuclease (SDN)“genome editing”
Genetic engineering has a similar function to introgression – “gene purification”
Elite tomato Poor tomato but disease resistant
Elite, disease resistant tomato
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
Source of gene (disease-resistant
plant)
Gene of interest
Isolate gene of interest using molecular biology methods
Recombine into recipient plant DNA
Once a gene is introduced into the plant genome it functions like any other gene
Molecular breeding
Why are GE methods used sometimes and molecular breeding others?
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
2. Genetic resources must be available
3. Plant should be propagated sexually
GM
1. Gene can come from any source or created
anew
3. Plant can be propagated vegetatively;
sex not needed
2. Genetic resources not required
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
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