Gene transformation techniques
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This document discusses various techniques for gene transformation, which is the process of altering or modifying genes. It begins by introducing the topic and describing early discoveries in bacterial genetic transformation in the 1920s. It then describes three main categories of gene transformation techniques: biological, physical, and chemical means. For each category, it provides examples of specific techniques and describes their mechanisms and applications. The physical means discussed include gene guns, microinjection, electroporation, and ultrasound. The document emphasizes that physical techniques allow for direct transformation without the use of carriers. It concludes by mentioning some applications of gene transfer techniques, such as for transgenic animals, crops, and gene therapy.
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Gene transformation techniques
- 3. Introduction: • Gene alteration or modification is known as gene transformation. • Frederick Griffith 1928 revealed genetic transformation in bacterial strains . • Gene transformation are of two types • Natural transformation • Artificial transformation
- 4. Natural transformation • Conjugation Genetic exchange by cellular contacts Transduction Genetic exchange through a vehicle Like plasmid or vector • Agrobacterium mediated gene transfer
- 5. Why gene transformation • science of transformation starts in 1970 with discovery of restriction enzymes by H-Smith • main purpose includes • Cloning of gene • Production of desired proteins • Enhancement of products • GMOs
- 6. Types • Stable transformation integration of gene permanently. e.g. agrobacterium mediated • Transient Temporary transformation Liposome mediated
- 7. Genetic transformation techniques • Three categories • Biological means • Physical means • Chemical means
- 8. Modes of gene transformation • Indirect method It uses carriers e.g. plasmids, vectors, liposome's, • Direct carrier free method e.g. microinjections Gene gun
- 9. Biological Means of transformation • This technique uses biological systems for gene transfer. • E.g. • Plasmids • Vectors • Cosmid • Phagemids • BACs and YACs • Modified Replication deficient viruses
- 10. Physical means of gene transformation
- 11. Introduction • It uses physical tools • Operates direct transformation • Auto regulation of genes i.e. auto integration • It overcomes the work-burden unlike biological means • No side effect and pathogenicity
- 12. Types • The well known physical means of gene transformation techniques include, • Gene Gun , Biolistic / Ballistic or Golden Gun method • Microinjections • Electroporation. • Laser beam technique • Ultrasound / Sonication etc
- 13. Gene gun/Golden gun Biolistics • Gene gun is an instrument used to shoot the DNA on target sites. • Invented by John c Sanford in 1985 • Also known as • Golden gun • Ballistics or Biolistics • Particle bombardment method
- 14. Gene gun Construction • Firing pin • Macro projectile • Micro projectile • Safety vents • Plates containing target cells.
- 15. Operating procedure The gene transfer is followed by four basic operating steps. • Preparation of DNA loaded beads • Preparation of plastic tubes. • Loading of Macro projectile • Shooting or Transformation
- 16. DNA Beads formation • 250mg gold particles in tube that are 1.6 micro meter in size. • Add spermadine • Prepare DNA solution • Mix (adhering occur) • Add IM Cacl2 • Centrifugation
- 17. Plastic tube preparation • Add polyvinyl pyridine to solution • Inert, dried tubes are used • Solution is transferred in to tube • Adhering occur • Rotation and refurnished • Incubation • Cutting of tubes
- 18. Loading in macro projectile • It act as cartridge • Specified DNA beads in tubes are loaded in it
- 19. Shooting • Pressurized gas is used • Distance and pressure ere optimized • Transfection of cell is done by shooting. • Ready for transfection. Success rate is 30 to 50 % Examples Nerve cells Liver cells were transformed
- 20. • *
- 21. Other methods
- 22. Micro injection • Introduced by Dr Marshall Barber • Physical technique of transformation • Direct transformation of both plant and animal cells • It uses micromanipulators • Syringes of 0.5µm-5µm in diameter size
- 24. Operating mechanism • Operate under observing lens • Micromanipulators hold plates containing target cells and injection pipettes.
- 25. Application Can be transferred to fertilized ovum e.g ;as in Dolly fish. Simple and Cheaper Method . Easy Handling Feasible Visible
- 26. Ultrasound • In 1988 Weber • It uses ultraviolet micro laser • (200-400nm) • Laser induce permeability in cell • Uptake of DNA • Example • Brassica napus • Less efficient
- 27. • Advantages and Disadvantages, • Physical methods of transformations Direct method • Efficient success rate. 30-40 percent. • Quick gene transfer. • The limitation are • Availability • cost effective tools. • requires expertise.
- 28. Electroporation • Physical mean • Dr Neumann 1982 • Uses electric pulse • Observed under microscope • known as electro-permeabilization • Usually applied for protoplast transformation
- 29. Operating procedure • Parameters are optimized • 60-75 Celsius. • Voltage of 150-160 volt
- 30. Description
- 31. Stem cell • Applied on Stem cells • Are totipotent • Uses physical tools • Direct transformation • Embryonic stem cell mediated gene transfer
- 32. Procedure • Isolation of Blastula cells • Transformation by physical method
- 33. • Success depend on transformation tools • confirmation of positive clone • Positive clones are cultures • Re implantation of positive clones in blastula
- 34. Advantage
- 35. Advantages Transgenic progeny Short time production Efficient success rate
- 36. Chemical means of gene transfer
- 37. Introduction • Uses chemical reagents • targeted delivery • Less side effects • Works on binding affinity
- 38. Examples • DEAE dextran • Calcium phosphate • Silicon carbide whiskers method. • Poly-l-ornithine • Polyethyelenimine • Polyethylene glycol method • Liposome’s mediated gene transfer
- 39. PEG • Used for plant transformation • Fusiogenic chemical • 40% w/V • Medium of mannitol • Chloroplast and protoplast transformation
- 40. Liposome's • Lipid in nature • Small in size 5µm-10 µm • Use for target delivery • Significance is bilayer • Lipid soluble layer • Core i.e. water soluble layer
- 41. Liposome
- 42. preparation • Preparation of core • Preparation of lipid bilayer • Sonication • preparation immune-protective specie • Addition of homing peptides • Confirmation
- 44. Applications of gene transfer • Transgenic animals • Transgenic crops • Gene therapy • Cloning
- 45. Examples • Onions : Agrobacterium • Barely (Hordeum vulgare) electroporation • Insulin by plasmids • Sorghum by biolistics • Sunflower by PEG
- 46. • . Bibliography The given facts and figure are sort out from different sources which are given below accordingly • GENETIC ENGINEERING principles, procedure and consequences Author: Gurbachan S.Migalani , published by Narosa • Genetic Transformation of Plants Edited by J.F.Jackson and H.F.Linksens by Springer (chapter 2) • Gene Transfer techniques by Hongbao Ma, Guozhong Chen [Nature and Science. 2005;3(1):25-31] • URL: https://www.google.com.pk/url?sa=t&rct=j&q=&esrc=s&source=web&cd=19&
- 47. Question? If any. Feel free to ask.