Download Molecular Strategies for detection of insertion of genes in transgenic

Molecular Strategies for
detection of insertion of
genes in transgenic plants
Dr Anil Kumar
Associate Professor & In charge
Dept. Of Molecular Biology &
Genetic Engineering, CBSH
GBPUAT Pantnagar Uttranchal
Pin-263245
Genes and
Proteins
transcription
mRNA
Gene
(a piece of DNA)
protein
trait
translation
Any agriculturally important trait can be introduced by
incorporation of a useful gene
Transgenic Technology : Art and
science of creating designer crops
• Identification of useful gene (s)
• Creation of a suitable gene construct
• Transfer of this construct in to plant cells/tissues in
vitro ( maintained as organized explants such as
immature embryos, stem sections, cotyledons etc.
Using tissue culture techniques, through a process
called transformation.
• Selection of transformed cell lines or seedling using
a suitable marker system and regeneration of fertile
plants from the transformed cells.
• Analysis of transformed plants for several aspects
including stable integration, expression and genetic
behavior of transgene(s)
DESIGNER CROPS OF SUPERIOR TRAITS
•
Agronomic Traits
– Biotic Stress
• Insect Resistance
• Disease Resistance
– Viral, Bacterial, Fungal, Nematode
• Weed, herbicide tolerance, parasitic weeds
– Abiotic Stress
• Drought, Cold, Heat, Salinity, Poor soils
•
Quality Traits
–
–
–
–
–
–
–
–
–
•
Yield- Nitrogen Assimilation, Starch Biosynthesis, O2 Assimilation
Processing
Shelf-life
Reproduction: sex barriers, male sterility ,seedless ness
Nutrients (Nutraceuticals)
• Macro: Protein, Carbohydrates, Fats, Fiber
• Micro: Vitamins, Minerals, Antioxidatnts, Isoflavonoids,
Phytoestrogens, Condensed tannins
• Anti-nutrients: Phytase, Allergen and Toxin removal
Taste
Fiber, quality, strength, natural colors
Architecture
Ornamentals: color, shelf-life, morphology, fragrance
Novel Crop Products
– Oils
– Proteins: nutraceuticals, therapeutics, vaccines
– Polymers
•
Renewable Resources, Biofuels, feedstocks for synthetics
How are Transgenic crops made ?
Locating Genes for Plant Traits
Identifying & locating genes for agriculturally important
traits: Most limiting step in transgenic process
Little is Known about specific genes
to enhance yield potential
improve stress tolerance
modify chemical properties of harvested products
otherwise affect plant characters
Identifying a single gene for a trait is not sufficient
Must understand how the gene is regulated
What other effects it might have on the plant
How it interacts with other genes active in the
same biochemical pathway
Designing Genes for Insertion
Once a gene has been isolated & cloned : it must be suitably
modified for effective insertion into a plant
A promoter sequence must be added for the gene to be correctly
expressed
Sometimes, the cloned gene is modified to achieve greater
expression in a plant
The termination sequence signals to the cellular machinery that
the end of the gene sequence has been reached
A selectable marker gene is added to the gene "construct" in order
to identify plant cells or tissues that have successfully integrated the
transgene.
Simplified representation of a constructed transgene, containing
necessary components for successful integration and expression
Promoters used for Transgene expression
It is the ON/OFF switch : controls when & where in the plant the
gene will be expressed
Most promoters used are CONSTITUTIVE : cause gene
expression throughout the life cycle of plant in most tissues.
CaMV 35S Promoter : Most commonly used & gives high degree
of expression in plants
Other promoters : More Specific : respond to cues in the plant’s
internal & external environment
Promoter from cab gene (encoding chlorophyll a/b
binding proteins) : Light – inducible
Promoters from Arabidopsis : Specifically & rapidly
induced by natural plant stress/wounding related
semiochemical cis-jasmone
How are Transgenic
Plants Produced?
Commonly Used Methods:
• Agrobacterium
tumefaciens
• Gene Gun / Biolistics
• Electroporation
Transformation System
Protoplast
Electroporation/
PEG mediated
transformation
Immature
embryo (IE)
Embryo derived
calli (EDC)
Biolistic
transformation
Agrobacterium
mediated
transformation
Selection in presence of
Selectable marker gene
Plant regeneration
Molecular characterization
event selection/gene expression/function of transgene
Development of transgenic plants
Transgenic Plants- From Lab to the field
Transgenic lines obtained through
Transformation and regeneration
Laboratory analysis to confirm
¾ Stable integration of transgene(s)
¾ Number of copies of the transgene(s)
¾ Expression of transgene(s)
Monitoring and analysis of transgenics in a
Containment (Glasshouse) Facility for
¾ Stability of transgene expression
¾ Agronomically desirable expression of transgenic trait
¾ Genetic behavior of transgene(s)
¾ Biosafety evaluation and risk assessment
Small plot (<500m2) field experiments
To evaluate agronomic performance
And to further analyze biosafety
Large scale field testing at multiple sites
Commercilization of transgenic variety
After risk assessment and evaluation of
Net benefit offered by the transgenic
Monitoring of transgenic variety during
commercialization
TRANSGENIC CROPS DEVELOPED
SO FAR AT PANTNAGAR
PRODUCTS
GENE
INTROGR
-ESSED
GENETICALLY ALTERED TRAITS
WHEAT
PAT
HERBICIDE RESISTANCE (BASTA)
BRASSICA
OSMOTIN
TOLERANCE TO ALTERNARIA BLIGHT AND
SALINITY
BRASSICA
ANNEXIN
MODULATION OF HYPERSENSITIVE RESPONSE
AGAINST ALTERNARIA BLIGHT AND POWDERY
MILDEW
TOMATO
CARROT
Pr M E
Pr M E
GUS
PRODUCTION OF EDIBLE VACCINE AGAINST
JAPANESE ENCEPHALITIS VIRUS
Core Characterization
•Gene(s)
– Source(s)
– Molecular characterization
– Insert / copy number / gene
integrity
•Protein(s)
– History of safe use and
consumption
– Function / specificity /
mode-of-action
– Levels
– Toxicology / allergenicity
testing
•Food/Feed
Composition
–
–
–
–
Proximate analysis
Key nutrients
Key anti-nutrients
Animal performance
assessment
•Environmental
– Host organism
– Safety to non-target
organisms
– Soil degradation, toxicity
– Outcrossing, weediness
METHODS TO CHECK GENE INTEGRATION
Gene that facilitates the detection of genetically modified plants
during development
Use of selectable marker genes-Antibiotic resistance markers
-Herbicide resistance markers
-Non-antibiotic based markers
These genes coming up
Use of a reporter genegene whose expression is easy to observe.
Used to “report” gene expression,
regulation and localization
•Luciferase (luc) gene
•Green fluorescent protein (GFP
•GUS (uidA) gene
Antibiotic/Herbicide
resistance Marker
Substrate for
selection
npt II (Neomycin
Phosphotransferase)
G418, Kanamycin,
Neomycin
hpt (Hygromycin
Phosphotransferase)
Hygromycin
dhfr (Dihydrofolate reductase)
Methotrexate,
Trimethoprim
ble
Bleomycin
gat (Gentamycin Acetyltransferase)
Gentamycin
bar & pat (Phosphinothricin
acetyltransferase)
Phosphenothricin
(Bialaphos, Basta)
epsps (5-enolpyruvylshikhimate-3phosphate synthase)
Glyphosate
Risks & Concerns of using Antibiotic/Herbicide
Resistance Genes
Horizontal Gene Transfer
Transfer of these genes from the transgenic plants to the
microbes
Make the bacteria in the guts of animals and humans
resistant to antibiotics
Make antibiotic medicines less effective
Transfer to soil microbes : A field study shows that
transgenic DNA persists for 2 years after the GM crop has
been harvested
Other genes used as Selectable Marker Genes
man A gene from E coli
encodes mannose phosphate transferase and confer
upon transformed cells ability to use mannose as a sole carbon
source
Ipt (isopentenyl transferase) gene from Agrobacterium
located on T-DNA and induces cytokinin synthesis. Plants
selected on the basis of their ability to produce shoots from
callus on medium lacking cytokinins.
Betaine aldehyde dehydrogenase gene (BADH) from Spinach
Used with chloroplast genome.Converts toxic betaine
aldehyde to non-toxic glycine betaine, which also serves as an
osmoprotectant and confer drought/salt tolerance
GUS (uidA) gene
β-glucuronidase
X-Gluc
Blue precipitate
5-bromo-4-chloro-3-inolyl
-β-D-glucuronic acid
gus expression in Roots
Green fluorescent protein (GFP)
Protein identified form luminescent jellyfish Aequorea victoria.
GFP has now been produced in a number of heterologous
cell types and there appears to be little requirement for specific additional
factors for post-translational modification of the protein, which may be
autocatalytic or require ubiquitous factors. Many structural variants now
available commercially (e.g. red fluorescent protein)
A. thaliana C24 wild type (left)
35S-mgfp4-ER transformed
(right)
Aequorea victoria
Luciferase (luc) gene
The glow is widely used as an assay for luc expression, which
acts as a "reporter" for the activity of any regulatory elements that
control its expression. Luciferase is particularly useful as a
reporter- low-light cameras can detect bioluminescence, in real
time and with high sensitivity, in living cells and organisms.
Plants (Arabidopsis)
Crystal structure of luciferase
Significance of Molecular
characterization
• Need for rigorous molecular characterization
of each transgenic plant submitted for review
for predicting the safety of a novel food.
• Molecular characterization of transgenic
plants provide attention to regulators about
the information in terms of food, feed or
environmental safety consideration.
Three Aspects to Consider
1. The transformation system
–
Agrobacterium-mediated
–
Protoplast system
–
Microparticle bombardment
2.
–
–
Molecular characterization of the inserted DNA
Insert number
Insert composition
3. Genetic stability of the introduced trait
–
Segregation analysis
–
Integron stability
WHY IT IS NEEDED?
Introgression of Transgene to
Product Development
Utility of Molecular Characterization
• May be able to address issues related to positional
effects, pleiotropic effects, and gene silencing
• Provides information on the composition and
integrity of the inserted DNA
• Ensures that the developer has appropriately
characterized the genetic modification
NUCLEUS
Transcription
Splicing
Translation
CYTOSOL
USE OF PCR TO CHECK GENE INTEGRATION
Different stages of hardening of annexin transformed Brassica plants.
(a) Rooted PCR positive shoots transferred in plastic pots covered with
polybags kept for hardening at transgenic glass house.
(b) A PCR positive plant growing in the pot.
(c) A hardened PCR positive plant growing in plantation pot after 2 months.
(d) Hardened PCR positive plants growing at transgenic glass house.
INHERITANCE OF ANNEXIN TRANSGENE IN T1 PLANTS
M
1
2
3
4
5
M
6
7
8
9
10
1.5 kb
1.0 kb
Annexin PCR of T1 progeny of T0 Brassica plant
Lane M = 100 bp ladder
Lanes 1-10 = T1 progeny plants of T0 plant
ƒ THIS EXPERIMENT HAS REVEALED THAT A TRANSFORMANT PLANT HAS MOSAICS OF GENE
WHEN GROWN IN VITRO FROM CALLUS.
• THE GENE MOSAICS COULD OCCUR AT BOTH ORGAN AND TISSUE OR CELLULAR LEVEL.
Edible vaccine against Japanese Encephalitis virus
Shoot initiation
Callusing
Different stages of regeneration in tomato
Rooting in solid
medium
Rooting in liquid
medium
Presence of Transgene in tomato
by PCR amplification
Transformed plant
kept for hardening
Transgene silencing
Reduced/abolished expression of foreign gene
Loss of expression : Not due to loss of transgene but due
to their inactivation
Concept of gene Space :
Genomes are made of isochores (long stretches of
DNA with high compositional homogeneity)
If a GC rich transgene is integrated into a GC isochore
or an AT rich transgene is integrated into an AT
isochore : It is Transcribed
If a GC rich transgene is integrated into the AT rich
gene space or vice versa : It is Inactivated, as there is
no compositional homogeneity with the neighboring
sequences
Position Dependent & Sequence Dependent Gene silencing
Transgene integrates into a genomic region containing
heterochromatin.
The repressive chromatin structure & DNA methylation can
spread into the transgenic locus from the flanking
genomic DNA
Homology Dependent Gene Silencing
Caused by Multiple copies of transgene (Repeat induced gene
silencing)
Affect not only the stability of transgene but also alter the
activity of endogenous gene (Cosuppression)
Transcriptional Gene Silencing
No mRNA is produced from silenced gene
Affected loci : nucleation points for heterochromatin
formation & DNA methylation
Post – Transcriptional Gene Silencing
Transcription is required for silencing to take place
Induce degradation of mRNA : very little accumulate
in cytoplasm
Transgene Expression
• So expression of transgenes has been
attributed to:
– copy number - the number of transgene
copies integrated into the host genome
– “positional effects” - the position of the T-DNA
integration site in the host genome may affect
the level of expression
– variable arrangements of transgene
sequences in the host genome e.g. multiple
copies in direct or inverted repeats
Detection of Insert Number
• Refers to the number of sites where the transgenic
element is incorporated into the host genome.
• This is deduced by digesting genomic DNA with a
restriction enzyme that does not cut within the
transgenic element followed by Southern blot
analysis with a probe specific to one or more of the
introduced genes.
• More than one band = more than one insertion site.
• This should be repeated with at least one other
restriction enzyme to confirm the number of inserts.
aattcc
32p-taagg
ttaagg
attcc-32p
gggccat
cccggta
32pt
Insert Number
Inserted Element
Gene A
32P-labelled
Gene B
cDNA probe
BamHI
Genomic DNA
BamHI digest
Southern
blot
Should yield a
single band if
one insert
Example: Insert Number
11 49 50 52 53 54 56 57 59 62 64 90 94 95 96 97 98 99 100102 NT PC
Fig. 1A
3.5 kb
109 135 136 146 147 150 151 152 154 155 156 157 NT PC
3.5 kb
Fig. 1B
Southern analysis showing the integration of crtI gene cassette in selected
PCR+ primary transgenics of BR29 (Golden indica rice) developed by Agrotransformation.
Enzyme = Eco RI, Probe = PCR generated 1.03 kb crtI fragment, Positive control
Eco RI released 3.5 kb fragment of pCaCar; lines showing single insertion are
marked by red rectangles (Datta et al., unpublished data).
Detection Of Transgene Copy
Number
• This is not the same as the number of insertion
sites
• Digestion with one or more (i.e. can be single or
double digests) restriction enzymes that either
do not cut within the transgenic element, or cut
only once but not within the sequence
complementary to the hybridization probe
• This should yield one band per inserted element.
• Usually this is done with more than one
restriction enzyme, or combination of enzymes.
Transgene Copy Number
Inserted Element
Gene A
32P-labelled
Gene B
cDNA probe
A
XhoI
EcoRI
Genomic
DNA
A
B
B
EcoRI digest
Southern
blot
XhoI digest
Southern
blot
A single
band from
each
digestion
indicates a
single copy
Transgene Rearrangements
•
•
•
•
Rearrangements of transgenic sequences may be observed in
Southern blot analyses as hybridizing fragments of a different
size than the full-length DNA insert.
Larger fragments are indicative of concatenation (head to head
or head to tail). Concatemers may be deduced by digesting
genomic DNA with a restriction enzyme that cuts at a single
site within the transgenic element; multiple copies of the DNA
insert will then be resolved by Southern blot analysis.
Larger than full length fragments of transgenic DNA may also
be caused by interspersion of inserted DNA with host DNA
(oats, rice).
Smaller than full-length fragments are evidence of deletions
and truncations
Gene Integrity
• The goal is to determine if the gene(s) of interest
are intact, or whether there have been
truncations/deletions
• Digest genomic DNA with restriction enzymes to
isolate the gene of interest
• Hybridize with a gene-specific probe.
• Resolved band should be the same size as that
isolated from the plasmid.
• Alternatively, PCR with 5’- and 3’-terminal
specific primers in order to amplify a fragment of
the same size as the inserted gene
Gene Integrity: MON 810
•Southern blot
analysis of plasmid
DNA (lane 1),
control genomic
DNA (lane 2), and
transgenic maize
DNA (lane 3)
digested with NcoI
and EcoRI and
hybridized with a
32P-labelled probe
specific to the
cry1Ab gene.
nptII
ori-pUC
BamHI 5336
cryIA(b)
MON810 Example: Plasmid
Backbone
Southern blot analysis of plasmid
DNA (lane 1), control genomic DNA
(lane 2), and transgenic maize DNA
(lane 3) digested with NcoI and EcoRI
and hybridized with 32P-labelled
probes specific for the nptII region
(lanes 1-3) or the ori-pUC region
(lanes 4-6).
nptII
ori-pUC
BamHI 5336
cryIA(b)
Genetic Stability
•
For each novel trait, the pattern and stability of inheritance
must be demonstrated as well as the level of expression of the
trait.
•
Serological techniques are generally used to measure trait
expression (gain of function) ( either qualitatively [e.g., Western
immunoblotting, enzyme linked immunosorbent assay (ELISA),
etc.] or quantitatively (e.g., ELISA, radioimmunoassay, etc.).
•
If the new trait is one that does not result in the expression of a
new or modified protein (e.g., transgenic plants containing
inserted antisense sequences i.e loss of function) then its
inheritance will have to be determined by examining the DNA
insert directly or by measuring RNA transcript production.
Transformation Vector Map (Partial),
Southern and Enzyme Assay
Fig 2 represents partial map of
transformation vector containing a
bacterial transgene (hph) encodes
hygromycinphosphotransferase
under the control of CaMV35S
constitutive promoter.
Fig 3 represents a Southern blot
analysis showing integration of
transgene (expected size) in To and
T1 offspring plants.
Fig 4 represents enzyme assay for
HPH of the same To and T1 plants
shown in the Southern blot
confirming inheritance and
functioning of the transgene.
Hazard Identification Requires
• Knowledge of which genes are expressed
• Characteristics, concentration and
localization of expressed products
• Consequences of expression – effects on
metabolite pools
• And, in cases where the modification results
in the production of antisense mRNA, the
consequences of altering the expression of
an endogenous gene must be evaluated
Characterize Expressed Protein
• Identity
– Immunological cross-reactivity (Western blotting,
ELISA)
– Physiochemical properties (size, stability to heat
and proteolytic digestion)
• Functionality
– Enzymatic activity in the case of introduced
enzymes [e.g. phosphinothricin acetyltransferase
(PAT)]
– Biological activity
• Where appropriate, similarity to products from
traditional sources
WESTERN BLOTTING TO CHECK GENE EXPRESSION
+ve electrode
Nitrocellulose
membrane
-ve electrode
Control
Gel
Transgenic
DOT BLOTTING TO CHECK GENE EXPRESSION IN
TRANSGENIC PLANTS
Control
transgenic
Patterns of Expression
• How, when, and where
• Based on the promoter used (constitutive,
tissue-specific, inducible, etc), is the novel
protein expressed in the expected tissues and
under the expected conditions?
• Presence or absence and amounts of expressed
protein should be determined for a range of
plant tissues (e.g. roots, leaves, seeds, pollen)
• And, from a food safety perspective, levels of
expression of the protein(s) in the edible
portions of the plant are critical
Microarrays for detecting gene and protein
expression
Normal
Transgenic
Microarrays for detecting gene
and protein expression