Methods of Gene Transfer
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Methods of Gene Transfer
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Methods of Gene Transfer
- 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. Transgenic versus Cloning Why transgenic plants ? Plant Genetic Engineering Process Methods of genetic transformation/ gene delivery OUTLINE
- 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. 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. 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. 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. Your own footer Your Logo Genetically modified plants
- 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. Plant Genetic Engineering Process Cell Extracted DNA Cell divisionTransgenic plant A single gene Transformation Plant cell
- 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. • 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. 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. 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. 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. 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. • 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. 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. 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. Physical Methods of Transformation •Microinjection •Biolistics - gene gun/Particle bombardment •Electroporation •Silica/carbon fibers •Lazer mediated
- 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. 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. This electroporator is for low-current applications such as those using small electrodes
- 23. Microinjection
- 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. 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
- 28. •Agrobacterium Tumefaciens •Agrobacterium Rhizogenes •Virus-mediated 2. Biological Methods
- 29. 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.
- 30. 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
- 31. Mechanism of Agrobacterium- mediated transformation
- 32. Mechanism of Agrobacterium- mediated transformation
- 33. Mechanism of Agrobacterium- mediated transformation
- 34. Chemical Transformation • PEG • Calcium phosphate • Artificial lipids • Proteins • Dendrimers
- 35. 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!
- 36. Meristem transformation Floral dip method Pollen transformation Non-tissue culture based In-Planta Transformation ♣ ♣ ♣
- 37. 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
- 38. 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
- 39. Analysis of T0 plants Yield characters Physiology Morphology GUS expression Gene expression Confirmation with selectable marker, Screenable marker, Negative & Positive control
- 40. GFP expression in soybean tissue Shows variability in expression pattern standard illumination on left – gfp illumination on right
- 41. • Few Examples of Transgenic crop
- 42. 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
- 43. • 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
- 44. 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
- 45. 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
- 46. Final Test of the Transgenic Consumer Acceptance RoundUp Ready Corn Before After
- 47. Thank you!
Editor's Notes
- Animated pointer and light-up text (Advanced) To reproduce the background effects on this slide, do the following: On the Home tab, in the Slides group, click Layout, and then click Blank. Right-click the slide background area, and then click Format Background. In the Format Background dialog box, click Fill in the left pane, select Solid fill in the right pane, and select White, Background 1 (first row, first option from the left). To reproduce the rectangle on this slide, do the following: On the Home tab, in the Drawing group, click Shapes, and then under Rectangles click Rounded Rectangle (second option from the left). On the slide, drag to draw a rounded rectangle. Select the rectangle. Drag the yellow diamond adjustment handle to the left to decrease the amount of rounding on the corners. With the rounded rectangle still selected, under Drawing Tools, on the Format tab, in the Size group, do the following: In the Shape Height box, enter 3.5”. In the Shape Width box, enter 0.25”. Under Drawing Tools, on the Format tab, in the bottom right corner of the Shape Styles group, click the Format Shape dialog box launcher. In the Format Shape dialog box, click Fill in the left pane. In the Fill pane, select Solid fill, click the button next to Color, and then under Theme Colors click White, Background 1, Darker 15% (third row, first option from the left). Also in the Format Shape dialog box, click Line Color in the left pane. In the Line Color pane, select No line. Also in the Format Shape dialog box, click Shadow in the left pane. In the Shadow pane, click the button next to Presets, under Outer select Offset Bottom (first row, second option from the left), and then do the following: In the Transparency box, enter 0%. In the Size box, enter 100%. In the Blur box, enter 8.5 pt. In the Angle box, enter 90°. In the Distance box, enter 1 pt. Also in the Format Shape dialog box, click 3-D Format in the left pane. In the 3-D Format tab, do the following: Under Bevel, click the button next to Top, and then under Bevel click Circle (first row, first option from the left). Next to Top, in the Width box, enter 5 pt, and in the Height box, enter 5 pt. Under Surface, click the button next to Material, and then under Standard click Matte (first row, first option from the left). Click the button next to Lighting, and then under Neutral click Soft (first row, third option from the left). On the slide, select the rounded rectangle. On the Home tab, in the Clipboard group, click the arrow to the right of Copy, and then click Duplicate. Select the duplicate rectangle. On the Home tab, in the Drawing group, do the following: Click the arrow next to Shape Fill, and then click No Fill. Click the arrow next to Shape Outline, and then click No Outline. Drag the second rectangle above the first rectangle until the lower edge overlays the top edge of the first rectangle. (Note: When the spinning animation effect is created later for these rectangles, the spin will center where the edges of the rectangles meet.) Press and hold CTRL, and then select both rectangles. On the Home tab, in the Drawing group, click Arrange, and do the following: Point to Align, and then click Align Selected Objects. Point to Align, and then click Align Center. Click Group. On the slide, drag the group until it is centered horizontally on the left edge of the slide (straddling the edge). On the Home tab, in the Drawing group, click Arrange, point to Align, and then do the following: Click Align to Slide. Click Align Middle. To reproduce the dashed arc on this slide, do the following: On the Home tab, in the Drawing group, click Shapes, and then under Basic Shapes click Arc (third row, 12th option from the left). On the slide, drag to draw an arc. Select the arc. Under Drawing Tools, on the Format tab, in the Size group, do the following: In the Shape Height box, enter 7.5”. In the Shape Width box, enter 7.5”. With the arc still selected, on the Home tab, in the Drawing group, click the arrow next to Shape Outline and then do the following: Under Theme Colors, click White, Background 1, Darker 15% (third row, first option from the left). Point to Dashes, and then click Dash (fourth option from the top). On the slide, drag the yellow diamond adjustment handle on the right side of the arc to the bottom of the arc to create a half circle. Drag the arc until the yellow diamond adjustment handles are on the left edge of the slide. With the arc still selected, on the Home tab, in the Drawing group, click the arrow under Arrange, point to Align, and then do the following: Click Align to Slide. Click Align Middle. To reproduce the half circle on this slide, do the following: On the slide, select the arc. On the Home tab, in the Clipboard group, click the arrow to the right of Copy, and then click Duplicate. Select the duplicate arc. Under Drawing Tools, on the Format tab, in the Size group, do the following: In the Shape Height box, enter 3.33”. In the Shape Width box, enter 3.33”. With the second arc still selected, under Drawing Tools, on the Format tab, in the Shape Styles group, click the arrow next to Shape Fill, and then under Theme Colors click White, Background 1, Darker 5% (second row, first option from the left). Under Drawing Tools, on the Format tab, in the Shape Styles group, click the arrow next to Shape Outline, and then click No Outline. Under Drawing Tools, on the Format tab, in the Shape Styles group, click Shape Effects, point to Shadow, and then click Shadow Options. In the Format Shape dialog box, click Shadow in the left pane. In the Shadow pane, click the button next to Presets , under Inner click Inside Right (second row, third option from the left), and then do the following: In the Transparency box, enter 86%. In the Blur box, enter 24 pt. In the Angle box, enter 315°. In the Distance box, enter 4 pt. On the slide, drag the second arc until the yellow diamond adjustment handles are on the left edge of the slide. On the Home tab, in the Drawing group, click Arrange, and then do the following: Point to Align, and then click Align to Slide. Point to Align, and then click Align Middle. Click Send to Back. To reproduce the button shapes on this slide, do the following: On the Home tab, in the Drawing group, click Shapes, and then under Basic Shapes click Oval (first row, second option from the left). On the slide, drag to draw an oval. Select the oval. Under Drawing Tools, on the Format tab, in the Size group, do the following: In the Shape Height box, enter 0.34”. In the Shape Width box, enter 0.34”. Under Drawing Tools, on the Format tab, in the Shape Styles group, click More, and then click Light 1 Outline, Colored Fill – Olive Green, Accent 3 (third row, first option from the left). Under Drawing Tools, on the Format tab, in the bottom right corner of the Shape Styles group, click the Format Shape dialog box launcher. In the Format Shape dialog box, click Fill in the left pane. In the Fill pane, select Solid Fill. Click the button next to Color, and then under Theme Colors click Olive Green, Accent 3, Lighter 80° (second row, seventh option from the left). Also in the Format Shape dialog box, click Line Color in the left pane. In the Line Color pane, select No line. Also in the Format Shape dialog box, click Shadow in the left pane. In the Shadow pane, click the button next to Presets, under Outer click Offset Bottom (first row, second option from the left), and then do the following: In the Transparency box, enter 0%. In the Size box, enter 100%. In the Blur box, enter 8.5 pt. In the Angle box, enter 90°. In the Distance box, enter 1 pt. Also in the Format Shape dialog box, click 3-D Format in the left pane, and then do the following in the 3-D Format pane: Under Bevel, click the button next to Top, and then under Bevel click Art Deco (third row, fourth option from the left). Next to Top, in the Width box, enter 5 pt, and in the Height box, enter 5 pt. Under Contour, click the button next to Color, and then under Theme Colors click White, Background 1 (first row, first option from the left). In the Size box, enter 3.5 pt. Under Surface, click the button next to Material, and then under Standard click Matte (first row, first option from the left). Click the button next to Lighting, and then under Neutral click Soft (first row, third option from the left). On the slide, select the oval. Under Drawing Tools, on the Format tab, in the bottom right corner of the Size group, click the Size and Position dialog box launcher. In the Format Shape dialog box, click Position in the left pane, and in the Position pane, do the following to position the third and fourth ovals: In the Horizontal box, enter 2.98”. In the Vertical box, enter 1.5”. Select the oval. On the Home tab, in the Clipboard group, click the arrow under Paste, and then click Duplicate. Select the duplicate oval. Under Drawing Tools, on the Format tab, in the bottom right corner of the Size group, click the Size and Position dialog box launcher. In the Format Shape dialog box, click Position in the left pane, and in the Position pane, do the following to position the third and fourth ovals: In the Horizontal box, enter 3.52”. In the Vertical box, enter 2.98”. Repeat step 9 two more times, for a total of four ovals. Under Drawing Tools, on the Format tab, in the bottom right corner of the Size group, click the Size and Position dialog box launcher. In the Format Shape dialog box, click Position in the left pane, and in the Position pane, do the following to position the third and fourth ovals: Select the third oval on the slide, and then enter 3.52” in the Horizontal box and 4.27” in the Vertical box. Select the fourth oval on the slide, and then enter 2.99” in the Horizontal box and 5.66” in the Vertical box. To reproduce the text on this slide, do the following: On the Insert tab, in the Text group, click Text Box, and then on the slide, drag to draw the text box. Enter text in the text box and select the text. On the Home tab, in the Font group, do the following: In the Font list, select Corbel. In the Font Size list, select 22. Click the arrow next to Font Color, and then under Theme Colors click White, Background 1, Darker 50% (sixth row, first option from the left). On the Home tab, in the Paragraph group, click Align Text Left to align the text left in the text box. On the slide, drag the text box to the right of the first oval. Select the text box. On the Home tab, in the Clipboard group, click the arrow to the right of Copy, and then click Duplicate. Click in the text box and edit the text. Drag the second text box to the right of the second oval. Repeat steps 5-7 to create the third and fourth text boxes, dragging them to the right of the third and fourth ovals. To reproduce the animation effects on this slide, do the following: On the Home tab, in the Editing group, click Select, and then click Selection Pane. In the Selection and Visibility task pane, select the rectangle group. On the Animations tab, in the Advanced Animation group, click Add Animation, and then under Emphasis click Spin. Also on the Animations tab, in the Animation group, click the Effect Options dialog box launcher. In the Spin dialog box, do the following: On the Effects tab, in the Amount list, in the Custom box, enter 123°, and then press ENTER. Also in the Amount list, click Counterclockwise. On the Timing tab, in the Duration box, select 1.00. On the Timing tab, in the Start list, select With Previous. On the slide, select the first oval. On the Animations tab, in the Advanced Animation group, click Add Animation, and then click More Emphasis Effects. In the Add Emphasis Effect dialog box, under Basic, click Fill Color. On the Animations tab, in the Animation group, click the Effect Options dialog box launcher. In Fill Color dialog box, the do the following: On the Effects tab, in the Fill Color list, click More Colors. In the Colors dialog box, on the Custom tab, enter values for Red:130, Green:153, Blue: 117. On the Timing tab, in the Duration box, , enter 0.50. On the Timing tab, in the Start list, select After Previous. On the slide, select the first text box. On the Animations tab, in the Advanced Animation group, click Add Animation, and then under Entrance click Fade. Also on the Animations tab, in the Timing group, do the following: In the Start list, select With Previous. In the Duration box, enter 0.50. In the Selection and Visibility task pane, select the rectangle group. On the Animations tab, in the Advanced Animation group, click Add Animation, and then under Emphasis click Spin. Also on the Animations tab, in the Animation group, click the Effect Options dialog box launcher. In the Spin dialog box, do the following: On the Effects tab, in the Amount list, in the Custom box, enter 22°, and then press ENTER. Also in the Amount list, click Clockwise. On the Timing tab, in the Start list, select On Click. On the Timing tab, in the Duration box, enter 0.50. On the slide, select the second oval. On the Animations tab, in the Advanced Animation group, click Add Animation, and then click More Emphasis Effects. In the Add Emphasis Effect dialog box, under Basic, click Fill Color. On the Animations tab, in the Animation group, click the Effect Options dialog box launcher. In the Fill Color dialog box, do the following: On the Effects tab, in the Fill Color list, click More Colors. In the Colors dialog box, on the Custom tab, enter values for Red:130, Green:153, Blue: 117. On the Timing tab, in the Start list, select After Previous. On the Timing tab, in the Duration box, enter 0.50. On the slide, select the second text box. On the Animations tab, in the Advanced Animation group, click Add Animation, and then under Entrance click Fade. Also on the Animations tab, in the Timing group, do the following: In the Start list, select With Previous. In the Duration box, enter 0.50. On the slide, select the third oval. On the Animations tab, in the Advanced Animation group, click Add Animation, and then click More Emphasis Effects. In the Add Emphasis Effect dialog box, under Basic, click Fill Color. On the Animations tab, in the Animation group, click the Effect Options dialog box launcher. In the Fill Color dialog box, do the following: On the Effects tab, in the Fill Color list, click More Colors. In the Colors dialog box, on the Custom tab, enter values for Red:130, Green:153, Blue: 117. On the Timing tab, in the Start list, select After Previous. On the Timing tab, in the Speed list, select Very Fast. On the slide, select the third text box. On the Animations tab, in the Advanced Animation group, click Add Animation, and then under Entrance click Fade. Also on the Animations tab, in the Timing group, do the following: In the Start list, select With Previous. In the Duration box, enter 0.50. On the slide, select the fourth oval. On the Animations tab, in the Advanced Animation group, click Add Animation, and then click More Emphasis Effects. In the Add Emphasis Effect dialog box, under Basic, click Fill Color. On the Animations tab, in the Animation group, click the Effect Options dialog box launcher. In the Fill Color dialog box, do the following: On the Effects tab, in the Fill Color list, click More Colors. In the Colors dialog box, on the Custom tab, enter values for Red:130, Green:153, Blue: 117. On the Timing tab, in the Start list, select After Previous. On the Timing tab, in the Duration box, enter 0.50. On the slide, select the fourth text box. On the Animations tab, in the Advanced Animation group, click Add Animation, and then under Entrance click Fade. On the Animations tab, in the Timing group, do the following: In the Start list, select With Previous. In the Duration box, enter 0:50.