Advanced Developmental
Biology
Experimental approaches and
techniques in Developmental Biology
Natalya Nikitina
Email: [Link]@[Link]
Office: Gate House 701
1. How research is done
Find interesting phenomenon
or unanswered question
Based on your knowledge, formulate
a hypothesis to explain the phenomenon
Refine Clearly state your assumptions
hypothesis
Thorough literature search: are the assumptions
valid? Is there new data I am overlooking?
2. Hypothesis is solid based on what
is currently known
Design experiments to test the
hypothesis
Refine
hypothesis
Perform experiments. Do results
validate or refute your hypothesis?
Phenomenon fully understood.
Explanation proposed
Publish your results!
3. If we want to study a role of a
novel gene in the development of an
organ, how would we approach it?
What questions would we ask?
4. Questions:
• Is the gene expressed in the organ/tissue of
interest? (RNA/protein)
• What happens to the embryo when we knock the
gene out/interfere with its translation/function
(loss-of-function experiments)?
• What happens when we express this gene where
it is not normally supposed to be (gain-of-
function, misexpression)?
5. Experimental approaches used to find out
where in the embryo a specific gene is
expressed:
• Immunocytochemistry (immunohistochemistry) – determining the
tissue localization/timing of expression of a specific protein
• In situ hybridization – determining the tissue localization/timing of
expression of a specific mRNA transcript (can be applied to non-
coding RNAs as well)
• RT-PCR/Northern blot (for mRNA) or Western blot (for proteins) on
dissected tissues – determines presence/absence/quantity/types of
target molecule in specific tissues.
6. Experimental controls
• When more than one explanation of the
experimental results is possible
• Serves to distinguish between the multiple
possible interpretations of the results
• Essential if you are to trust your data!
• Example?
7. Immunohistochemistry
A: Fluorescent
Antibody staining
B: Color antibody staining
8. ICC: colour vs fluorescent detection
• Fluorescent • Colour
+ relatively cheap, signal
+ can see co-localization of several
stable over several years
proteins in the same cell
-longer protocol, can’t
+well-established protocols,
do multiple overlapping
convenient, specific
colours, can produce
-Fluorescently-labelled Ab
non-specific background
expensive, signal will photobleach
9. Immunohistochemistry: Controls
A: Fluorescent
Antibody staining 1. No Primary Ab
control
2. Positive tissue
control: tissue with
known expression
3. Negative tissue
control: tissue that
should not express.
B: Color antibody staining
10. RNA in situ hybridization (1)
mRNA probe
11. RNA in situ hybridization (2)
Colour substrates:
NBT (nitroblue
tetrazolium) +
BCIP (5-bromo-4-
chloro-3-indolyl
phosphate)
Figure 3.31 In situ hybridization (Part 3)
12. In situ hybridization: representative result
• Single • Double
+ signal stable over several years; protocol well
established
-can do more than 1 gene on the same embryo,
but poor resolution in the overlap area
13. In situ controls
• Sense probe
• Positive tissue control
• Negative tissue control
14. Loss-of-function experimental
approaches
• Mutants or transgenic animals (mouse, zebrafish)
• shRNA/miRNA injection into early embryo
(Xenopus, zebrafish)
• Introduction of a dominant-negative construct
(works for receptors and some TFs)
• Morpholinos (mouse, chick)
• Modern tools of genome engineering (ZFN,
TALENs, CRISPR-Cas)
15.
16. miRNA/shRNA
• Cause transcriptional silencing or mRNA
degradation of a specific gene
• Can be delivered as short duplexes,
hairpins, transcribed off a plasmid, a viral
vector (labels)
• - off-target effects (sequence, saturation of
the pathway), RNA degradation
• Controls:
– 2nd shRNA that binds to a different sequence in
the target gene
– Negative control: 5-mismatch shRNA or a verified
random RNA that does not target anything in the
genome of the organisms of interest
– Rescue experiment to demonstrate specificity:
co-inject shRNA with the target mRNA lacking the
shRNA binding site (not always possible if the
target site is within mRNA coding sequence)
17. Dominant-negative constructs
• Mutated form of a receptor
that can bind a signalling
molecule but can’t initiate
the signal transduction
cascade.
• -only works for receptors or
members of multiprotein
complexes; loss-of-function
not always 100%.
18. Morpholinos
• RNA analogue
• Translation inhibition or blocking of
splice sites (“splicing Mo”)
• +stable, non-toxic, can be labeled
(fluorescein)
• -off-target effects possible,
gets diluted out with growth
Controls:
– 2nd morpholino that binds to a different sequence in
the target gene
– Negative control: 5-mismatch morpholino or a verified
random morpholino that does not target anything in
the genome of the organisms of interest
– Rescue experiment to demonstrate specificity: co-
inject the morpholino with the target mRNA lacking
the Mo binding site (frequently possible as Mo binding
sites are usually in the 5’ UTR)
20. Figure 3.34 CRISPR/Cas9-mediated gene editing
21. CRISPR-Cas
• Novel tool for precise
genome editing
• Introduces DSB in
sequence-specific manner
– NHEJ: insertions/deletions
– HDR: specific mutations,
tagged alleles, conditional
alleles
– Base editing
• +efficient, specific, low off-
target effects, most of
genome can be modified,
relatively easy to design,
multiplexing
• -mozaics possible
22. Gain-of-function experimental
approaches
• Transgenic animals (mouse)
• Using viruses or transposons to insert a gene into
genome to study its function (chick, Drosophila)
• RNA injection into an early embryo (zebrafish,
Xenopus)
• Extrachromosomal DNA constructs introduced by
electroporation (chick, mouse)
• Modern tools of genome engineering (ZFN,
TALENs, CRISPR-Cas)
23. Introducing DNA via electroporation
24: Tools for promoter/enhancer analysis
• Reporter constructs:
lacZ or GFP under the
control of a
promoter/enhancer
combination to be
studied
25. Figure 3.32 Chromatin immunoprecipitation-sequencing (ChIP-Seq)
28. Learning objectives – you should be able to
• Formulate a hypothesis to explain a phenomenon based
on your previous knowledge ( from the course material)
• Based on your hypothesis, predict a likely outcome of an
experiment
• Specify the assumptions that your hypothesis is based on
• Assess the validity of your hypothesis when provided
with actual experimental results
• Explain why controls are necessary (using specific
examples)
• Discuss all of the experimental approaches described in
this lecture. Explain their advantages and shortcomings.
Explain what controls are necessary and why.
