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Met hods of Gene Transf er
Pravin V Jadhav, PhD
Assistant Professor,
Biotechnology Centre, Dr. PDKV, Akola
E-mail: jpravee...
Transgenic versus Cloning
Why transgenic plants ?
Plant Genetic Engineering Process
Methods of genetic transformation/ gen...
Your own footer Your Logo
Transgenic versus Cloning
 Transgenic : creation of transgenic animal or plant
(introduction of...
What is a transgenic?
Transgenic
An organism containing a transgene
introduced by technological (not
breeding) methods
Tra...
Why transgenic plants ?
Why do we need transgenic
plants ?
•Improvement of agricultural value of plant
(resistance to herb...
Transgenic Plants
Advantages & Disadvantages
• Advantages:
- Plant cells are totipotent: whole plant can be regenerated fr...
Your own footer Your Logo
Genetically modified plants
What About the Term Genetic Engineering?
Genetic engineering is the basic tool set of biotechnology
Genetic engineering in...
Plant Genetic Engineering Process
Cell
Extracted DNA
Cell divisionTransgenic plant
A single
gene
Transformation
Plant cell
Production of
transgenic plants
Isolate and clone gene of interest
Add DNA segments to initiate or enhance
gene expression...
• Prepare tissue for transformation
• Tissue must be capable of developing into normal
plants
• Leaf, germinating seed, im...
Plant transformation
• DNA delivery systems must be
• Simple
• Efficient and preferably inexpensive
• The method must be a...
A. Cell culture and plant regeneration system
B. Cloned DNA to be introduced
1. selectable marker gene
• kanamycin or G148...
Selectable marker gene
Screening & Selection of
transformant
Positive selection
PMI (phospho- mannose
isomerase) Plant cel...
Transformation Cassettes
Contains
1. Promoter
• Regulatory sequence/initiation site
2. Gene of interest
• The coding regio...
• Constitutive promoter
•CaMV 35S : suitable for expression of foreign genes in dicots:
•The maize ubiquitin promoter, als...
Selectable Markers
•allow the selection of transformed cells, or tissue explants
•by ability to grow in the presence of an...
Plant
Transformation
Methods
• PEG
• Calcium phosphate
• Artificial lipids
• Proteins
• Dendrimers
ChemicalPhysical
• Agro...
Physical Methods of
Transformation
•Microinjection
•Biolistics - gene gun/Particle bombardment
•Electroporation
•Silica/ca...
• 1970s, 1990 versatile method – in vivo (skin and muscles)
• short pulses of high voltage to carry DNA across the cell
me...
Drawbacks
•Limited effective range of ~1 cm between the electrodes
•Surgical procedure is required to place the electrodes...
This electroporator is for low-current applications such as those using small electrodes
Microinjection
MAJOR LIMITATIONS:
shallow penetration of particles
associated cell damage
the inability to deliver the DNA systemicall...
Particle gun
Your Logo
For particle bombardment, tungsten or gold
particles are coated with DNA and accelerated
towards ta...
DNA
Delivery
Agrobacterium Particle gun
•Agrobacterium Tumefaciens
•Agrobacterium Rhizogenes
•Virus-mediated
2. Biological Methods
Agrobacterium- mediated
• In the laboratory, bacteria are co-
cultured or inoculated with plant
tissue and the bacteria tr...
Agrobacterium tumefaciens
• Wild type Tk plasmid = 200 kb – too large for cloning
• Intermediate shuttle plasmid is used t...
Mechanism of Agrobacterium- mediated transformation
Mechanism of Agrobacterium- mediated transformation
Mechanism of Agrobacterium-
mediated transformation
Chemical Transformation
• PEG
• Calcium phosphate
• Artificial lipids
• Proteins
• Dendrimers
PEG mediated
• It is the oldest (direct DNA) reliable method for plant transformation. In
the first report (Krens et al. 1...
Meristem transformation
Floral dip method
Pollen transformation
Non-tissue culture based
In-Planta Transformation
♣
♣
♣
Vacuum Infiltration
– Plant leaf disks are placed in a suspension of bacteria and
vacuum pulled
– Air is release like a sp...
Floral Dip
– Simple submersion of plant into bacterium suspension
– No vacuum is needed
– Conducted with plants grown unti...
Analysis of T0 plants
Yield characters
Physiology
Morphology
GUS expression
Gene expression
Confirmation with selectable
m...
GFP expression in soybean tissue
Shows variability in expression pattern standard illumination
on left – gfp illumination ...
• Few Examples of Transgenic
crop
Golden Rice Synthesis
Two Daffodil genes and one bacterial gene Erwinia
uredovora were cloned into agrobacterium T DNA
and...
• Golden rice contains increased levels of pro-vitamin
A .
• Traditional rice is white (a).
• The prototype of golden rice...
World's First Blue Roses On Display In Japan
Danielle Demetriou, Daily Telegraph, October 31, 2008, See the rose at
http:/...
Tearless Onion
Dr Eady
Crop & Food Research in New Zealand
and his collaborators in Japan
As onions are sliced, cells are ...
Final Test of the Transgenic
Consumer Acceptance
RoundUp Ready Corn
Before After
Thank you!
Methods of Gene Transfer
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Methods of Gene Transfer

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Methods of Gene Transfer

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Methods of Gene Transfer

  1. 1. Met hods of Gene Transf er Pravin V Jadhav, PhD Assistant Professor, Biotechnology Centre, Dr. PDKV, Akola E-mail: jpraveen26@yahoo.co.in
  2. 2. Transgenic versus Cloning Why transgenic plants ? Plant Genetic Engineering Process Methods of genetic transformation/ gene delivery OUTLINE
  3. 3. Your own footer Your Logo Transgenic versus Cloning  Transgenic : creation of transgenic animal or plant (introduction of foreign gene into organism) • transgenic organisms produced by introduction of foreign gene into germ line (transgenic offspring!!!) • introduction of gene into somatic cells -> gene therapy  Cloning : obtaining an organism that is genetically identical to the original organism • such as Dolly the sheep • asexual propagation of plants (taking cuttings)
  4. 4. What is a transgenic? Transgenic An organism containing a transgene introduced by technological (not breeding) methods Transgene The genetically engineered gene added to a species
  5. 5. Why transgenic plants ? Why do we need transgenic plants ? •Improvement of agricultural value of plant (resistance to herbicides, resistance to insect attack, Bacillus thuringiensis toxin) •living bioreactor, produce specific proteins •studying action of genes during development or other biological processes (knock-out plants, expression down- regulated)
  6. 6. Transgenic Plants Advantages & Disadvantages • Advantages: - Plant cells are totipotent: whole plant can be regenerated from a single cell (engineered cells - engineered plants) - Plants have many offspring: rare combinations and mutations can be found - Transposons used as vectors • Disadvantages: - Large genomes (polypoid - presence of many genomes in one cell) - plants regenerating from single cells are not genetically homogenous (genetically instable)
  7. 7. Your own footer Your Logo Genetically modified plants
  8. 8. What About the Term Genetic Engineering? Genetic engineering is the basic tool set of biotechnology Genetic engineering involves: Isolating genes  Modifying genes so they function better  Preparing genes to be inserted into a new species  Developing transgenes
  9. 9. Plant Genetic Engineering Process Cell Extracted DNA Cell divisionTransgenic plant A single gene Transformation Plant cell
  10. 10. Production of transgenic plants Isolate and clone gene of interest Add DNA segments to initiate or enhance gene expression Add selectable markers Introduce gene construct into plant cells (transformation) Select transformed cells or tissues Regenerate whole plants
  11. 11. • Prepare tissue for transformation • Tissue must be capable of developing into normal plants • Leaf, germinating seed, immature embryos • Introduce DNA • Agrobacterium or gene gun • Culture plant tissue • Develop shoots & Roots • Screening of putative transformants • Field test the plants Developing Transgenic Plant Introducing the Gene Selection of transformant s Introduction of the gene Create transformati on cassette STEPS
  12. 12. Plant transformation • DNA delivery systems must be • Simple • Efficient and preferably inexpensive • The method must be available for use either because it is in the public domain or because it can be licensed • System of choice depends on • the target plant • its regeneration system
  13. 13. A. Cell culture and plant regeneration system B. Cloned DNA to be introduced 1. selectable marker gene • kanamycin or G148 resistance: neomycin, phosphotransferase (NPTII), hygromycin B: hygromycin phosphotransferase (HygB)gentamicin: gentamicin acetyltransferase • streptomycin: streptomycin phosphotransferase • Bialophos: BAR 2. promoter (constituitive or inducible), coding region C. Method of delivery of DNA into the cell D. Proof of transformation of plant Requirements for plant transformation
  14. 14. Selectable marker gene Screening & Selection of transformant Positive selection PMI (phospho- mannose isomerase) Plant cells without this enzyme are unable to survive in a tissue culture medium containing mannose-6- phosphate as a sole carbon source. Positive selection Removable selectable marker gene Genes using the Cre-lox system or transposable elements Selectable marker gene 211
  15. 15. Transformation Cassettes Contains 1. Promoter • Regulatory sequence/initiation site 2. Gene of interest • The coding region and its controlling elements 3. Selectable marker • Distinguishes transformed/untransformed plants 4. Insertion sequences • Aids Agrobacterium insertion P G M TATA
  16. 16. • Constitutive promoter •CaMV 35S : suitable for expression of foreign genes in dicots: •The maize ubiquitin promoter, also a constitutive promoter which •drives strong expression of transgenes in monocots. • Organ/ tissue specific promoters •Vicilin and phytohemaglutinin, glutenin promoters seed specific expression •a-amylase promoter for expression in the aleurone of cereal grains; •Patatin promoter for tuber specific expression in potatoes and the RuBisCo promoter for green tissue specificity Commonly used promoters
  17. 17. Selectable Markers •allow the selection of transformed cells, or tissue explants •by ability to grow in the presence of an antibiotic or a herbicide. •frequently used - kanamycin and hygromycin Screen able markers •encode gene products whose enzyme activity can be easily assayed •allowing not only the detection of transformants •also estimation of the levels of foreign gene expression in transgenic tissue • markers such as GUS, luciferase or β-galactosidase allow screening for enzyme activity by histochemical staining or fluorimetric assay of individual cells •can be used to study cell-specific as well as developmentally regulated gene expression Marker gene screen able marker & selectable marker
  18. 18. Plant Transformation Methods • PEG • Calcium phosphate • Artificial lipids • Proteins • Dendrimers ChemicalPhysical • Agrobacterium Tumefaciens • Agrobacterium Rhizogenes • Virus-mediated Biological & In-planta • Microinjection • Biolistics - gene gun/Particle bombardment • Electroporation • Microinjection • Silica/carbon fibers • Lazer mediated Physi cal Chemical Biological In planta
  19. 19. Physical Methods of Transformation •Microinjection •Biolistics - gene gun/Particle bombardment •Electroporation •Silica/carbon fibers •Lazer mediated
  20. 20. • 1970s, 1990 versatile method – in vivo (skin and muscles) • short pulses of high voltage to carry DNA across the cell membrane • to assist the uptake of useful molecules such as a DNA vaccine into a cell • Parameters, electrical field strength [V/cm], pulse length Electroporation
  21. 21. Drawbacks •Limited effective range of ~1 cm between the electrodes •Surgical procedure is required to place the electrodes deep into the internal organs •High voltage applied to tissues can result in irreversible tissue damage as a result of thermal heating electron-avalanche transfection Electroporation Technique Duracell DNA containing the gene of interest Plant cell Protoplast Power supply DNA inside the plant cell The plant cell with the new gene
  22. 22. This electroporator is for low-current applications such as those using small electrodes
  23. 23. Microinjection
  24. 24. MAJOR LIMITATIONS: shallow penetration of particles associated cell damage the inability to deliver the DNA systemically the tissue to incorporate the DNA must be able to regenerate and the expensive equipment . • Simplest method of direct introduction of therapeutic DNA into target cells • Looks like a pistol but works more like a shotgun with “Golden pellets” • First described as a method of gene transfer into plants • John Sanford at Cornell University in 1987 • Particle bombardment -physical method of cell transformation in which high density and sub-cellular sized particles are accelerated to high velocity in order to carry DNA or RNA into living cells Particle gun
  25. 25. Particle gun Your Logo For particle bombardment, tungsten or gold particles are coated with DNA and accelerated towards target plant tissues. In the early days, the force used to accelerate the particles was a .22 caliber blank. Today, most devices use compressed helium. The DNA-coated particles can end up either near or in the nucleus, where the DNA comes off the particles and integrates into plant chromosomal DNA. The particles punch holes in the plant cell wall and usually penetrate only 1-2 cell layers. Particle bombardment is a physical method for DNA introduction and the biological incompatibilities associated with Agrobacterium are avoided. ✓ ✓ ✓ 1 2 3
  26. 26. DNA Delivery Agrobacterium Particle gun
  27. 27. •Agrobacterium Tumefaciens •Agrobacterium Rhizogenes •Virus-mediated 2. Biological Methods
  28. 28. Agrobacterium- mediated • In the laboratory, bacteria are co- cultured or inoculated with plant tissue and the bacteria transfer part of their DNA into plant cells. • Most of the native transferred bacterial DNA is replaced with genes of interest • Agrobacterium is a soil borne gram- negative bacterium, that has a unique ability to introduce part of its DNA into plant cells.
  29. 29. Agrobacterium tumefaciens • Wild type Tk plasmid = 200 kb – too large for cloning • Intermediate shuttle plasmid is used to cut in Gene of Interest • VIR genes must be removed for genetic engineering • LB and RB are required for insertion and recombination with plant genome • Insertion into plant host is random (sort of) • First cloned gene – luciferase in tobacco plant
  30. 30. Mechanism of Agrobacterium- mediated transformation
  31. 31. Mechanism of Agrobacterium- mediated transformation
  32. 32. Mechanism of Agrobacterium- mediated transformation
  33. 33. Chemical Transformation • PEG • Calcium phosphate • Artificial lipids • Proteins • Dendrimers
  34. 34. PEG mediated • It is the oldest (direct DNA) reliable method for plant transformation. In the first report (Krens et al. 1982 Nature 296:72), Agrobacterium Ti plasmid was introduced into petunia protoplasts. Formation of tumors, opine synthesis and Southern blot provided the verification, which is an extensive and complete analysis to show success of transformation. • The first report of generating transgenic plants using this method was provided by Paszkowski et al. (1984). They regenerated transformed protoplasts into plants that were kanamycin (drug) resistant. • This method has been very useful and applied to several plant species. • But it is a tedious procedure!
  35. 35. Meristem transformation Floral dip method Pollen transformation Non-tissue culture based In-Planta Transformation ♣ ♣ ♣
  36. 36. Vacuum Infiltration – Plant leaf disks are placed in a suspension of bacteria and vacuum pulled – Air is release like a sponge being squeezed – Vacuum is released and solution floods tissue – Plant disk is cultured
  37. 37. Floral Dip – Simple submersion of plant into bacterium suspension – No vacuum is needed – Conducted with plants grown until just flowering – Progeny seeds are harvested and germinated using selective antibiotic
  38. 38. Analysis of T0 plants Yield characters Physiology Morphology GUS expression Gene expression Confirmation with selectable marker, Screenable marker, Negative & Positive control
  39. 39. GFP expression in soybean tissue Shows variability in expression pattern standard illumination on left – gfp illumination on right
  40. 40. • Few Examples of Transgenic crop
  41. 41. Golden Rice Synthesis Two Daffodil genes and one bacterial gene Erwinia uredovora were cloned into agrobacterium T DNA and inserted into rice genome to generate needed enzymes Two Daffodil genes and one bacterial gene Erwinia uredovora were cloned into agrobacterium T DNA and inserted into rice genome to generate needed enzymes Phytoene synthase & Lycopene-b-cyclase Carotene desaturaseT DNA Germ-line transformation with agrobacterium X Cross T-formed rice with genes T-formed rice with gene Progeny rice plant with complete b carotene pathway
  42. 42. • Golden rice contains increased levels of pro-vitamin A . • Traditional rice is white (a). • The prototype of golden rice was developed in 2000 and is a light yellow color (b). It contains 1.6 mg/g of carotenoid. • In 2005, new transgenic lines were developed that dramatically increased the amount of carotenoid synthesized, making the rice a deep golden color (c). • This latest form contains 37 mg/g of carotenoid, of which 84% is b-carotene – trial Golden Rice
  43. 43. World's First Blue Roses On Display In Japan Danielle Demetriou, Daily Telegraph, October 31, 2008, See the rose at http://www.telegraph.co.uk/news/worldnews/asia/japan/3327043/Worlds- first-blue-roses-on-display-in-Japan.html Tokyo, Japan - World's first blue roses have been unveiled to the public for the first time at an international flower fair in Japan, following nearly two decades of scientific research. The blue-hued blooms are genetically modified and have been implanted with a gene that simulates the synthesis of blue pigment in pansies. Its scientists successfully pioneered implanting into the flowers the gene that produces Delphinidin, the primary plant pigment that produces a blue hue but is not found naturally in roses. The world's first genetically modified blue roses were unveiled in the laboratory four years ago, although further research was required to make them safe to grow in nature. The Blue Rose was developed by Suntory Flowers
  44. 44. Tearless Onion Dr Eady Crop & Food Research in New Zealand and his collaborators in Japan As onions are sliced, cells are broken, alliinases - break down aa sulphoxides - generate sulphenic acids - unstable - rearrange into a volatile gas - syn-propanethial-S-oxide – diffuses by air - reaches the eye - reacts with the water to form a diluted solution of sulphuric acid - Tear glands produce tears to dilute and flush out the irritant
  45. 45. Final Test of the Transgenic Consumer Acceptance RoundUp Ready Corn Before After
  46. 46. Thank you!

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