Gene Gun ppt.pptx

I. INDIRECT
a) BIOLOGICAL
Agrobacterium mediated Virus mediated
II. DIRECT
PHYSICAL
Gene gun/biolistics
Micro/Macro injection Electrophoresis Pressure
Laser mediated Using pollen tubes
Silica/ carbon fibers (fiber mediated DNA delivery)
CHEMICAL
Artificial lipids (lipofection)
PEG
Proteins Dendrimers Dextran
II. DIRECT GENE TRANSFER
Is a Vector less DNA transfer systems
Naked DNA is introduced into the plant/animal cells
DNA can be introduced by the following methods;
Chemical
Microinjection
Electroporation
Particle bombardment (Biolistic)
Gene gun
A biolistic particle delivery system, originally designed for plant transformation,
Device for delivering exogenous DNA (transgenes) to cells
It was invented and used by John C Sanford, Ed Wolf and Nelson Allen at Cornell university, and Ted Klein of Dupont, between 1983 and 1986, to transform epidermal cells of Allium cepa.
This method is mainly used for cereal transformation
Steps
Prepare the tungsten particles and coat them with DNA.
Load the particles onto macrocarriers.
Prepare the target cells.
Decide on your parameters and set the gene gun accordingly for distance from target, pulse duration, and CO2 pressure.

1. Gene Gun By Allah Nawaz Class: BS Botany Presented to Dr NOSHIA ARSHAD Subject: Genetic Engineering

2. TRANSFORMATION TECHNIQUE AGROBACTERIUM MEDIATED GENE TRANSFER (INDIRECT) DIRECT GENE TRANSFER Used for engineering DICOTS For MONOCOT 2 methods are used Chemical Physic a l Electroporation Biolis t i cs Micro/Macro injection 6

3. Or the methods used for producing transgenic plant s can be categorized as…………… I. INDIRECT a) BIOLOGICAL Agrobacterium mediated Virus mediated II. DIRECT PHYSICAL Gene gun/biolistics Micro/Macro injection Electrophoresis Pressure Laser mediated Using pollen tubes Silica/ carbon fibers (fiber mediated DNA delivery) CHEMICAL Artificial lipids (lipofection) PEG Proteins Dend r i mers Dextran 7

4. II. DIRECT GENE TRANSFER Is a Vector less DNA transfer systems Naked DNA is introduced into the plant/animal cells DNA can be introduced by the following methods; Chemical Microinjection Electroporation Particle bombardment (Biolistic )

5. d) Gene Gun Method A biolistic particle delivery system, originally designed for plant transformation, Device for delivering exogenous DNA (transgenes) to cells It was invented and used by John C Sanford , Ed Wolf and Nelson Allen at Cornell university , and Ted Klein of Dupont , between 1983 and 1986, to transform epidermal cells of Allium cepa. This method is mainly used for cereal transformation 22

6. The basic steps to use the gene gun are: Prepare the tungsten particles and coat them with DNA. Load the particles onto macrocarriers . Prepare the target cells. Decide on your parameters and set the gene gun accordingly for distance from target, pulse duration, and CO 2  pressure.

7. DNA is coated with gold or tungsten How DNA is coated with gold or tungsten particles? This can be achieved with ballistic labeling, which refers to the delivery of a cell label by means of carrier particles (tungsten or gold) propelled from a pressurized gun. Ballistic delivery can reach many dispersed cells in one shot and can deploy a wide variety of cell markers to neurons in diverse preparations .

8. Load the particles onto macrocarriers . Loading of coated gold particles onto macrocarriers for bombardment in a PDS-10 0 0/He Biolistic Particle Delivery System. A) Macrocarrier with coated gold microparticles in a drying chamber. The yellow line shows the area where the coated particles should be applied. B) Macrocarriers with dried DNA-coated microparticles . C) Position of endosperm culture plate on the target plate shelf of the biolistic bombardment system. D) setup of the bombardment chamber.

9. Decide on your parameters and set the gene gun accordingly for distance from target, pulse duration, and CO 2  pressure. High-pressure solenoid ($75) Adjustable relay that provides pulses in the 10–500 millisecond range ($30–$60 on eBay) A bicycle CO 2  inflator to attach the CO 2  cartridge to the gene gun ($22) A valve to adjust the pressure ($21) A pressure gauge that reads from 0–1,000 psi ($15 on eBay) A fitting to suspend the tungsten particles above the target in front of the target A compressed gas discharge ( macrocarrier ) ($5) Pipe fittings to hook up the Solenoid, CO 2 , and nozzle ($30)

10. The four key parts that are needed are the relay, the solenoid, the valve, and a CO 2  tire inflator. Other than that, the rest of the parts are 1/8″–1/2″ pipes and fittings available from any hardware store. See  Figure 1-1 .

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12. APPLICATION Herbicide resistance Insect resistance Virus resistance Altered oil content Delayed fruit ripening Drough t, cold, salinity resistance Pollen control Enhanced shelf life Pharmaceutical & edible vaccines Biotic & Abiotic stress tolerance Nutritional quality 23

13. 1. HERBICIDE RESISTANCE a. Bromoxynil Resistance A gene enc o ding the enz yme B romo x yni l nitri l ase ( B XN ) is t r ansf e rr e d fr o m K lebsie l la pneumoniae bacteria to plants. Nitrilase inactivates the bromoxynil before it kills the plant. Sulfonylurea Kills plants by blocking an enzyme needed for synthesis of the amino acids valine, leucine, and isoleucine. Resistance generated by mutating a gene in tobacco plants, and transferring the mutated gene into crop plants . INSECT RESISTANCE The Bt toxin isolated from Bacillus thuringiensis has been used in plants. The gene has been placed in corn, cotton, and potato, and has been marketed. Alkaline protein degrades gut wall of lepidopteran larvae Corn borer catepillars Cotton bollworm catepillars Tobacco hornworm catepillars Gypsy moth larvae Sprayed onto plants – but will wash off 24

14. 3. VIRUS RESISTANCE Chemicals are used to control the insect vectors of viruses, but controlling the disease itself is difficult because the disease spreads quickly. Plants may be engineered with genes for resistance to viruses, bacteria, and fungi. Virus-resistant plants have a viral protein coat gene that is overproduced, preventing the virus from reproducing in the host cell, because the plant shuts off the virus’ protein coat gene in response to the overproduction. Coat protein genes are involved in resistance to diseases such as cucumber mosaic virus, tobacco rattle virus, and potato virus x. ALTERED OIL CONTENT Oil content in plants are altered by modifying an enzyme in the fatty acid synthesis pathway (oils are lipids, which fatty acids are a part of). Varieties of canola and soybean plants have been genetically engineered to produce oils with better cooking and nutritional properties. Genet i cally e ng i neere d p lan t s may also be able t o prod uce o i ls t h at are use d i n 25 detergents, soaps, cosmetics, lubricants, and paints.

15. 5. DELAYED FRUIT RIPENING Allow for crops, such as tomatoes, to have a higher shelf life. Tomatoes generally ripen and become soft during shipment to a store. Tomatoes are usually picked and sprayed with the plant hormone ethylene to induce ripening, although this does not improve taste Tomatoes have been engineered to produce less ethylene so they can develop more taste before ripening, and shipment to markets. POLLEN CONTROL Hybrid crops are created by crossing two distantly related varieties of the same crop plant. The method may generate plants with favorable traits, such as tall soybean plants that make more seeds and are resistant to environmental pressures. For success, plant pollination must be controlled. This is usually done by removing the male flower parts by hand before pollen is released. Also, sterilized plants have been genetically engineered with a gene from the bacteria Bacillus amyloliqueifaciens (barnase gene) . This gene is dominant gene for male sterility 26

16. RISKS OF GMO/GMCs …….. Can be dangerous & cause allergies E.g.. Soybean containing gene of brazil nut Indirectly promote Antibiotic resistance (Resistance of microbes) Weed shows herbicide resistance & resistance to viral disease Change in chemistry of soil Genetically engineered plant cross pollinate non- engineered plants 27

17. EXAMPLES OF GM CROPS ……… . Corn Canola. Cotton. Papaya, rice, Tomato, Sugar beet, and Red heart chicory. Golden rice Transgenic technology produced a type of rice that accumulates beta-carotene in rice grains. Once inside the body, beta-carotene is converted to vitamin A. Normal Rice Golden Rice 28

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