Methods of Gene Transfer

1. Methods of Gene Transfer

2. 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) Your own footer Your Logo

3. 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

4. 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)

5. 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)

6. Genetically modified plants Your own footer Your Logo

7. 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

8. Cell Plant cell A single gene Extracted DNA Transformation Cell division Transgenic plant Plant Genetic Engineering Process

9. 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

10. Introducing the Gene Developing Transgenic Plant STEPS • Prepare tissue for transformation • Tissue must be capable of developing into normal plants • Leaf, germinating seed, immature embryos Create transformation cassette • Introduce DNA • Agrobacterium or gene gun • Culture plant tissue • Develop shoots & Roots Introduction of the gene Selection of transformants • Screening of putative transformants • Field test the plants

11. 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

12. Requirements for plant transformation 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: hygromycinphosphotransferase(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

13. Screening & Selection of transformant Selectable marker gene Selectable marker gene Positive selection 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. Removable selectable marker gene Genes using the Cre-lox system or transposable elements 1 2

14. Transformation Cassettes Contains P TA G M TA • 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

15. Commonly used promoters • 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

16. Marker gene screen able marker & selectable marker 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 -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

17. Plant Transformation Methods Chemical Biological & In-planta Physical • Agrobacterium Tumefaciens • Agrobacterium Rhizogenes • Virus-mediated In planta • Microinjection • Biolistics - gene gun/Particle bombardment • Electroporation • Microinjection • Silica/carbon fibers • Lazer mediated • PEG • Calcium phosphate • Artificial lipids • Proteins • Dendrimers Chemical Biological Physical

18. Physical Methods of Transformation • Microinjection • Biolistics - gene gun/Particle bombardment • Electroporation • Silica/carbon fibers • Lazer mediated

19. Electroporation • 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

20. Plant cell Duracell Protoplast The plant cell with the new gene DNA inside the plant cell DNA containing the gene of interest Electroporation Technique Power supply • 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

21. This electroporator is for low-current applications such as those using small electrodes

22. Microinjection

23. Particle gun • 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 • 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 .

24. Particle gun ✓ 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. 1 ✓ 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. 2 ✓ 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. 3 Your Logo

26. DNADelivery Particle gun Agrobacterium

27. 2. Biological Methods • Agrobacterium Tumefaciens • Agrobacterium Rhizogenes • Virus-mediated

28. Agrobacterium- mediated • Agrobacterium is a soil borne gram-negative bacterium, that has a unique ability to introduce part of its DNA into plant cells. • Most of the native transferred bacterial DNA is replaced with genes of interest • In the laboratory, bacteria are co-cultured or inoculated with plant tissue and the bacteria transfer part of their DNA into plant cells.

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. Mechanism of Agrobacterium- mediated transformation

31. Mechanism of Agrobacterium- mediated transformation

32. Mechanism of Agrobacterium- mediated transformation

33. Chemical Transformation • PEG • Calcium phosphate • Artificial lipids • Proteins • Dendrimers

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. In-Planta Transformation Non-tissue culture based ♣ Meristem transformation ♣ Floral dip method ♣ Pollen transformation

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. 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. Analysis of T0 plants Confirmation with selectable marker, Screenable marker, Negative & Positive control Morphology Physiology Gene expression GUS expression Yield characters

39. GFP expression in soybean tissue Shows variability in expression pattern standard illumination on left – gfp illumination on right

40. Few Examples of Transgenic crop

41. Golden Rice Synthesis Two Daffodil genes and one bacterial gene Erwiniauredovora were cloned into agrobacterium T DNA and inserted into rice genome to generate needed enzymes Phytoene synthase & Lycopene-b-cyclase Carotene desaturase T DNA Germ-line transformation with agrobacterium X Cross T-formed rice with gene T-formed rice with genes Progeny rice plant with complete b carotene pathway

42. Golden Rice • 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

43. World's First Blue Roses On Display In JapanDanielle 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.htmlTokyo, 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. Tearless Onion DrEady Crop & Food Research in New Zealand and his collaborators in Japan As onions are sliced, cells are broken, alliinases - break down aasulphoxides - 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. Before After Final Test of the Transgenic Consumer Acceptance RoundUp Ready Corn

46. Thank you!