BI 117Recitation Session 1

Part 1: Methods Using DNA, RNA, and Protein

Jon or jevKerckhoff 017

jev@caltech.edu

Techniques in Developmental Biology

The life codes

Methods with DNA and RNA

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

DNA = A, T, C, G

cytosineguanine

adenine

thymine

D: deoxyriboseP: phosphate

Coding sequence of SpBra

exonsNon-coding sequences (intergenic and intronic) in the vicinity of SpBra

TSSGene A

Regulatory element binding sites

DNA Amplification- Polymerase Chain Reaction (PCR)

Denaturing

Annealing

Elongation

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

DNA Cloning into Plasmids

-cloning design: -use PCR to make insert-ligate insert and plasmid together and transform into bacteria or yeast

Purpose: to generate a superabundance of copies of your DNA fragment

Cloning is useful for many downstream applications!

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

cDNA library

Gene expression analysis

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

Southern Blot

Gilbert, 2006

Movie

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

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)

in situ Hybridization

• Used to detect the spatial and temporal expression pattern of RNA in an embryo or any fixed tissue

Stathopoulos, 2005

How it Works

Anti-sense RNA

Anti-sense RNA

mRNA

P

P

labeled dUTP (digoxigenin, biotin, fluorescein, etc.)

Anti-DIG-AP

Alkaline phosphatase

P Substrate for Alkaline phosphatase

Methods with Proteins

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

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

antigen

primary antibody

secondary antibody

Often conjugated to a flourescent molecule

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.

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

Western Blot (immunoblot)-an analytical technique used to detect specific proteins in a given

sample of tissue homogenate or extract.

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, 1213-1223, July 2004

Movie-The inner life in a cell

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

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

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

RNAi Pathway

• Method of Delivery:• C. elegans- feeding or injection•Other organisms- injection

RNAi Movie1

Movie2

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

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

Embryology 101: An introduction to physical manipulations

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

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

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

• 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

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

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

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

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

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!

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

Compare and Contrast of Model Organisms

Sea Urchin Frog Chick

Invertebrate Vertebrate Vertebrate

Embryo size Small Big Moderate

Embryo clutch Big Big Small

Embryo Transparency

Yes No, it is opaque. No, it is opaque.

Access to Embryo Easy Easy Relatively difficult

DNA/RNA Introduction

Microinjection Microinjection Microinjection and Electroporation

Transgenic lines ? ? ?

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

Cytoskeletal Perturbations

– 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

Nature Reviews Genetics