4 DNA->RNA->Protein - the flow of biological information
5 Coding sequence of SpBra DNA = A, T, C, GcytosineguanineadeninethymineD: deoxyriboseP: phosphateCoding sequence of SpBraexonsNon-coding sequences (intergenic and intronic) in the vicinity of SpBraTSSGene ARegulatory element binding sites
6 DNA Amplification - Polymerase Chain Reaction (PCR) DenaturingAnnealingElongation
7 RT-PCR (Reverse Transcriptase- PCR) Can be used to amplify and quantify the amount of RNA in tissueUse reverse transcriptase to make cDNA from mRNAthe cDNA is used as the template for PCR
8 DNA Cloning into Plasmids Purpose: to generate a superabundance of copies of your DNA fragmentcloning design:use PCR to make insertligate insert and plasmid together and transform into bacteria or yeastCloning is useful for many downstream applications!
9 Genomic Library Genomic Library Plates of bacteria, in which each well contains sheered chromosomal DNA that was inserted into a cloning vector, usually large plasmid (i.e., a BAC [Bacterial Artificial Chromosome])Can amplify and maintain entire genome of source organism in vectorsthe collection of cloned DNA molecules represents the entire genome of the source organism
12 Southern and Northern Blots Southern- use DNA probe to detect DNACan be used to find if there is a homolog of a certain gene in other speciesNorthern- use DNA probe to detect RNACan be used to see if a gene is expressed in a specific tissue or stage in development
14 MicroarrayCan be used to see if all coding genes are turned on in a specific location or stage during developmentFirst, extract the mRNA from the tissue of interest. Then use RT-PCR to convert mRNAs into their complementary DNAs (cDNAs). Each cDNA is then cloned and amplified by PCR. A robot will print the cDNAs into glass slides in a particular order. Now it is your Microarray.But with ISH, one can only detect a limited number of genes at a time. Microarray allows to observe gene expression at a mass scale. For example, you can see all of the genes that are turned on in the heart at a particular time point. The most common application of DNA/oligonucleotide microarray is gene expression analysis. In this technique, RNA isolated from two samples are labelled with two different fluorescent dyes, one green and one red, before being hybridised to a microarray consisting of large numbers of cDNAs/oligonucleotides orderly arranged onto a glass microscope slide. After hybridisation under stringent conditions, a scanner records, after excitation of the two fluorochromes at given wavelengths, the intensity of the fluorescence emission signals is proportional to transcript levels in the biological samples. The microarray data are analysed using specific softwares that will cluster genes with similar expression patterns, assuming that they share common biological functions.
15 Quantitative-PCR (QPCR) Achieves an accurate estimation of DNA and RNA targetsTwo quantitative requirements:Absolute-Requires standard whose concentration is known absolutelyRelative-Standard curve or comparative CT and endogenous reference (e.g. 18S ribosomal RNA)
16 in situ HybridizationUsed to detect the spatial and temporal expression pattern of RNA in an embryo or any fixed tissueAnother method of detecting RNA is in situ. The word in situ comes from Latin, it means “in the original place”. Like Northern and Southern Blots, ISH indicates the presence of a particular RNA or DNA sequence, but ISH differs from blots in that the labeled probe reveals the actual location of the sequence in the cells in an organism. So you don’t need to section the organism. But since the organism is intact, the RNA sequence you are trying to detect is at a lower concentration, and will be masked because of associated protein, or protected within a cell or cellular structure. Therefore, in order to probe the tissue or cells of interest you have to increase the permeability of the cell and the visibility of the nucleotide sequence to the probe without destroying the structural integrity of the cell or tissue. Here are some examples of whole mount in situ. On the left panel is a chick embryo. The RNA in interest is visualize with color substrates. On the right panel, it shows multi-color fluorescent labeling of genes in a fly embryo.Stathopoulos, 2005
17 How it Works Anti-sense RNA mRNA Plabeled dUTP (digoxigenin, biotin, fluorescein, etc.)Anti-DIG-APAlkaline phosphataseSubstrate for Alkaline phosphataseSo how does it work? First you make an antisense RNA of the DNA sequence or a gene that you are interested in. This RNA sequence is tagged with a dye, could be a color dye or a fluorescent dye, As I mentioned previously, the permeability of cells is low. To increase cell permeability, the samples are treated with proteinase K. you then leave your RNA probe to bind with mRNAs . This usually takes overnight, or at least 8 hours. The second day, you add .antibody-phosphatase to bind to RNA-probe for 2 hours at room temperature or overnight at 4C. At last, you add alkaline phosphatase to stain the antibody and visualize under microscope. The whole process takes about 2 to 3 days. But it is worth it tells you the spatial and temporal expression pattern of a gene.
19 Antibodies-What are they? Antibodies recognize antigens on proteinsNormally the immune system uses them to recognize bacteria and viruses, biologists use them as a probe for proteinsMonoclonal means it recognizes one site on the antigen (vs polyclonal) = specificityThe most commonly application of protein in the lab is antibodies. They can recongize antigens on proteins. It is present in our bodies to recognize bacteria and toxins as a mechanism of body defense. But we biologists use them as a probe for proteins. Here is how an antibody looks like, its has two chains, the heavy chain and the light chain, and the antigen binding sites.
20 Immunohistochemistry Use antibodies to visualize the location of specific proteins in embryosTo visualize, amplification is needed, generally a two step procedure:1. Primary antibody2. Secondary antibodyImmunohistchemistry is very much like ISH, but it detects the location of proteins that you are interested to study.Now why do we want to do immunostaining? ISH tells location of gene expression. Why bother doing immunostaining?Right, they tell you the different steps of DNA or gene expression. One is to detect the presence of mRNA, whereas one is the detect the production of protein. A gene can be activated, transcribed but never translated into protein. Like say you see a phenotype when knocking out a gene, you have to sort out at which point does it go wrong. That’s why we need to use many different techniques and approaches to answer the question.When you read a paper, you would always see a list of techniques being used to answer one question.Often conjugated to a flourescent moleculesecondary antibodyprimary antibodyantigen
21 Example of an immunohistochemistry figure Embryos were stained at room temperature with the following primary antibodies: mouse monoclonal anti-Ftz, mouse monoclonal anti-Engrailed (a gift of N. H. Patel), and rabbit polyclonal anti-Even-skipped (a gift of M. Frasch). The primaries were visualized with Cy3 anti-mouse and FITC anti-rabbitExamples of an immunostaining. These are drosophila embryos. I aopologize I don’t remember the stage but they look like they are at gastrula stage. Certainily not cleavage or blastula. Am I right?Anyway, these embryos are stained with two different antibodies, tagged with two different fluorescent dyes. In figures A to C, green is eveskip and red is Ftz. You see expression patterns of the two proteins are very discrete. They make these strips one after another they never overlap. The rest of the figures are co-labeled with eveskip and engrailed. They have some overlapped domain. See the orange color? Here and here. Beautiful figures.Genetics September; 168(1): 161–180.
22 Immunoprecipitation (IP) The technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein. Purpose: to isolate a specific protein from a lysate or crude extractMovie
23 Western Blot (immunoblot) -an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract.
24 Example of a Western Blot Histone H1 accumulation in sktl salivary glands. Salivary glands from wild-type (wt) and sktl drosophila larvae were dissected, analyzed by SDS-PAGE, and immunoblotted with histone H1 and tubulin antibodies. (Top) Antihistone H1 immunoblot. Histone H1 antibody recognizes both the phosphorylated (32 kD) and nonphosphorylated (31 kD) forms of histone H1. Nonphosphorylated histone H1 is completely absent in sktl mutant salivary glands. (Bottom) Same blot probed with tubulin antibody as a loading control.From: Genetics, Vol. 167, , July 2004
26 Loss of function vs Gain of Function Loss of Function- can show if a gene or protein is necessary for a certain eventKnockout or knockdown protein or gene, if this gets rid of the event then it is necessary for the event to occurGain of Function- can show if a gene or protein is sufficient for a certain eventExpress gene or protein in area where the event does not occur naturally, if the event occurs then the protein or gene is sufficient for the event
27 Loss of Function (LOF)Knockdown of ß-Catenin in Xenopus results in a loss of dorsal structuresConclude ß-Catenin necessary for dorsal structuresWays to knockout/knockdown a gene/proteinFunction blocking antibodiesMorpholino- antisense oligo-nucleotide analog binds to mRNA & doesn’t allow translation machinery to bindRNAi- double stranded RNA targets mRNA for degradationGenetic knockouts
28 Morpholino 6 member morpholino ring makes them resistant to nucleases Block initiation of translation, so usually made to recognize 5’UTRDelivered through injectionThey are very stable and can function for a long time after being injected
29 RNAi Pathway Method of Delivery: C. elegans- feeding or injection Other organisms- injectionRNAi Movie1Movie2
30 Gain of Function (GOF)Express myocardin (co-factor that activates expression of cardiac specific genes) in non-muscle cell types observe expression of cardiac genesConclude myocardin sufficient for expression of those cardiaic specific genesWays to do gain of functionInject protein or mRNAExpress protein using tissue specific promoterTransfect cell line with construct
31 Transgenics Mouse Extract and culture embryonic stem cells Clone desired gene or construct into stem cells,Can make knockout mice by inserting neomycin resistance gene into middle of gene you want to knockoutconstruct will replace the gene you are knocking out by homologous recombination
32 Embryology 101: An introduction to physical manipulations
33 Embryological Techniques Single-cell perturbationsDissociations and cell cultureCell ablations, transplantationsCell labelingCytoskeletal perturbationsDissections, grafts, and transplantsAnimal caps and neural tube cultures, tissue recombinationsOrganizer graftsTissue transplants: neural tube, somites, limbElectroporationsMicroinjectionsTime-lapse imaging
34 Dissociation/Cell Culture Testing developmental potential - do you need cell-cell contacts?Xenopus:Dissect piece of ectodermal tissue from animal poleCulture in a solution lacking calcium and magnesium (inhibits cadherins), pipet, culture cells in salineCan reaggregate cells by centrifuging!Kuroda et al., 2005
35 Dissociation/Cell Culture Mammalian cells:Dissect tissue of interest (ex- neural tube or piece of skin ectoderm)Treat tissue with digestive enzyme to remove cell contacts, pipetCulture on a dish coated with fibronectin or collagen substrateAdd growth media containing serum, growth factors, antibiotics, etcReplate to prevent overgrowth
36 Cell Ablations Testing conditional vs autonomous specification Feasible in embryos with very large cells (ex - C. elegans, zebrafish)Point laser beam at a cell nucleus - create double-stranded breaks in DNA apoptosisCell fusion - Point laser beam at cell membrane between two cells - fuse cytoplasmic contents
37 Cell Transplantations Testing behavior of cells in different environmentdifferent location, different timing, or behavior of mutant cells in wild-type contextDepending on cell size, can transplant single or group of cells (ex - zebrafish)Can fluorescently label cells, remove from one embryo (by suction with glass micropipette), inject into region of interest in another embryo
38 Single Cell Labeling Fate mapping/lineage tracing Following progeny - what does a particular cell give rise to later in development?Labeling cells for transplantation - visualizing donor cellsFluorescent dextrans - big hydrophilic molecules conjugated to fluorescent dyesdiI, diO - dye incorporates into cell membranes - long-term labeling but in intensityCaged fluorescein - fluorescent molecule that is activated by laser pulse - can inject early in devt when cells are large and activated laterGFP - tissue-specific if use promoter/enhancer reporter construct
39 Tissue CultureSpecific tissues may be easier to manipulate than whole embryoTest specification vs commitment or challenge with different factors - developmental potentialXenopus - animal cap assayDissect ectoderm from animal pole, culture in saline solution (can inject animal cap with mRNA of interest first)Can add growth factors (BMP, FGF, Wnt, noggin)Induction assays
40 Organizer GraftsTest inductive potential of signaling tissue and capacity of area to respondTransplant organizer to area that usually does not receive signalTransplant organizer from older embryo to a younger embryo and vice versa (heterochronic)Xenopus blastopore lip, zebrafish shield, chick and mouse nodeHeterospecies grafts work! (same signals - conserved)LabelingDifferently pigmented donor and host (Xenopus)Fluorescent labelsGFP transgenics
41 ElectroporationUseful in organisms that are not amenable to genetics or single-cell injections (ex chicken)Introduce of DNA, RNA, or morpholino into cells using electrical currentPlace solution around cells, place electrodes on both sides of target tissueApply several small-voltage pulsesPulses make tiny holes in cell membranesSlightly charged solution (DNA - neg) enters the cellsCannot target specific cells, and not every single cell is electroporated!
42 Time-lapse Live Imaging Visualize developmental processes dynamicallyFollow behavior/migration of specific cellsMake really cool movies!!ConditionsEmbryo must survive during imaging time (hydration, temperature, CO2)Must be optically clearCells must be labeled - transgenic GFP, reporter, fusion protein, etc
43 Compare and Contrast of Model Organisms Sea UrchinFrogChickInvertebrateVertebrateEmbryo sizeSmallBigModerateEmbryo clutchEmbryo TransparencyYesNo, it is opaque.Access to EmbryoEasyRelatively difficultDNA/RNA IntroductionMicroinjectionMicroinjection and ElectroporationTransgenic lines?
44 Tissue CultureOther tissues - neural tube, brain, somites, limb, lens (eye), gut, etcCulture in growth media on a substrate of fibronectin or collagen gelAdd growth factors to mediaRecombination assayCulture 2 different tissues together to study inductive interactionsDoes combination give rise to a 3rd different tissue type?Ex: Xenopus animal cap + vegetal mass mesoderm (Nieuwkoop assay)Ex: Chick intermediate neural tube + skin ectoderm neural crest
45 Cytoskeletal Perturbations Nature Reviews GeneticsCytoskeleton necessary for cleavage, rearrangement of cells/tissuesBest example - Xenopus - affect dorso-ventral patterningCan soak or irradiate the whole eggUV lightcross-links microtubules (GTP bound to tubulin - cant polymerize)D2O - heavy waterstabilizes microtubules and randomizes their arrayNocodazole, colchicinechemical agents that depolymerize microtubules