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