Gene transfer technology.
- 1. GENE TRANSFER TECHNOLOGY R.NAGALAKSHMI ASST. PROF THE OXFORD COLLEGE OF PHARMACY BANGALORE
- 2. •INTRODUCTION • Gene transfer is defined as a technique to efficiently and stably introduce foreign genes into the genome of target cells . It is the subsequent stable integration & expression of a foreign DNA into the genome. • The directed desirable gene transfer from one organism to another genome is referred as genetic transformation. The transferred gene is known as trans gene and the organism that develop after a successful gene transfer is known as transgenic. History: During the 1970’s Rogers made it became possible to introduce exogenous DNA constructs into higher eukaryotic cells in vitro. • In 1990’s, first approved gene therapy case in The United States took place on 14th September 1990, at the national institute of health, under the direction of professor William French Anderson. • In 2012, Glybera (Alipogene tiparvovec) became the first gene therapy treatment designed to reverse LIPOPROTEIN LIPASE DEFECIENCY(LPLD) a rare inherited disease of pancreatitis. It was first approved for clinical use in either Europe or The United States after its endorsement by the European commission.
- 3. METHODS OF GENE TRANSFER DNA transfer by natural methods DNA Transfer by artificial methods Physical methods Chemical method •Conjugation •Transformation •Transduction •Transposition •Retroviral transduction •Agrobacterium mediated transfer •Electro poration* •Electro fusion •Particle Bombardment* or Biolistics transformation. •Microinjection* •Microinjection* •Microlaser •Ultrasound Mediated Transfer •Impalefection •Magnetofection •DNA transfer by calcium phosphate co-precipitation method •Liposome mediated transfer DNA transfer by PEG mediated method •Silicon carbide fiber (scf) mediated transfer •DEAE dextran method •DMSO polycation •Rubidium Chloride Mediated DNA Transfer. •Viral delivery systems:
- 4. • CONJUGATION : It was discovered by Joshua Lederberg and Edward Tatum in 1946 in Escherichia coli. • This process involved the transfer of DNA through a direct link between the bacterial cells in the form of proteinaceous tube known as a pilus and the plasmid is known as the F (for fertility) factor. • The plasmid determine the F− to an F+ phenotype. • In some cases, however, the F plasmid could integrate into the bacterial chromosome, and conjugation could result in the transfer of chromosomal genes. This process, which was used to construct the first genetic map of E. coli, was termed sexduction. • 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. A conjugation event occurs when the male cell extends his sex pilli and one attaches to the female. This attached pilus 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).
- 6. TRANSFORMATION: It is the 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.
- 7. • TRANSDUCTION: Gene transfer from a donor to a recipient by way of a bacteriophag. • 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
- 8. AGROBACTERIUM MEDIATED TRANSFER : Agrobacterium tumefaciens is a soil borne gram negative bacterium. It invades many dicot plants when they are injured at the soil level and causes crown gall disease. This disease is associated with the presence of the Ti (tumour inducing) plasmid within the bacterial cell. • Co-cultivate with the Agrobacterium: • —Small pieces of leaf tissue placed into a culture of Agrobacterium for about 30 mins. The explants then placed on MS medium without selective agent. • Incubate explants with Agrobacterium for 2 days to allow transfer of the T-DNA. • Kill the Agrobacterium with a suitable antibiotic: • The explants are removed from the medium and washed in cefotaxime. • Select for transformed plant cells: The explant are transferred to a selective (kanamycin) medium with cefotaxime. Auxin, Cytokinin are used to encourage the regeneration of by organogenesis. • Regeneration of whole plant: —The shoot can be rooted by placing them on solid medium containing a high auxin to cytokinin ratio.
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- 10. ELECTROPORATION/ELECTRIC FIELD-MEDIATED MEMBRANE PERMEABILIZATION: Microscopic pores are induced in biological membrane by the application of high volt of electric pulse. These pores are known as electropores which allow the molecules, ions and water to pass from one side of the membrane to another and allow to accept exogenous DNA. • Electroporation has been reported to enhance the level of gene expression and can be used to increase efficiency of transformation or transfection of bacterial cells. It significantly improve immune responses elicited to DNA vaccines in both large and small animals. • General applications of electroporation: Introduction of exogeneous DNA into animal cell lines, plant protoplast, yeast protoplast and bacterial protoplast. • Wheat, rice, maize, tobacco have been stably transformed with frequency upto 1% by this method. Electroporation of early embryo may result in the production of transgenic animals. • Hepatocytes, epidermal cells, haematopoietic stem cells, fibroblast, mouse T and B lymphocytes can be transformed by this technique. • Naked DNA may be used for gene therapy by applying electroporation device on animal cells.
- 11. • Procedure: • During electroporation, protoplast or intact plant cells are taken in electroporation chamber fitted with parallel steel electrodes. • The chamber is initially filled with buffer containing DNA of interest and high initial field strength of 1000-1500 volts with a short decay time in microseconds in applied. • Plant materials is incubated in a buffer solution containing DNA and subjected to high-voltage electric pulse is applied by discharge of the capacitor across the cell. • —The DNA then migrates through high-voltage-induced pores in the plasma membrane and integrates into the genome. • —It can be used to deliver DNA into plant cells and protoplasts. • Even other tissues such as callus and immature embryos are suggested. Several methods have been suggested to increase transformation efficiency.
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- 13. Advantages: Efficient transformation. • Large number of transformed cells can be obtained and least number of cells deaths. • Method is fast and Low equipment cost. • Does not require experties individual. • Simultaneously a large number of cell can be treated. • High percentage of stable transformants can be produced. Disadvantages: • Difficulties associated with regeneration of plants from protoplast. • Rise of obtaining genetic variation in protoplast mediated regenerated plants. • ~40 to 50% incubated cells receive DNA • —~50% of the transformed cells can survive
- 14. •PARTICLE BOMBARDMENT /BIOLISTICS /MICROPROJECTILES / GENE GUN METHOD : It is a physical method that uses accelerated micro projectiles to deliver DNA or other molecules into intact tissues and cells. Firstly used by Klein et al (1987) & Sanford et al (1987). Gene gun is developed to enable penetration of the genetic material containing a gene of interest in the cell. 1-2μm tungsten or gold particles (micro-projectiles)are used, coated with the DNA. Acceleration is given to enter the micro-projectiles into the plant cells. •The coated beads are then attached to the end of the plastic bullet and loaded into the firing chamber of the gene gun. An explosive force fires the bullet down the barrel of the gun towards the target cells that lie just beyond the end of the barrel. •When the bullet reaches the end of the barrel it is caught and stopped, but the DNA coated beads continue on toward the target cells. Some of the beads pass through the cell wall into the cytoplasm of the target cells. Here the bead and the DNA dissociate and the cells become transformed. Once inside the target cells, the DNA is solubilised and may be expressed.
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- 17. • General applications of biolistics.: It is used successfully to transfor soyabean, cotton, spruce, sugarcane, papaya, sunflower, rice, maize, wheat, tobacco etc. • Genomes of subcellular organelles have been accessible to genetic manipulation by biolistic method. • Method can be applied to filamentous fungi and yeast (mitochondria). • The particle gun has also been used with pollen, early stage embryoids, meristems and somatic embryos. • Advantages: Requirement of protoplast can be avoided and this method can be use to transform all plant species. • Manipulation of genome of subcellular organelles can be achieved. • Limitations: High cost of the equipment and microcarriers. • Intracellular target is random (cytoplasm, nucleus, vacuole, plastid, etc.). • Transfer DNA is not protected.
- 18. • MICROINJECTION : Microinjection where the DNA is directly injected into plant protoplasts or cells (specifically into the nucleus or cytoplasm) using fine tipped (0.5 - 1.0 micrometer diameter) glass needle or micropipette. • This method of gene transfer is used to introduce DNA into large cells, normally performed under a specialized optical microscope setup called a micromanipulator. • The process is frequently used as a vector in genetic engineering and transgenetics to insert genetic material into a single cell. • Computerized control of holding pipette, needle, microscope stage and video technology has improved the efficiency of this technique. • Applications of microinjection: Process is applicable for plant cell as well as animal cell but more common for animal cells and is ideally useful for producing transgenic animal quickly. • Procedure is important for gene transfer to embryonic cells. Applied to inject DNA into plant nuclei such as cells and protoplast of tobacco, alfalfa etc. • Microinjection is potentially a useful method for simultaneous introduction of multiple bioactive compounds such as antibodies, peptides, RNAs, plasmids, diffusion markers, elicitors, Ca2+ as well as nucleus and artificial micro or Nano particles containing those chemicals into the same target single-cells.
- 19. • Procedure: During microinjection, plant protoplast or partially synthesized cells are fixed to glass cover slips with the help of poly L. lysine. • If any cell type is reluctant to attach to cover slips by binding agent, holding pipette can be an essential factor in microinjection. • These cell types are firmly retained on fixed place by blunt holding pipette. • The exogenous DNA of 1 pm is taken in micro-injector and the cells or protoplasts are firmly immobilized by holding pipette by exerting suction pressure. • Microinjection containing approximate dosage of DNA is then directly delivered inside the cells. • In microinjection, it is possible to microinject 200-350 protoplasts intra nuclearly and transformation frequency has been demonstrated with 20-60% success . • By means of reference marking on the coverslip, it is possible to locate microinjected cells/protoplast by recording with a video camera, which enables to work more freely from one microinjected cell to next one without interception. • Earlier microinjection studies were restricted to insect fluorescent dye and introduction of virus. Microinjection of protoplast for transformation purpose is a recent achievement. It was however, reasonably believed that injection of DNA directly into the nucleus accelerates transformation frequency.
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- 21. • Advantages of microinjection: Method is effective in transforming primary cells as well as cells in established cultures. • The DNA injected in this process is subjected to less extensive modifications. • The amount of DNA delivered can be optimized. • Precise and predictable delivery of DNA. • Small cell structures like microspores, callus and proembyros can be precisely targeted. • Micro-culture is accomplished. • Limitations of microinjection: • Costly, • Sophisticated equipment. • Handling of protoplast for microinjection requires skilled personal required. • Knowledge of mating timing and Only one cell receives DNA per injection. • Method is useful for protoplasts and not for the walled cells.
- 22. • MACRO INJECTION • Macroinjection is the method tried for artificial DNA transfer to cereals plants that show inability to regenerate and develop into whole plants from cultured cells. • Needles used for injecting DNA are with the diameter greater than cell diameter (>10-100um). • DNA injected with conventional syringe into region of plant which will develop into floral tillers. • Around 0.3 ml of DNA solution is injected at a point above tiller node until several drops of solution came out from top of young inflorescence. • Timing of injection is important and should be fourteen days before meiosis. • This method was found to be successful with rye plants. • It is also being attempted for other cereals plants.
- 23. • Advantages • This technique does not require protoplast. • Instrument will be simple and cheap. • Methods may prove useful for gene transfer into cereals which do not regenerate from cultured cell easily. • Limitations • Less specific, • Less efficient, • Frequency of transformation is very low (0.07%)
- 24. LIPOSOME MEDIATED GENE TRANSFER /LIPOFECTION • Gene transfer mediated by liposomes was first described by Fengler in 1980 • Liposomes are microscopic vesicles developed in a laboratory environment. Each liposome is a spherical ball like structure made up of phospholipid bilayers with a hollow central space, allowing liposomes to interact directly with cells. • Such vesicles when mixed with cells in culture, fuse with the cell membrane and deliver DNA directly into the cytoplasm. • It is a first non-viral technique devised specifically for in vivo DNA transfer. • A liposome can fuse with the cell membrane of the taken host cell and can deliver its content to it. The recombinant DNA enclosed in the liposome vesicles penetrates into the protoplast of the host cell. • These are artificial vesicles that can act as delivery agents for exogenous materials including transgenes. Cationic lipids are those having a positive charge are used for the transfer of nucleic acid. • Liposomes are able to interact with the negatively charged cell membrane more readily than uncharged liposomes. Due to fusion between cationic liposome and cell surface results in the delivery of DNA directly across the plasma membrane.
- 25. • Procedure: • In this technique the recombinant DNA, which is negatively charged at a near neutral pH because of its phospho-diester backbone, is mixed with the lipid molecules with positively charged (cationic) head groups. The lipid molecules form a bilayer around the recombinant DNA molecules. • This results in the formation of liposomes which are further mixed with the host cells. Most eukaryotic cells are negatively charged at their surface, so the positively charged liposomes interact with the cells. • Cells take up the lipid-recombinant DNA complexes, and some of the transfected DNA enters the nucleus. Fusion of liposomes will be resulted at the point of attachment of DNA or plasmid DNA while entering the cell. This technique has no obvious advantages over any other gene transfer methods. • DNA containing liposomes can be directly microinjected into the vacuole, releasing the content of liposome into the cytoplasm. However, micro-injected vacuole led to fusion with tonoplast. This indicates that they could be used to transform even vacuolated cells. Although this method is elegant on certain criteria, unfortunately, regeneration plants are problematic with high vacuolated cells.
- 26. • Advantages • High degree of reproducibility. • Long term stability. • Protection of nucleic acid from degradation. • Low toxicity. • Disadvantage • preparation of DNA-containing liposomes is complicated and labor- intensive.
- 27. • CALCIUM CHLORIDE (CACL2) MEDIATED DNA TRANSFER: This is used for the transformation of prokaryotic host cells. • Principle: In the process of transformation all bacterial cells cannot uptake the exogenous DNA molecule. CaCl2 makes the cell wall of the bacteria more permeable to the exogenous DNA and thus increases the competence of the host cell. • The process of transfection involves the admixture of isolated DNA (10-100ug) with solution of calcium chloride and potassium phosphate under condition which allow the precipitate of calcium phosphate to be formed. • Cells are then incubated with precipitated DNA either in solution or in tissue culture dish. 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 %. It can be increased if very high purity DNA is used and the precipitate allowed to form slowly.
- 28. • Procedure: Growing E. Coli cells are isolated and suspended in 50 mM CaCl2 at a concentration of 108-1010 cells/ml. • The cells may be incubated for 12- 24 hr. to increase the frequency of transformation. The recombinant DNA (10-100ug) is then added. • Efficient transformation takes only a few minutes and the cells are plated on a suit- able medium for the selection of transformed clones. • The frequency of transformed cells is 106-107 per mg of plasmid DNA; this is about one transformation per 10,000 plasmid molecules. • The transformed cells are suitably diluted and spread thinly on a suitable medium so that each cell is well separated and produces a separate colony. • Generally, the medium is so designed that it permits only the transformed cells to divide and produce colonies. • This frequency can be further improved by using special E. Coli strains, e.g., SK1590, SK1592, X1766, etc.
- 29. Limitations • Frequency is very low. Integrated genes undergo substantial modification. • Many cells do not like having the solid precipitate adhering to them and the surface of their culture vessel. • Due to above limitations transfection applied to somatic gene therapy is limited. RUBIDIUM CHLORIDE MEDIATED DNA TRANSFER: In this method a variant of the calcium chloride method that offers somewhat higher competency. The process followed is same as before but just the CaCl2 is replaced with RbCl2. This is also used in the transformation of the prokaryotic host cell.
- 30. • POLY ETHYLENE GLYCOL (PEG) MEDIATED TRANSFORMATION: • Poly ethylene glycol (PEG) is inert, least toxic to cells and protoplast. Polyethylene glycol (PEG), in the presence of divalent cations (using Ca2+), destabilizes the plasma membrane of protoplasts and renders it permeable to naked DNA. • PEG in complex with divalent cation can disturb molecular organization of the plasma membrane of the protoplast. • Positive charges of the calcium are attracted by the negative charge of the protoplast membrane and alter its zeta potential and destabilize it. Finally DNA makes entry inside the cell and integrates into the genome. • The technique not only helps in assessment of transformation, but also involve in regulating gene transfer into the plant cells. Once DNA gains entry inside the cell, it is susceptible for degradation inside cytoplasm. • Culture of protoplasts is taken into a tube and to this tube 40% PEG 4000 (w/v) dissolved in mannitol and calcium nitrate is added slowly. Then incubated for few min.
- 31. • Process • Protoplast suspended in medium (Mg and Ca ions) • Heat shock treatment (5min, 45 ˚C) • PEG added (20-28% conc.) • Incubation (calcium conc. enhenced) • Cultured • Advantages • A large number of protoplasts can be simultaneously transformed. • Can successfully use for a wide range of plant species. • Limitations • The DNA is susceptible for degradation. • Random integration of foreign DNA into genome may result in undesirable traits. • Regeneration of plants from transformed protoplasts is a difficult task.
- 32. ULTRASOUND MEDIATED TRANSFER/SONOPORATION: • The uptake of foreign DNA by protoplast or cells can be facilitated by imposing ultrasound. It involves the exposure of cells to a rapidly oscillating probe, such as the tip of a sonicator. Test tube containing cells or protoplast in a buffer is made to contact by inserting tip of ultrasonic device. The ultrasonic pulse generated by ultrasonicator of 0.4 m/cm2 acoustic intensity is applied for 20-25 min. • Vigorous vibration in the medium and violent collpase of bubbles generates high hydrostatic pressure and shock wave may result in sporadic localized rupture in the membrane and it can facilitates uptake of exogenous DNA. • The transient appearance of such cavities allows DNA to cross the membrane into the cytoplasm. It has been shown that the application of low-frequency ultrasound allows the efficient delivery of nucleic acids into mammalian cells both in vitro and in vivo, because the plasmid DNA is left structurally intact. • Furthermore, the ultrasound waves appear to have no adverse effects when focused on different anatomic locations in the human body. • Hence, ultrasound-mediated gene delivery raises no safety concerns. Gene transfer in vivo is generally achieved by injection followed by the application of a focused ultrasound device.
- 33. • DNA TRANSFER BY DAE-DEXTRAN METHOD : DEAE-dextran was the first transfection reagent to be developed and was very widely used until the advent of lipofection reagents in the 1990s. • It is a soluble polycationic carbohydrate that forms aggregates with DNA through electrostatic interactions. It provides the entire complex with a net positive charge, which allows it to interact with the negatively charged cell membrane and promotes uptake by endocytosis. • Like the calcium phosphate method, the reagents are inexpensive and the procedure is simple and efficient. • DEAE-dextran-mediated transfection is not particularly efficient for the production of stably transformed cell lines. • If DEAE-Dextran treatment is coupled with Dimethyl Sulphoxide (DMSO) shock, then upto 80% transformed cell can express the transferred gene. • It is known that serum inhibits this transfection so cells are washed nicely to make it serum free. Stable expression is very difficult to obtain by this method. Treatment with chloroquinine increases transient expression of DNA. The advantage of this method is that, it is cheap, simple and can be used for transient cells which cannot survive even short exposure of calcium phosphate.
- 34. • SILICON CARBIDE FIBER (SCF) MEDIATED TRANSFER: SCF does not require any specialized equipment. • In this approach, silicon carbide fibres in average of 0.4-0.6 µm in diameter and 10- 90 µm long are taken along with DNA in vortex tube. • Plant cells or embryos are then introduced and vortexed gently. • Entry of DNA into the cell is probably due to the penetration through the cell wall and plasma membrane. • Vortexing process results in the adhering DNA to silicon carbide fibres and gained access to inside the nucleus and eventually stable integration into the nucleus genome. Thus, passing of the DNA across the cell wall has advantage over other methods. • This approach does not involve regeneration of protoplast. Presently this technique is applicable to a particular species, which produce friable nature of callus. • Many cereals cannot be transformed by SCF as they produce non friable brittle nature of callus.
- 35. • MICROLASER: Micro laser mediated gene transfer offers advantage only in specific cases where other methods are not advantageous. • This technique involves focusing micro laser beam into the light path or microscope used to burn holes into the cell wall as membrane DNA uptake is possible through penetrated cells during incubation. • Several instances have shown that DNA gets adsorbed to the cell wall material even before its entry inside the cell. • IMPALEFECTION: Impalefection is a method of gene delivery using Nano materials, such as carbon Nano fibres, carbon nanotubes, nanowires, etc. This technique is used for the transfection of plant and animal cells. In this technique needle-like nanostructures are synthesized perpendicularly to the surface of a sub- strate. Recombinant DNA is attached to the nanostructure surface. A chip with arrays of these needles is then pressed against cells or tissue. • MAGNETOFECTION: Magnetofection, or Magnet assisted transfection is a method, which uses magnetic force to deliver recombinant DNA into target host cells. Nucleic acids are first associated with magnetic nanoparticles. Then, application of magnetic force drives the nucleic acid particle complexes towards and into the target host cells, where the cargo is released. This has been successfully used to transfect the plant and animal cells.
- 36. • VIRAL DELIVERY SYSTEMS: Viruses are naturally evolved vehicles that efficiently transfer their genes into host cells. • Viral vectors that have been extensively studied and genetically manipulated for safety concerns in laboratory research and for in vivo gene transfer protocols include retroviruses, adenoviruses, herpes simplex viruses, lentiviruses, adeno associated viruses and Sindbis viruses. • Choice of viral vectors is dependent on gene transfer efficiency, capacity to carry foreign genes, toxicity, stability, immune responses towards viral antigens and potential viral recombination. • There is a wide variety of vectors used to deliver DNA or oligo nucleotides into mammalian cells, either in vitro or in vivo. • Other viral vectors that are currently under development are based on lenti viruses, human cytomegalovirus (CMV), Epstein-Barr virus (EBV), poxviruses, negative- strand RNA viruses (influenza virus), alpha viruses etc. • The three commonly used viral gene transfer systems are • Retrovirus (RV), Adenovirus (AV), Adeno Associated Virus (AAV).
- 38. References: • Transposition - CoGepedia.mhtml • https://www.takarabio.com/learning-centers/gene-function/viral- transduction/retrovirus/retroviral-products • Physical Methods of Gene Transfer _ Genetics.mhtml THANK YOU