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Gene Transfer Methods
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- 2. Gene • A geneis the basic unit of heredity in a living organism • Genes are working subunit of DNA/RNA
- 3. Gene Transfer – Theinsertion of genetic material into a cell – It is defined simply as a technique to efficiently and stably introduce foreign genes into the genome of target cells – The insertion of unrelated, therapeutic genetic information in the form of DNA into target cells Bacteria (or) Virus Animal Plant
- 5. Applications of GeneTransfer- Bacteria
- 6. Applications of GeneTransfer- Plants • Provide resistance against viruses. • Acquire insecticidal resistance. • To strengthen the plant to grow against bacterial diseases. • Develop the plants to grow in draught. • Engineering plants for nutritional quality. • Make the plants to grow in various seasons. • Herbicide resistant plant can be made. • Resistance against fungal pathogens. • Engineering of plants for abiotic stress tolerance. • Delayed ripening can be done.
- 7. • Clinical genetransfer applications • Vaccine Development • Production of transgenic animals • Treatment of Cancer, AIDS • Gene Discovery • Gene Therapy • GMO Applications of Gene Transfer- Animals
- 8. Types of genetransfer • Natural method – Conjugation – Transformation – Transduction – Vector mediated – Transposon • Artificial method – Physical method – Chemical method – Electrical method
- 9. Artificial method • Physicalmethod – Microinjection – Macroinjection – Gene gun (Biolistic) – Laser transfection – Sonication
- 10. Artificial method • Chemicalmethod –Calcium Phosphate –PEG –Liposome –DEAE Dextran (Diethylaminoethyl) • Electrical method –Electroporation –Electrofusion
- 11. Conjugation • Requires thepresence of a special plasmid called the F plasmid. • Bacteria that have a F plasmid are referred to as as F+ or male. Those that do not have an F plasmid are F- of female. • The F plasmid consists of 25 genes that mostly code for production of sex pilli. • A conjugation event occurs when the male cell extends his sex pilli and one attaches to the female.
- 12. Conjugation • This attachedpilus is a temporary cytoplasmic bridge through which a replicating F plasmid is transferred from the male to the female. • When transfer is complete, the result is two male cells. • When the F+ plasmid is integrated within the bacterial chromosome, the cell is called an Hfr cell (high frequency of recombination cell).
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- 14. Transformation • Transformation isthe direct uptake of exogenous DNA from its surroundings and taken up through the cell membrane . • Transformation occurs naturally in some species of bacteria, but it can also be effected by artificial treatment in other species. • Cells that have undergone this treatment are said to be competent. • Any DNA that is not integrated into he chromosome will be degraded.
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- 16. Transduction • Gene transferfrom a donor to a recipient by way of a bacteriophage • Two way of integration • If the lysogenic cycle is adopted, the phage chromosome is integrated (by covalent bonds) into the bacterial chromosome, where it can remain dormant for thousands of generation • The lytic cycle leads to the production of new phage particles which are released by lysis of the host.
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- 18. Vector Mediated GeneTransfer • Carrying molecule related gene transfer –Plant vector- CaMV, TMV •Agrobacterium mediated gene transfer –Animal vector- Sv40, Pox Viral vector –Bacterial vector- pSC 101
- 19. Agrobacterium Mediated GeneTransfer • The Agrobacterium system was historically the first successful plant transformation system, marking the break through in plant Genetic engineering in 1983. • Large plasmids (140–235 kbp) in these bacteria are called tumour inducing (Ti plasmid) and root inducing (Ri plasmid) respectively. • Virulence genes aid in the transfer of T-DNA into the host plant cell. Ti plasmid contains 35 vir genes arranged in 8 operons. • Integrated in the plant nuclear DNA at random site
- 20. Agrobacterium Mediated GeneTransfer Advantages • Agrobacterium is capable of infecting infect plant cells, tissue and organs. • Capable of transfer of large fragments of DNA very efficiently • Integration of T DNA is a relative precise process. • The stability of gene transferred in excellent. Limitations : • Host specificity • Somaclonal variation • Slow regeneration • Inability to transfer multiple genes
- 22. SV 40 • Simianvirus 40 vector belongs to family polyoma virus family. • This virus induces tumours to animal cells and used for gene transfer into eukaryotic cells • Sv40 diameter is 45nm and its capsid contains 72 proteins • It doesn’t have enzymes in structure • Genome of sv40 is single molecule of double stranded DNA • It contain 5243bp in genome structure
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- 24. Transposons • Transposon, classof genetic elements that can “jump” to different locations within a genome. • In addition, most transposons eventually become inactive and no longer move.
- 25. Microinjection •DNA introduced intocells or protoplasts with use of very fine needles or glass micropipettes •T. P. Lin in 1966 (mouse eggs) •The DNA solution is injected directly inside the cell using capillary glass micropipettes with the help of micromanipulators of a microinjection assembly. •It is easier to use protoplast than cells since cell wall interferes with the process of microinjection. •The protoplast are usually immobilized in agarose (or) on a glass slides coated with polylysine or by holding them under suction by a micropipette. •The process of microinjection is technically demanding and time consuming a maximum of 40-50 protoplasts can be microinjected in one hour. •The transformation frequency ranging between 14 to 66 %.
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- 27. Microinjection •Advantages – Stable integrationbetter than other method. –DNA injected in this Process less modification only occur –Effective transforming primary cells and protoplast. •Limitations –Cost effective –Random integration –Only one cell at a time can be manipulated. –Method not useful for the walled cells –Skilled person required
- 28. Macroinjection •DNA Macroinjection employingneedles each with diameter greater than cell diameter •De la Pena et al., 1987 •The injection of DNA into the cells or tissues using hypodermic syringe is known as macro injection. •It is hypothesized that the DNA is taken up by microspores during some specific stage of their development. •This approach is also very simple and easy; the only problem concerns the frequency (0.07%) and the consistency of stable transformants obtained.
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- 30. Macroinjection •Advantages –Instrument simple andcheap. –Does not require Protoplast. –Technically simple. •Limitations –Less specific –Less efficient –Frequency of transformation is very low.
- 31. Gene gun • Biolisticsmethod/Microprojectile/Particle bombardment • Sanford and coworkers in 1987 (onion epidermal) • Klein et al., transferred genomic RNA (TMV) • The process of transformation employees foreign DNA coated with (0.2-0.7 μm ) gold (or) are tungsten particles to deliver into target plant cells. • Two procedures have been used to this techniques – i. By using pressurized helium gas. – ii. By electro static energy released by a droplet of water exposed to a high voltage.
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- 33. Gene gun • Advantages –Need of protoplast obtaining can be avoided – Walled intact cells can be penetrated – Genome of subcellular organelles can be manipulated. • Limitations – Random injection – Need of equipment – Damage cell
- 34. Laser • It ismethod of introducing DNA into plant cells with a laser micro beam. • Small pores in the membrane are created by laser micro beam. • Lasers puncture holes in the cell membrane through which DNA may enter into the cell cytoplasm. • This method also used in Plant cells as well as Animal cells. • Lasers punctures holes in cell membrane through which DNA may enter into the cell cytoplasm. • But, There is no information/report on gene expression or stable integration.
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- 36. Sonication • Sonoporation involvesthe use of ultrasound for temporary permeabilization of the cell membrane allowing the uptake of DNA, drugs or other therapeutic compounds from the extracellular environment. • This method leaves the compound trapped inside the cell after ultrasound exposure. • It employs of micro bubbles for enhancing the delivery of large molecules like DNA. The micro bubbles form complex with DNA followed by injection and ultrasound treatment to deliver DNA into the target cells. • Unlike other methods of transfection, sonoporation combines the capability to enhance gene and drug transfer.
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- 38. Sonication Advantages • Simple andhighly efficient gene transfer method. • No significant damage is cause to the target tissue. Limitations • Not suitable for tissues with open or complex structures. • High exposure to low-frequency (<MHz) ultrasounds result in complete cellular death (rupture of the cell). • Thus cellular viability must be taken into consideration while employing this technique.
- 39. Calcium Phosphate • Transfectionis most common method • Graham and Vander E.B. in 1973. • A fraction of cells will take up the calcium phosphate DNA precipitate by endocytosis. • Transfection efficiencies using calcium phosphate can be quite low, in the range of 1-2 % . • This technique is used for introducing DNA into mammalian cells.
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- 41. Advantages –Mostly in mammaliancells. –Along with other method Limitations –Small fraction of cells is stably integrated –Frequency very Low –Gene modification –1-5% DNA enter into the nucleus. –Toxic especially to primary cells Calcium Phosphate
- 42. • This methodis utilized for protoplast only. • Polyethylene glycol stimulates endocytosis and therefore DNA uptake occurs. • Protoplasts are kept in the solution containing polyethylene glycol (PEG). • The molecular weight of PEG used is 8000 Dalton having the final concentration of 15%. • CaCl2 or sucrose or Glucose – osmotic buffer reagent . • Mostly plant system (petunia) PEG
- 43. • Advantages –More efficientthan electroporation –Useful for plant • Limitations –Transformation not high –Applicable only protoplast –Increases nucleases effect. PEG
- 44. • Artificial phospholipidvesicles • Lipofection. • Fraley in 1988 • DNA complex with liposomes • Liposome +ve charage • Membrane – membrane fusion • Endocytosis Liposome
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- 46. • Advantages –Simplicity –Long termstability –Low toxicity –Protection of nucleic acid from degradation • Limitations –DNA passes from cytoplasm to the nucleus unknown Liposome
- 47. • Polycationic highmolecular weight • Endocytosis • Along with DMSO and PMMA (Polymethyl- methacrylate) - high • Viral infection in cell lines. • 80% transformed can express. • Transfer efficiency increased by Glycerol. DEAE Dextran
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- 49. • Advantages –Simple –Cheap –Not survivecalcium phosphate use this method • Limitations –Stable expression is difficult –Degrade cells DEAE Dextran
- 50. • Zimmermann etal., 1983 • Electrical shock (Charge) • Suspending solution • Electroinjection • Types –High voltage short duration –Low voltage long duration Electroporation
- 51. • Advantages –Method isapplicable to variety of cell lines –Method quick –Large number cells handle simultaneously –Less cost • Limitations –Degradation of DNA –Cell structure –Clumping of cell Electroporation
- 53. • Mostly protoplast •Mixed fusion • Two types fusion (Cybrid, Hybrid) • Along with PEG • Sensitive to high PEG Electrofusion
- 54. Cybrid Formation Hybrid Formation DifferentCells Electrofusion
- 55. • Advantages –Fusion veryeasy –Fuse more cells • Limitations –Degradation of DNA –Cell structure –Clumping of cell –Integrated DNA may be modified Electrofusion