TRANSFER OF RECOMBINANT DNA TRANSFER OF RECOMBINANT DNA TRANSFORMATION TRANSFECTION TRANSFORMATION  Transformation can be defined as the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material from its surroundings through cell membrane.  This can be done to bacteria fungi plants animals  One of the processes for horizontal gene transfer  For transformation to take place the recipient should be in state of competence HISTORY OF TRANSFORMATION  1928 – Transformation was first demonstrated by Frederick Griffith  1944 – Transformation principle was identified by Oswald Avery, Cocin MacLeod, and Maclyn McCarty  1970 – Morton Mandel and Akiko Higa shoed that E.coli may be induced to take up DNA after treatment with CaCl2  1972 – Stanley Norman Cohen, Annie Chang, Leslie Hsu showed treatment with cacl2 is effective for transformation  Later Douglas Hanahan improved Mandel and Higa’s method of transformation PROCEDURE 1.Several successive washes in cold NaCl, MgCl2, or CaCl2 2.Incubation in Cacl2 at 0ºC with DNA 3. A short incubation at some higher temp 4.Dilution into growth medium or selection APPLICATIONS  To make multiple copies of DNA, called DNA cloning.  To make large amounts of specific human proteins, for example, human insulin, which can be used to treat people with Type I diabetes.  To genetically modify a bacterium or other cell LIMITATIONS  Size of the genome  Might disrupt the existing gene at the point of insertion  the new gene may be a selfish replicator, e.g. a transposable element that duplicates itself many times within the host genome or a virus that destroys the infected cell. TRANSFECTION  Transfection is the transfer of foreign DNA into a cultured host cells mediated through chemicals.  this typically involves opening transient pores in the cell plasma membrane to allow uptake of the material.  Charged chemical substances such as cationic liposomes, calcium phosphate or DEAE dextran are used. TRANSFECTION BY CALCIUM PHOSPHATE  One of the cheapest methods uses calcium phosphate, originally discovered by F. L. Graham and A. J. van der Eb in 1973. Saline solution 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. This process has been a preferred method of identifying many oncogenes. TRANSFECTION BY CATIONIC POLYMER  Another method is the use of cationic polymers, which structurally may either be linear or highly branched  Commonly used cationic polymers are histones, polyamidoamine dendrimers and protamine.  The negatively charged DNA binds to the polycation and the complex is taken up by the cell via endocytosis. LIPOFECTION  Lipofection (or liposome transfection) is a technique used to inject genetic material into a cell by means of liposomes, which are vesicles that can easily merge with the cell membrane since they are both made of a phospholipid bilayer.  Lipofection generally uses a positively charged lipid (cationic liposomes or mixtures) to form an aggregate with the negatively charged (anionic) genetic material.  This transfection technology performs the same tasks as other biochemical procedures utilizing polymers, DEAE-dextran, calcium phosphate, and electroporation.  The efficiency of lipofection can be improved by treating transfected cells with a mild heat shock. LIMITATIONS  Factors such as serum in media and cell confluency affect the transfection efficiency.  For protocol using calcium phosphate, it is important to have consistency in reagent properties, such as pH, to avoid compromised efficiency.  The calcium phosphate method does not work for cells grown in media with high phosphate levels, TRANSFORMATION VS TRANSFECTION  Direct uptake and incorporation of exogenous genetic material form the surroundings through cell membrane.  Introduces foreign DNA into plant, yeast or bacterial cells.  Uses chemical transformation, electroporation  Process of deliberately introducing naked or purified nucleic acids into eukaryotic cells.  Introduces foreign DNA into mammalian cells.  Uses calcium phosphate coprecipitation, liposomes, cationic polymers THANK YOU