Week 7-8 Lecture PDF

LABORATORY METHODS A/PROF DONALD WLODKOWIC BIOLOGICAL MODELS A BASIC INTRODUCTION IN VITRO MODELS BIOCHEMICAL AND MOLECULAR ANALYSIS In vitro (meaning: in the glass) Petri dish studies are performed with biological molecules outside their normal biological context or even with livi...

LABORATORY METHODS A/PROF DONALD WLODKOWIC BIOLOGICAL MODELS A BASIC INTRODUCTION IN VITRO MODELS BIOCHEMICAL AND MOLECULAR ANALYSIS In vitro (meaning: in the glass) Petri dish studies are performed with biological molecules outside their normal biological context or even with living microorganisms, cells, or tissues. Colloquially called "test-tube experiments", these studies in biology and its subdisciplines are traditionally done in various labware such as test tubes, flasks, Petri dishes, and microtiter plates. IN VITRO MODELS BIOCHEMICAL AND MOLECULAR OMICS TECHNIQUES IN VITRO CELL CULTURE KEEPING SINGLE CELLS AND TISSUES IN THE LAB In vitro studies as mentioned can be also are performed with microorganisms, plant, animal and human cells and even tissue samples that are kept outside of their organism or biological environment. Colloquially cell culture refers to keeping and growing cells in special media to perform various experiments on them. CELLS CAN BE ISOLATED FROM TISSUES AND GROWN IN LABS Source: cloud-clone.com IN VITRO CELL CULTURE MULTIPLE EXPERIMENTS CAN BE CARRIED ON CELLS Cell culture of human cells Source: oboeipr.com Examples of in vitro experiments how cells respond to drugs how cells proliferate how cells move and migrate genetic modifications of cells how cell communicate Source: unisense.com IN VITRO CELL CULTURE FROM BACTERIA TO HUMAN CELLS Bacteria Yeast Plant / Animal / Human (c) various eukaryotic cells extracted from multicellular organisms grown in special media and flasks in the lab widely used in drug discovery, toxicology and cell biology IN VIVO MODELS TRADITIONAL ANIMAL MODELS IN BIOLOGY In vivo studies and in vivo models are those in which the effects of drugs or biological agents are tested on intact, living organisms, usually animals, including humans, and plants. Animal testing and clinical trials are major elements of in vivo research. In vivo testing is often preferred over in vitro because of it superior physiological relevance. ALTERNATIVE IN VIVO MODELS SMALL MODEL ORGANISMS IN VIVO SMALL MODEL ANIMALS Source: Kokel and Peterson 2008 IN VIVO MODELS ALTERNATIVE ANIMAL MODELS IN BIOSCIENCES Fruit fly Nematode Zebrafish (e) Danio rerio (zebrafish) complex multicellular organism with physiology similar to humans many transgenic models and molecular tools available widely used in drug discovery, toxicology, developmental biology and neurobiology THE MIGHTY FISH ZEBRAFISH DANIO RERIO MODELS IN MODERN BIOMEDICINE Transgenic Fli1:EGFP line (EGFP expressed in vasculature and neural crest) Control Drug Treated WHAT IS A PHYSIOME? INTEGRATING GENES, PROTEINS, CELLS, TISSUES, ORGANS The physiome of an individual's or species' physiological state is the description of its functional behaviour. The physiome describes the physiological dynamics of the normal intact organism and is built upon genetic information. The physiome integrates genomes, proteomes, and morphomes. Studying physiomes allows phenotypic bioassays within an intact physiological milieu. PHENOMICS - THE NEXT FRONTIER GENES + ENVIRONMENT = MODIFIABLE OUTCOME OF PHYSIOME PHYSIOME Source: http://phenomecentre.org/about-us/what-is-phenomics/ OMICS AS A SCALAR APPROACH TO UNCOVER STRUCTURE, FUNCTION AND DYNAMICS OF LIFE PHENOMICS PHYSIOMICS BIOLOGICAL COMPLEXITY CELLOMICS METABOLOMICS PROTEOMICS GENOMICS TRANSCRIPTOMICS EPIGENOMICS PHYSIOLOGICAL RELEVANCE OMICS IN DRUG DISCOVERY INCREASING PHYSIOLOGICAL RELEVANCE OF BIOASSAYS PHENOMICS / PHYSIOMICS TISSUEOMICS COST OF ANALYSIS CELLOMICS STUDYING PHENOTYPES AND PHYSIOMES GENOMICS ALLOWS BIOASSAYS WITHIN AN INTACT PROTEOMICS P H Y S I O LO G I C A L M I L I E U. I T A L S O ALLOWS DRUG DISCOVERY WITHOUT AN A PRIORI KNOWLEDGE OF THE MOLECULAR TARGET. PHYSIOLOGICAL RELEVANCE LABORATORY METHODS MICROSCOPY SEEING CELLS MICROSCOPY: SEEING CELLS STAPLE OF CELL BIOLOGY TECHNIQUES Source: azooptics.com MICROSCOPY STAPLE OF CELL BIOLOGY TECHNIQUES The optical microscope, also called the light microscope, is the oldest type of microscope which uses visible light and lenses in order to magnify images of very small samples. It is a standard tool frequently used in biology. The first basic optical microscopes were developed in the 17th century. Today, there are many variations available that enable high level resolutions and sample contrasts. With the help of computer-aided optical design and automated grinding methodology, image quality has improved immensely. Source: azooptics.com TYPES OF MICROSCOPY FROM LIGHT TO ELECTRON MICROSCOPY DIFFERENT TYPES OF LIGHT MICROSCOPY EXPLOIT DIFFERENT PROPERTIES OF LIGHT CELL PREPARATION BEFORE MICROSCOPY CELLS NEED TO BE OFTEN FIXED, STAINED AND SECTIONED IMMUNOFLUORESCENCE STAINING OF SPECIFIC CELL COMPONENTS WITH ANTIBODIES Cells are made of thousands of different types of molecules that make up a wide variety of cellular structures. With so many different molecules present, how can researchers determine the presence and location of one specific type of molecule within a cell? IMMUNOFLUORESCENCE UTILISES FLUORESCENT PROBES, ANTIBODIES AND MICROSCOPY Immunofluorescence is a technique in which a fluorescent molecule (fluorochrome, fluorophore) is attached to an antibody, which recognizes and binds to one specific complementary target molecule, known as its antigen. Using a fluorescence or confocal microscopy, a researcher can then identify and locate the specific target molecule within the cell. FLUOROCHROMES FLUORESCENT MOLECULES WITH SPECIAL CHARACTERISTICS ANTIBODIES DEFENCE PROTEINS PRODUCED BY IMMUNE SYSTEM ANTIBODIES ARE PRODUCED BY LYMPHOCYTES Antibodies (Ab) known as an immunoglobulins (Ig), are large, Y- shaped proteins produced mainly by white blood cells called B lymphocytes. Antibodies are used by the immune system to neutralize pathogens such as pathogenic bacteria and viruses. Antibodies do not directly kill pathogens, but by binding to antigens, they interfere with pathogen activity or mark pathogens in various ways for inactivation or destruction. Consider, for example, neutralisation, a process in which antibodies bind to proteins on the surface of a virus ANTIGENS STRUCTURES SPECIFICALLY BOUND BY ANTIBODIES Antigens are molecular structures "targeted" by antibodies. Each antibody is specifically produced by the immune system to match an antigen after cells in the immune system come into contact with it; this allows a precise identification or matching of the antigen and the initiation of a tailored response. In most cases, antibody can only react to and bind one specific antigen; in some instances, however, antibodies may cross- react and bind more than one antigen. ANTIBODIES CAN DETECT AND BIND TO SPECIFIC ANTIGENS Antibody has a very characteristic structure. It contains a constant region (C) that is the same for all antibodies of a particular type, and variable regions (V) that are identical to each other but unique for each antibody. The unique V regions at the tips of the Y contain a binding pocket into which only one specific antigen will fit. Antigens (Ag) are structures (aka substances) specifically bound by antibodies (Ab) MONOCLONAL ANTIBODIES CAN BE MASS PRODUCED AGAINST ANY ANTIGEN Monoclonal antibodies (mAb) are antibodies that are made by identical immune cells that are all clones of a unique parent cell. Monoclonal antibodies have monovalent affinity, in that they bind to the same epitope (the part of an antigen that is recognized by the antibody). Such antibodies can be generated in the laboratory by injecting a foreign protein or other macromolecule into an animal host, such as a rabbit or mouse, producing antibodies that will bind selectively to virtually any protein that a scientist wishes to study. IMMUNOFLUORESCENCE PRIMARY (DIRECT) IMMUNOFLUORESCENCE TECHNIQUE Using primary (or direct) immunofluorescence, antibody molecules are labeled with a fluorescent dye, known as a fluorophore, that is covalently linked to the C region of each antibody molecule. The antibody recognizes and binds to the target molecule, which can then be detected using fluorescence or confocal microscopy. IMMUNOFLUORESCENCE PRIMARY (DIRECT) IMMUNOFLUORESCENCE TECHNIQUE IMMUNOFLUORESCENCE SECONDARY (OR INDIRECT) IMMUNOFLUORESCENCE TECHNIQUE In secondary (or indirect) immunofluorescence antibody that is not labeled with dye. This antibody, called the primary antibody, attaches to specific anti- genic sites within the tissue or cell. A second type of antibody, called the secondary antibody, is labeled with a fluorescent dye and then added to the sample, where it attaches to the primary antibody. FLUORESCENT MICROSCOPY EMPLOYS A SET OF FILTERS FOR DETECTION OF SIGNALS FLUORESCENT MICROSCOPY EMPLOYS A SET OF FILTERS FOR DETECTION OF SIGNALS FLUORESCENT MICROSCOPY CAN IMAGE MORE THAN ONE STAINED MOLECULE AT A TIME By using different combinations of antibodies and dyes, more than one molecule in a cell can be labeled at the same time. Different dyes can be imaged using different combinations of fluorescent filters, and the dif- ferent images can be combined to generate striking pictures of cellular structures. Source: micro.magnet.fsu.edu CELLS IN MULTIPLE COLOURS FLUORESCENT MICROSCOPY Source: cellimagelibrary.org LIVING CELL FLUORESCENT MICROSCOPY ENABLES US TO STUDY LIVING CELLS IN CULTURE Division of a human stem cell: Programmed cell death: orange stain - mitochondria different colours are markers labelling different phases of cell death LIMITATIONS OF LIGHT MICROSCOPY CAN ONLY RESOLVE DETAILS 200 NM APART LIMITATIONS OF LIGHT MICROSCOPY DIFFRACTION OF LIGHT LIMITS THE RESOLUTION TO 200 NM An optical microscope cannot resolve two objects located closer than λ/2NA, where: λ is the wavelength of light NA is the numerical aperture of the imaging lens. In other words, diffraction limits the ability of the microscope to distinguish between two objects divided by a lateral distance of less than half the wavelength of light used to image the sample. ELECTRON MICROSCOPY SEEING IN NANOMETER SCALE USING ELECTRONS TRANSMISSION ELECTRON MICROSCOPY BEAM OF ELECTRONS IS TRANSMITTED THROUGH A SPECIMEN TO FORM AN IMAGE SCANNING ELECTRON MICROSCOPY BEAM OF ELECTRONS IS SCATTERED BY A SURFACE OF SPECIMENS NEXT GEN ELECTRON MICROSCOPY ION ABRASION SCANNING ELECTRON MICROSCOPY MOLECULAR METHODS ANALYSING BIOMOLECULES CELL FRACTIONATION ISOLATING BIOMOLECULES CELL FRACTIONATION INDIVIDUAL CELL COMPARTMENTS CAN BE SEPARATED Cell fractionation is the process used to separate cellular components while preserving individual functions of each component. This process involves: cell/tissue homogenisation differential centrifugation CELL FRACTIONATION HOMOGENISATION Step 1 - Homogenisation Tissue is typically homogenized in a buffer solution that is isotonic to stop osmotic damage. Mechanisms for homogenization include grinding, mincing, chopping, pressure changes, osmotic shock, freeze-thawing, and ultra-sound. The samples are then kept cold to prevent enzymatic damage. It is the formation of homogenous mass of cells (cell homogenate or cell suspension). CELL FRACTIONATION DIFFERENTIAL CENTRIFUGATION CELL FRACTIONATION DIFFERENTIAL CENTRIFUGATION Step 2 - Differential centrifugation The sequential increase in gravitational force results in the sequential separation of organelles according to their density. After each centrifugation the pellet is removed and the centrifugal force is increased. Finally, purification may be done through equilibrium sedimentation, and the desired layer is extracted for further analysis. CELL FRACTIONATION DIFFERENTIAL CENTRIFUGATION In differential centrifugation, you can isolate and enrich the following cell components, in the separating order: Whole cells and nuclei; Mitochondria, chloroplasts, lysosomes, and peroxisomes; Microsomes (vesicles of disrupted endoplasmic reticulum); and Ribosomes and cytosol. DIFFERENTIAL CENTRIFUGATION SEDIMENTATION SPEED In differential centrifugation, the relative size and density of an organelle or macromolecule is expressed in Svedberg units (S), which describe its sedimentation coefficient. This is a measure of how rapidly the particle sediments when subjected to centrifugation. Larger and/or denser particles have higher sedimentation coefficients. For example, human ribosomes are slightly larger than bacterial ribosomes and have larger sedimentation coefficients. DENSITY GRADIENT CENTRIFUGATION ALLOWS FOR MORE PRECISE SEPARATION In differential centrifugation, the particles are uniformly distributed throughout the solution prior to centrifugation, in density gradient centrifugation homogenate is placed instead as a thin layer on top of a gradient of solute, typically sucrose. This gradient has an increasing concentration of solute—and therefore density—from the top of the tube to the bottom. When centrifuged, particles differing in size and density move downward as discrete zones, or bands, that migrate at different rates.  CHROMATOGRAPHY ISOLATING SPECIFIC BIOMOLECULES COLUMN CHROMATOGRAPHY CAN BE USED TO SEPARATE / FILTRATE MOLECULES Column chromatography is frequently used by to purify proteins. A sample of water soluble mature of proteins is loaded onto a column of adsorbant, such as silica gel or alumina. A solvent continuously flows down through the column. Components of the sample separate from each other by partitioning between the stationary packing material and the mobile solvent. Molecules with different chemical characteristics can be fractionated and isolated from one another because they move through the column at different rates. COLUMN CHROMATOGRAPHY CAN BE USED TO SEPARATE / FILTRATE MOLECULES Column chromatography is frequently used by to purify proteins. A sample of water soluble mature of proteins is loaded onto a column of adsorbant, such as silica gel or alumina. A solvent continuously flows down through the column. Components of the sample separate from each other by partitioning between the stationary packing material and the mobile solvent. Molecules with different chemical characteristics can be fractionated and isolated from one another because they move through the column at different rates. AFFINITY CHROMATOGRAPHY CAN BE USED TO ISOLATE SPECIFIC MOLECULES Affinity chromatography employs specific molecules e.g. antibodies attached to the surface of the surface of the column that preferentially binds to the protein being purified. One very common form of affinity chromatography is immunoaffinity chromatography. In this approach, an antibody that is specific for a protein of interest is attached to beads. The bound protein can then be eluted from the column using special buffer The chromatin immunoprecipitation technique for identifying DNA-binding proteins relies on this approach. STUDYING PROTEINS PROTEIN GEL ELECTROPHORESIS (PAGE) PROTEIN SEPARATION BY SIZE Polyacrylamide gel electrophoresis (PAGE), describes a technique widely used in biochemistry, forensics, genetics, molecular biology and biotechnology to separate biological macromolecules, usually proteins or nucleic acids, according to their electrophoretic mobility. Mobility is a function of the length, conformation and charge of the molecule. SDS-PAGE PROTEIN ELECTROPHORESIS IN DENATURATING CONDITIONS Proteins may be run in their native state, preserving the molecules' higher-order structure. This method is called Native-PAGE. Alternatively, a chemical denaturant may be added to remove this structure and turn the molecule into an unstructured molecule whose mobility depends only on its length and mass-to-charge ratio. This procedure is called SDS-PAGE. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a method of separating molecules based on the difference of their molecular weight. ISOELECTRIC FOCUSSING PROTEIN SEPARATION BY CHARGE Proteins can separated in a polyacrylamide tube gel using isoelectric focusing. Isoelectric focusing relies on separating proteins by charge using a pH gradient. Proteins stop migrating at a pH at which their net charge is zero, a point known as that protein’s isoelectric point TWO DIMENSIONAL GEL ELECTROPHORESIS 2D SDS-PAGE COMBINES ISOELECTRIC FOCUSING AND SDS-PAGE Step 1 - Isoelectric focusing Step 2 - SDS-PAGE WESTERN (IMMUNO) BLOTTING SEPARATED PROTEINS ARE DETECTED WITH ANTIBODIES Western blots are performed by first separating the proteins by size using SDS-PAGE. The separated proteins are then blotted onto a nitrocellulose or nylon membrane using an electric current. The next step is similar in logic to immunostaining: after the transfer is complete, the blot is washed, incubated with a primary antibody, washed again, and then exposed to a secondary antibody. The secondary antibody is conjugated to an enzyme whose presence can be detected either using a colored precipitation reaction product or using chemiluminescence, the generation of light through a chemical reaction. STUDYING ENZYMES ENZYME KINETICS IS MEASURED USING A SPECTROPHOTOMETER The rate of product formation can be determined using a spectrophotometer. The spectrophotometer measures how much light of a particular wavelength is absorbed by a solution. If a reactant or product absorbs light at a characteristic wavelength, an increase in the products or a decrease in the reactants provides a measure of the progress of the reaction. ENZYME KINETICS EFFECTS OF SUBSTRATE CONCENTRATION By determining v (initial reaction velocity) in a series of experiments at varying substrate concentrations, the dependence of v on [S] can be shown experimentally to be that of a hyperbola. As [S] tends toward infinity, v approaches an upper limiting value known as the maximum velocity (V). ENZYME KINETICS EFFECTS OF SUBSTRATE CONCENTRATION At low [S], a doubling of [S] will double v. But as [S] increases, each additional increment of substrate results in a smaller increase in reaction rate. As [S] becomes very large, increases in [S] increase only slightly, and the value of v reaches a maximum. ENZYME KINETICS SATURATION This Vmax value depends on the number of enzyme molecules and can therefore be increased only by adding more enzyme. The inability of increasingly higher substrate concentrations to increase the reaction velocity beyond a finite upper value is called saturation. At saturation, all available enzyme molecules are operating at maximum capacity. ENZYME KINETICS DOUBLE-RECIPROCAL PLOT The hyperbolic plot can be converted to a double-reciprocal one because neither Vmax nor Km can be easily determined from the values as plotted on a hyperbolic MM plot. Reciprocals are calculated for each value of [S] and v by dividing 1 by each value. ENZYME KINETICS IMPORTANCE FOR BIOCHEMISTRY AND MEDICINE The lower the Km value for a given enzyme and substrate, the lower the [S] range in which the enzyme is effective Vmax is important as a measure of the potential maximum rate of the reaction By knowing Vmax, Km, and the in vivo substrate concentration, we can estimate the likely rate of the reaction under cellular conditions DNA RECOMBINATION TECH RESTRICTION NUCLEASES ENZYMES THAT CLEAVE DNA MOLECULES AT SPECIFIC SITES Restriction endonucleases (also called restriction enzymes), are DNA cutting defence enzymes isolated from bacteria. The cutting action of a restriction enzyme generates a specific set of DNA pieces called restriction fragments. Each restriction enzyme cleaves double-stranded DNA only in places where it encounters a specific recognition sequence, called restriction site, that is usually four or six (but may be eight or more) nucleotides long. RESTRICTION FRAGMENTS CAN BE SEPARATED BY SIZE DNA electrophoresis is a common lab technique used to identify, quantify, and purify nucleic acid fragments derived from treatment of DNA with restriction enzymes. Samples are loaded into wells of an agarose or acrylamide gel and subjected to an electric field, causing the negatively charged nucleic acids to move toward the positive electrode. Shorter DNA fragments with lower molecular mass will travel more rapidly, whereas the longest fragments with higher molecular mass will remain closest to the origin of the gel, resulting in separation based on size. DNA GEL ELECTROPHORESIS SEPARATION OF NUCLEIC ACIDS FRAGMENTS BY SIZE SOUTHERN BLOTTING IDENTIFIES SPECIFIC RESTRICTION FRAGMENTS FROM A MIXTURE Southern blotting is a common lab technique using a special nucleic acid probe (a single-stranded molecule of DNA) that can identify a desired DNA sequence by complementary base-pairing. Nucleic acid probes are labeled either with radioactivity or with some other chemical group that allows the probe to be easily visualized by producing light that can expose X-ray film. SOUTHERN BLOTTING IDENTIFIES SPECIFIC RESTRICTION FRAGMENTS FROM A MIXTURE First, DNA is digested with a restriction enzyme. The resulting restriction fragments are then separated from each other by gel electrophoresis. Because the DNA often contains many, many types of DNA fragments of different sizes, hundreds or thousands of bands might appear at this stage if all were made visible. Here is where Southern’s procedure comes in. A special kind of “blotter” paper (nitrocellulose or nylon) is pressed against the completed gel, allowing the separated DNA fragments to be transferred to the paper. SOUTHERN BLOTTING IDENTIFIES SPECIFIC RESTRICTION FRAGMENTS FROM A MIXTURE Then a nucleic acid probe is added to the blot. The bound probe is made visible by exposing the blot to X-ray film. Southern blotting has in many cases been replaced by PCR- based techniques for studying DNA, but related techniques are still very important for analyzing RNA. SOUTHERN BLOTTING IDENTIFIES SPECIFIC RESTRICTION FRAGMENTS FROM A MIXTURE RECOMBINANT DNA TECHNOLOGY JOINING DNA FROM DIFFERENT SPECIES Since the development of recombinant DNA technology in the 1970s, scientists have been able to join segments of DNA from one source (e.g one organism) together with any other piece of DNA. A central feature of recombinant DNA technology is the ability to produce specific pieces of DNA in large enough quantities for research and other uses. RESTRICTION NUCLEASES ARE USED TO CREATE RECOMBINANT DNA Creating Recombinant DNA Molecules. A restriction enzyme that generates sticky ends (in this case EcoRI restriction enzyme) is used to cleave DNA molecules from two different sources. The complementary ends of the resulting fragments join by base pairing to create recombinant molecules containing segments from both of the original sources. DNA CLONING GENERATING MANY COPIES OF SPECIFIC DNA FRAGMENTS The process of generating many copies of specific DNA fragments is called DNA cloning. In biology, a clone is a population of organisms that is derived from a single ancestor and hence is genetically homogeneous, and a cell clone is a population of cells derived from the division of a single cell. By analogy, a DNA clone is a population of DNA molecules that are derived from the replication of a single molecule and hence are identical to one another. DNA CLONING IN PLASMID VECTORS PRODUCES MILLIONS OF IDENTICAL DNA COPIES DNA cloning is accomplished by putting the DNA of interest to the DNA of a cloning vector, which can replicate autonomously when introduced into a cell grown in culture—in many cases, a bacterium such as E. coli. The cloning vector can be a circular piece of DNA known as a plasmid or the DNA of a virus. The vector’s DNA “passenger” is copied every time in many replicates POLYMERASE CHAIN REACTION (PCR) RAPID DNA AMPLIFICATION FOR CLONING AND BEYOND New methods for DNA cloning use a PCR method that simply requires that you know part of the base sequence of the gene you wish to amplify. The next step is to synthesize short, single-stranded DNA primers that are complementary to sequences located at opposite ends of the gene. These primers are then used to target the intervening DNA for amplification. PCR can be used to rapidly generate sufficient quantities of DNA for cloning. POLYMERASE CHAIN REACTION (PCR) UTILISES SPECIAL TAQ DNA POLYMERASE Each reaction cycle begins with a short period of heating to near boiling (95°C) to denature the DNA double helix into its two strands. The DNA solution is then cooled to 50°C to allow the primers to base- pair to complementary regions on the DNA strands being copied. The temperature is then raised to 72°C, and the Taq DNA polymerase (derived from hot spring bacterium Thermus aquaticus) goes to work, adding nucleotides beginning at the 3′ end of the primer. POLYMERASE CHAIN REACTION (PCR) UTILISES SPECIAL TAQ DNA POLYMERASE The specificity of the primers ensures the selective copying of the stretches of downstream template DNA. It takes no more than a few minutes for the Taq polymerase to completely copy the targeted DNA sequence. The reaction mixture is then heated again to melt the new double helices, more primer binds to the DNA, and all steps are repeated. POLYMERASE CHAIN REACTION (PCR) MULTIPLE REPEATED CYCLES TO MASS PRODUCE DNA POLYMERASE CHAIN REACTION (PCR) A SUMMARY TRANSGENIC ORGANISMS PRACTICAL USE OF RECOMBINANT DNA TECHNOLOGIES TRANSGENIC ORGANISMS PRACTICAL USE OF RECOMBINANT DNA TECHNOLOGIES FLUORESCENT PROTEINS FLUORESCENT TAGS DEVELOPED USING RECOMBINANT TECH TRANSGENIC ZEBRAFISH POWERFUL MODELS IN MODERN DRUG DISCOVERY Transgenic Fli1:EGFP line (EGFP expressed in vasculature and neural crest) SUMMARY OMICS AS A SCALAR APPROACH TO UNCOVER STRUCTURE, FUNCTION AND DYNAMICS OF LIFE PHENOMICS PHYSIOMICS BIOLOGICAL COMPLEXITY CELLOMICS METABOLOMICS PROTEOMICS GENOMICS TRANSCRIPTOMICS EPIGENOMICS PHYSIOLOGICAL RELEVANCE THANK YOU