Transcript BI117 Recitation Session 1

BI 117
Recitation Session 1
Techniques in Developmental Biology
Part 1: Methods Using DNA, RNA, and
Protein
Jon or jev
Kerckhoff 017
[email protected]
The life codes
Methods with DNA and RNA
DNA->RNA->Protein
- the flow of biological information
DNA = A, T, C, G
cytosine
adenine
guanine
thymine
D: deoxyribose
P: phosphate
Coding sequence of SpBra
Gene A
TSS
exons
Non-coding sequences (intergenic and intronic)
in the vicinity of SpBra
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
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!
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
Movie
Gilbert, 2006
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
labeled dUTP (digoxigenin, biotin, fluorescein, etc.)
Anti-sense RNA
P
P
Anti-sense RNA
Anti-DIG-AP
Alkaline phosphatase
P
Substrate for Alkaline phosphatase
mRNA
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
Often conjugated to a flourescent
molecule
secondary
antibody
primary
antibody
antigen
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-Evenskipped (a gift of M. Frasch).
The primaries were
visualized with Cy3 antimouse 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 organismsinjection
RNAi Movie1
Movie2
Gain of Function (GOF)
• Express myocardin (co-factor that activates
expression of cardiac specific genes) in nonmuscle 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
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–
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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
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