Methods of introducing gene in target cells are allexcept:
FISH Fluorescent In Situ Hybridization (FISH) is a method to detect or comfirm gene or chromosome abnormalities. It utilizes fluorescently labeled DNA probes. The sample DNA is first denatured (a process that separates the complimentary strands within the DNA double helix structure) then fluorescently labeled probe of interest is added to the denatured sample mixture and hybridizes with the sample DNA at the target site as it reanneals (or reforms itself) back into a double helix. The probe signal can then be seen through a fluorescent microscope and the sample DNA scored for the presence or absence of the signal. Gene therapy is a technique for correcting defective genes responsible for disease development. inseed into a nonspecific location within the genome to replace a nonfunctional gene. A carrier molecule called a vector is used to deliver the therapeutic gene to the patient's target cells. Currently, the most common vector is a virus that has been genetically altered to carry normal human DNA. Besides virus-mediated gene-delivery systems, there are several nonviral options for gene delivery. The simplest method is the direct introduction of therapeutic DNA into target cells. This approach is limited in its application because it can be used only with ceain tissues and requires large amounts of DNA. Transfection is defined as the process of deliberately introducing nucleic acids into cells. The term is used notably for non-viral methods "Transduction" is the term often used to describe virus-mediated DNA transfer. Methods of Gene delivery There are various methods of introducing foreign DNA into a eukaryotic cell: some rely on physical treatment (electroporation, nanopaicles, magnetofection), other on chemical materials or biological paicles (viruses) that are used as carriers. Chemical-based transfection Therapeutic DNA can get inside target cells by chemically linking the DNA to a molecule that will bind to special cell receptors. Once bound to these receptors, the therapeutic DNA constructs are engulfed by the cell membrane and passed into the interior of the target cell. Methods used are: a) Calcium phosphate: (This method is dependent upon the use of a calcium chloride-containing HEPES-buffered saline solution to deliver plasmid DNA. HEPES-buffered saline solution (HeBS) containing phosphate ions is combined with a calcium chloride solution containing the DNA to be transfected. When the two are combined, a fine precipitate of the positively charged calcium and the negatively charged phosphate will form, binding the DNA to be transfected on its surface. The suspension of the precipitate is then added to the cells to be transfected (usually a cell culture grown in a monolayer). By a process not entirely understood, the cells take up some of the precipitate, and with it. the DNA.) b) Dendrimers: are highly branched organic compounds. used to bind the DNA and get it into the cell. C) Liposomes: are small, membrane-bounded bodies that are in ways similar to the structure of a cell and can actually fuse with the cell membrane, releasing the DNA into the cell. d) Cationic polymers: such as DEAE-dextran or polyethylenimine. The negatively charged DNA binds to the polycation and the complex is taken up by the cell endocytosis. Non chemical methods a) Electroporation: is a technique by which plasma membrane permeability is transiently increased using a small electric current so that it allows extracellular macromolecules (including DNA) to be taken up into the cell. b) Sonoporation: Similar to electroporation Sonoporation, is the use of sound (typically ultrasonic frequencies) for modifying the permeability of the cell plasma membrane. c)Optical transfection: is a method where a tiny (--1 pm diameter) hole is transiently generated in the plasma membrane of a cell using a highly focused laser. d) Irnpalefection: is a method of gene delivery using nanomaterials, such as carrbon nanofibers, carbon nanolubes, nanowires e)Hydrodynamic delivery: is dependent on the rapid injection of a large aqueous volume containing DNA, RNA or other molecules into the blood vessels. Paicle-based methods Gene gun: A direct approach to transfection is the gene gull, where the DNA is coupled to a nanopaicle of an ine solid (commonly gold) which is then "shot" directly into the target cell's nucleus. Magnetofection: or Magnet assisted transfection is a transfection method, which uses magnetic force to deliver DNA into target cells. Nucleic acids are first associated with magnetic nanopaicles. Then, application of magnetic force drives the nucleic acid paicle complexes towards and into the target cells, where the cargo is released.
Electroporation
Transfection
Site directed recombination
FISH