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Part 1: Methods Using DNA, RNA, and Protein

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1 Part 1: Methods Using DNA, RNA, and Protein
BI 117 Recitation Session 1 Techniques in Developmental Biology Part 1: Methods Using DNA, RNA, and Protein Jon or jev Kerckhoff 017

2 The life codes

3 Methods with DNA and RNA

4 DNA->RNA->Protein - the flow of biological information

5 Coding sequence of SpBra
DNA = A, T, C, G cytosine guanine adenine thymine D: deoxyribose P: phosphate Coding sequence of SpBra exons Non-coding sequences (intergenic and intronic) in the vicinity of SpBra TSS Gene A Regulatory element binding sites

6 DNA Amplification - Polymerase Chain Reaction (PCR)
Denaturing Annealing Elongation

7 RT-PCR (Reverse Transcriptase- PCR)
Can be used to amplify and quantify the amount of RNA in tissue Use reverse transcriptase to make cDNA from mRNA the cDNA is used as the template for PCR

8 DNA Cloning into Plasmids
Purpose: to generate a superabundance of copies of your DNA fragment cloning design: use PCR to make insert ligate insert and plasmid together and transform into bacteria or yeast Cloning 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 vectors the collection of cloned DNA molecules represents the entire genome of the source organism

10 cDNA library

11 Gene expression analysis

12 Southern and Northern Blots
Southern- use DNA probe to detect DNA Can be used to find if there is a homolog of a certain gene in other species Northern- use DNA probe to detect RNA Can be used to see if a gene is expressed in a specific tissue or stage in development

13 Southern Blot Movie Gilbert, 2006

14 Microarray Can be used to see if all coding genes are turned on in a specific location or stage during development First, 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 targets Two quantitative requirements: Absolute-Requires standard whose concentration is known absolutely Relative-Standard curve or comparative CT and endogenous reference (e.g. 18S ribosomal RNA)

16 in situ Hybridization Used to detect the spatial and temporal expression pattern of RNA in an embryo or any fixed tissue Another 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
P labeled dUTP (digoxigenin, biotin, fluorescein, etc.) Anti-DIG-AP Alkaline phosphatase Substrate for Alkaline phosphatase So 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.

18 Methods with Proteins

19 Antibodies-What are they?
Antibodies recognize antigens on proteins Normally the immune system uses them to recognize bacteria and viruses, biologists use them as a probe for proteins Monoclonal means it recognizes one site on the antigen (vs polyclonal) = specificity The 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 embryos To visualize, amplification is needed, generally a two step procedure: 1. Primary antibody 2. Secondary antibody Immunohistchemistry 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 molecule secondary antibody primary antibody antigen

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-rabbit Examples 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 extract Movie

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

25 Movie-The inner life in a cell

26 Loss of function vs Gain of Function
Loss of Function- can show if a gene or protein is necessary for a certain event Knockout or knockdown protein or gene, if this gets rid of the event then it is necessary for the event to occur Gain of Function- can show if a gene or protein is sufficient for a certain event Express 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 structures Conclude ß-Catenin necessary for dorsal structures Ways to knockout/knockdown a gene/protein Function blocking antibodies Morpholino- antisense oligo-nucleotide analog binds to mRNA & doesn’t allow translation machinery to bind RNAi- double stranded RNA targets mRNA for degradation Genetic knockouts

28 Morpholino 6 member morpholino ring makes them resistant to nucleases
Block initiation of translation, so usually made to recognize 5’UTR Delivered through injection They 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- injection RNAi Movie1 Movie2

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 genes Conclude myocardin sufficient for expression of those cardiaic specific genes Ways to do gain of function Inject protein or mRNA Express protein using tissue specific promoter Transfect 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 knockout construct will replace the gene you are knocking out by homologous recombination

32 Embryology 101: An introduction to physical manipulations

33 Embryological Techniques
Single-cell perturbations Dissociations and cell culture Cell ablations, transplantations Cell labeling Cytoskeletal perturbations Dissections, grafts, and transplants Animal caps and neural tube cultures, tissue recombinations Organizer grafts Tissue transplants: neural tube, somites, limb Electroporations Microinjections Time-lapse imaging

34 Dissociation/Cell Culture
Testing developmental potential - do you need cell-cell contacts? Xenopus: Dissect piece of ectodermal tissue from animal pole Culture in a solution lacking calcium and magnesium (inhibits cadherins), pipet, culture cells in saline Can 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, pipet Culture on a dish coated with fibronectin or collagen substrate Add growth media containing serum, growth factors, antibiotics, etc Replate 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  apoptosis Cell fusion - Point laser beam at cell membrane between two cells - fuse cytoplasmic contents

37 Cell Transplantations
Testing behavior of cells in different environment different location, different timing, or behavior of mutant cells in wild-type context Depending 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 cells Fluorescent dextrans - big hydrophilic molecules conjugated to fluorescent dyes diI, diO - dye incorporates into cell membranes - long-term labeling but  in intensity Caged fluorescein - fluorescent molecule that is activated by laser pulse - can inject early in devt when cells are large and activated later GFP - tissue-specific if use promoter/enhancer reporter construct

39 Tissue Culture Specific tissues may be easier to manipulate than whole embryo Test specification vs commitment or challenge with different factors - developmental potential Xenopus - animal cap assay Dissect 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 Grafts Test inductive potential of signaling tissue and capacity of area to respond Transplant organizer to area that usually does not receive signal Transplant organizer from older embryo to a younger embryo and vice versa (heterochronic) Xenopus blastopore lip, zebrafish shield, chick and mouse node Heterospecies grafts work! (same signals - conserved) Labeling Differently pigmented donor and host (Xenopus) Fluorescent labels GFP transgenics

41 Electroporation Useful in organisms that are not amenable to genetics or single-cell injections (ex chicken) Introduce of DNA, RNA, or morpholino into cells using electrical current Place solution around cells, place electrodes on both sides of target tissue Apply several small-voltage pulses Pulses make tiny holes in cell membranes Slightly charged solution (DNA - neg) enters the cells Cannot target specific cells, and not every single cell is electroporated!

42 Time-lapse Live Imaging
Visualize developmental processes dynamically Follow behavior/migration of specific cells Make really cool movies!! Conditions Embryo must survive during imaging time (hydration, temperature, CO2) Must be optically clear Cells must be labeled - transgenic GFP, reporter, fusion protein, etc

43 Compare and Contrast of Model Organisms
Sea Urchin Frog Chick Invertebrate Vertebrate Embryo size Small Big Moderate Embryo clutch Embryo Transparency Yes No, it is opaque. Access to Embryo Easy Relatively difficult DNA/RNA Introduction Microinjection Microinjection and Electroporation Transgenic lines ?

44 Tissue Culture Other tissues - neural tube, brain, somites, limb, lens (eye), gut, etc Culture in growth media on a substrate of fibronectin or collagen gel Add growth factors to media Recombination assay Culture 2 different tissues together to study inductive interactions Does 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 Genetics Cytoskeleton necessary for cleavage, rearrangement of cells/tissues Best example - Xenopus - affect dorso-ventral patterning Can soak or irradiate the whole egg UV light cross-links microtubules (GTP bound to tubulin - cant polymerize) D2O - heavy water stabilizes microtubules and randomizes their array Nocodazole, colchicine chemical agents that depolymerize microtubules

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