molecular strategies for detection of insertion of genes in
TRANSCRIPT
Molecular Strategies for Molecular Strategies for detection of insertion of detection of insertion of genes in transgenic plants genes in transgenic plants
Dr Anil KumarAssociate Professor & In chargeDept. Of Molecular Biology &Genetic Engineering, CBSHGBPUAT Pantnagar UttranchalPin-263245
Genes and Genes and ProteinsProteins
Gene(a piece of DNA)
traitprotein
translationtranslation
mRNA
transcriptiontranscription
Any agriculturally important trait can be introduced by incorporation of a useful gene
Transgenic Technology : Art and Transgenic Technology : Art and science of creating designer cropsscience 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 TraitsIdentifying & locating genes for agriculturally important traits: Most limiting step in transgenic process
Little is Known about specific genesto enhance yield potentialimprove stress tolerancemodify chemical properties of harvested productsotherwise affect plant characters
Identifying a single gene for a trait is not sufficientMust understand how the gene is regulatedWhat other effects it might have on the plantHow 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 suitablymodified 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, containingnecessary 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 How are Transgenic Plants Produced?Plants Produced?
•• AgrobacteriumAgrobacteriumtumefacienstumefaciens
•• Gene Gun / Gene Gun / BiolisticsBiolistics•• ElectroporationElectroporation
Commonly Used Methods:Commonly Used Methods:
Protoplast Immatureembryo (IE)
Embryo derivedcalli (EDC)
Electroporation/PEG mediatedtransformation
Biolistictransformation
Agrobacteriummediated
transformationSelection in presence ofSelectable marker gene
Plant regeneration
Molecular characterizationevent selection/gene expression/function of transgene
Development of transgenic plants
Transformation System
Transgenic Plants- From Lab to the field
Transgenic lines obtained throughTransformation and regeneration
Laboratory analysis to confirmStable integration of transgene(s)Number of copies of the transgene(s)Expression of transgene(s)
Monitoring and analysis of transgenics in aContainment (Glasshouse) Facility for
Stability of transgene expressionAgronomically desirable expression of transgenic traitGenetic behavior of transgene(s)Biosafety evaluation and risk assessment
Small plot (<500m2) field experimentsTo evaluate agronomic performanceAnd to further analyze biosafety
Large scale field testing at multiple sites
Commercilization of transgenic varietyAfter risk assessment and evaluation ofNet benefit offered by the transgenic
Monitoring of transgenic variety during commercialization
PRODUCTS GENE INTROGR-ESSED
GENETICALLY ALTERED TRAITS
WHEAT
BRASSICA
BRASSICA
TOMATOCARROT
PAT
OSMOTIN
ANNEXIN
Pr M EPr M EGUS
HERBICIDE RESISTANCE (BASTA)
TOLERANCE TO ALTERNARIA BLIGHT AND SALINITY
MODULATION OF HYPERSENSITIVE RESPONSE AGAINST ALTERNARIA BLIGHT AND POWDERY MILDEW
PRODUCTION OF EDIBLE VACCINE AGAINST JAPANESE ENCEPHALITIS VIRUS
TRANSGENIC CROPS DEVELOPED SO FAR AT PANTNAGAR
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 INTEGRATIONGene 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 gene-gene 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/Herbicideresistance 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 (Phosphinothricinacetyltransferase)
Phosphenothricin(Bialaphos, Basta)
epsps (5-enolpyruvylshikhimate-3-phosphate synthase)
Glyphosate
Risks & Concerns of using Antibiotic/HerbicideResistance Genes
Horizontal Gene TransferTransfer 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 coliencodes mannose phosphate transferase and confer
upon transformed cells ability to use mannose as a sole carbon source
Ipt (isopentenyl transferase) gene from Agrobacteriumlocated 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 SpinachUsed 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
X-Gluc Blue precipitate5-bromo-4-chloro-3-inolyl-β-D-glucuronic acid
β-glucuronidase
gus expression in Roots
Green fluorescent protein (GFP)
Protein identified form luminescent jellyfish Aequorea victoria.GFP has now been produced in a number of heterologouscell types and there appears to be little requirement for specific additionalfactors for post-translational modification of the protein, which may beautocatalytic or require ubiquitous factors. Many structural variants nowavailable 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.
Crystal structure of luciferasePlants (Arabidopsis)
Significance of Molecular Significance of Molecular characterizationcharacterization
• 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 ConsiderThree Aspects to Consider1. 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 Introgression of TransgeneTransgene to to Product DevelopmentProduct Development
Utility of Molecular CharacterizationUtility 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
Transcription
Splicing
Translation
NUCLEUS
CYTOSOL
USE OF PCR TO CHECK GENE INTEGRATIONUSE 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.
Annexin PCR of T1 progeny of T0 Brassica plantLane M = 100 bp ladderLanes 1-10 = T1 progeny plants of T0 plant
1.5 kb1.0 kb
M 1 2 3 4 5 M 6 7 8 9 10
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.
INHERITANCE OF ANNEXIN TRANSGENE IN TINHERITANCE OF ANNEXIN TRANSGENE IN T11 PLANTSPLANTS
Edible vaccine against Japanese Encephalitis virusEdible vaccine against Japanese Encephalitis virus
Callusing Shoot initiation
Rooting in solid medium
Rooting in liquid medium
Different stages of regeneration in tomato
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 isochoreor 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 neighboringsequences
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 ExpressionTransgene 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 NumberDetection 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.
gggccat
ttaaggattcc-32p
aattcc32p-taagg
cccggta
32pt
Insert NumberInsert Number
Gene A Gene B
BamHI
Inserted Element
Genomic DNA BamHI digest
32P-labelled cDNA probe
Southern blot
Should yield a single band if one insert
3.5 kb
11 49 50 52 53 54 56 57 59 62 64 90 94 95 96 97 98 99 100102 NT PC
3.5 kb
109 135 136 146 147 150 151 152 154 155 156 157 NT PC
Southern analysis showing the integration of crtI gene cassette in selected PCR+ primary transgenics of BR29 (Golden indica rice) developed by Agro-transformation.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).
Fig. 1A
Fig. 1B
Example: Insert NumberExample: Insert Number
Detection Of Detection Of TransgeneTransgene Copy Copy NumberNumber
• 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 NumberTransgene Copy Number
Gene A Gene B
EcoRI
Inserted Element
32P-labelled cDNA probe
GenomicDNA
EcoRI digest Southern blotA
A
B
BXhoI
XhoI digest Southern blot
A single band from each digestion indicates a single copy
Transgene RearrangementsTransgene 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 IntegrityGene 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 810Gene Integrity: MON 810•Southern blot analysis of plasmid DNA (lane 1), control genomic DNA (lane 2), and transgenic maize DNA (lane 3) digested with NcoIand EcoRI and hybridized with a 32P-labelled probe specific to the cry1Ab gene.
nptII
ori-pUC
cryIA(b)
BamHI 5336
MON810 Example: Plasmid MON810 Example: Plasmid BackboneBackbone
Southern blot analysis of plasmid DNA (lane 1), control genomic DNA (lane 2), and transgenic maize DNA (lane 3) digested with NcoI and EcoRIand hybridized with 32P-labelled probes specific for the nptII region (lanes 1-3) or the ori-pUC region (lanes 4-6).
nptII
ori-pUC
cryIA(b)
BamHI 5336
Genetic StabilityGenetic 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), Transformation Vector Map (Partial), Southern and Enzyme AssaySouthern and Enzyme Assay
Fig 2 represents partial map of transformation vector containing a bacterial transgene (hph) encodes hygromycinphosphotransferaseunder 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 RequiresHazard 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 ProteinCharacterize 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 WESTERN BLOTTING TO CHECK GENE EXPRESSION
Nitrocellulose membrane
Gel-ve electrode
+ve electrode
Control Transgenic
Control transgenic
DOT BLOTTING TO CHECK GENE EXPRESSION INDOT BLOTTING TO CHECK GENE EXPRESSION INTRANSGENIC PLANTS TRANSGENIC PLANTS
Patterns of ExpressionPatterns 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
TransgenicNormal
Microarrays for detecting gene and protein expression