BI 117 Recitation Session 1

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

2. The life codes

3. Methods with DNA and RNA

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

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

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

7. RT-PCR (Reverse Transcriptase- PCR) • RT-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

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 http://www.microarray.lu/images/overview_1.jpg

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 Stathopoulos, 2005

17. labeled dUTP (digoxigenin, biotin, fluorescein, etc.) Anti-sense RNA P P Anti-sense RNA Anti-DIG-AP mRNA Alkaline phosphatase Substrate for Alkaline phosphatase P How it Works

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

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 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 Genetics. 2004 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 sktldrosophila 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, 1213-1223, 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 http://www.bseinquiry.gov.uk/report/volume2/fig1_6.htm

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

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