Molecular tool box for studying gene function in neural development

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Molecular tool box for studying gene function in
neural development

• For Eye Development Course

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Studying gene function in neural development
Differential Gene Expression
(The Paradigm of Developmental Biology)
approx. 30000 human genes, but
developing roof plate cells express BMP4 signal
protein
developing floor plate cells express Shh signal
protein
differentiated neurons express N-tubulin protein
oligodendrocytes express myelin-associated
glycoprotein
lens cells express crystallin protein
Differential gene expression from genetically
identical nuclear DNA creates different cell
types in different parts of the embryo.
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Studying gene function in neural development
Gene Expression
(The Central Dogma of Molecular Biology)
DNA
RNA
PROTEIN
GENE FUNCTION
GENE
GENE PRODUCTS
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Studying gene function in neural development
Experimental Design
(gene A functions in process B)

1. Investigation of gene expression gene A is
   expressed in the appropriate tissue and at the
   appropriate stage to mediate process B.
2. Gain-of-function experiment gene product of gene
   A is sufficient for mediating process B.
3. Loss-of-function experimentgene A or gene
   product of gene A is normally required for
   process B.
4. Analysing of experiment by investigating the
   resulting phenotype or marker gene expression.

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Studying gene function in neural development
Methods tool box

1. Methods for analysing gene expression (Protein,
   RNA, Reporter gene (DNA))
2. Methods for expressing gene products (ectopic
   expression, over-expression, expression rescue of
   Protein, RNA or DNA)
3. Methods for inhibiting gene function (inhibiting
   Protein function, disrupting or inhibiting RNA,
   disrupting gene (DNA))

(IV. Analyse experiments with tissue-specific
molecular markers.)
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I. Methods for analysing gene expression
Detecting protein expression/localisation

• Antibodies
• gel-based methods (biochemistry) Western Blot,
  Proteomics
• tissue-based methods (in situ)
  immuno-histochemistry (whole-mount, histology
  sections)
• Protein fusion constructs
• artificial chimera protein fuse endogenous
  protein coding sequence to
• (lacZ (gene) ?-gal (protein))
• GFP etc.

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Western Blot
Protein A
linked to the enzyme Alkaline Phosphatase (AP) or
HRP
(chemiluminescent)
Protein extraction
st. 10
st. 20
st. 30
st. 50
st. 0
st. 5
Protein gel
Blot
Film
Protein A is expressed from stages 10 to 30
during Xenopus development
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Immunohistochemistry
endogenous protein in fixed embryonic tissue
Protein A
linked to the enzyme Alkaline Phosphatase (AP)
or linked to fluorescent molecule
coloured precipitate
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I. Methods for analysing gene expression
Detecting RNA expression

• Gel-based methods (Molecular Biology)
• Northern Blot (qualitative, detecting different
  gene products)
• RNase Protection Assay (quantitative), antisense
  RNA probe forms dsRNA with target mRNA and
  protects it from RNaseH digest (RNaseH only
  digests ssRNA).
• Microarrays (Analysing the RNA expression of
  hundreds of genes)
• RTPCR (high sensitivity, qualitative, advanced
  methods are quantitative)
• Tissue-based methods (in situ)
• RNA in situ hybridisation (whole-mount, histology
  sections)

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Northern Blot
endogenous mRNA of gene A
AAAAAAAAAAAA
radioactively labelled DNA probe
RNA extraction
st. 10
st. 20
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st. 0
st. 5
zygotic RNA
maternal RNA
RNA gel
Blot
Film
Gene A is expressed maternally and zygotically
from stages 10-30 during Xenopus development
Maternal and zygotic mRNA from gene A is
different size
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RT-PCR
RNA extraction
endogenous mRNA of gene A
AAAAAAAAAAAA
reverse transcription
AAAAAAAAAAAA
cDNA of gene A
TTTTTTTTT
PCR with radioactive nucleotides
TTTTTTTTT
radioactive DNA PCR product
gene-specific PCR primers
st. 10
st. 20
st. 30
st. 50
st. 0
st. 5
DNA gel
Film
Gene A is expressed from stages 20 onward during
Xenopus development
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RNA in situ hybridisation
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I. Methods for analysing gene expression
Reporter gene DNA constructs

• cis-regulatory DNA elements drive expression of
  reporter protein.
• Lac Z
• very sensitive but only in fixed dead tissue
• Green Fluorescent Protein (GFP)
• less sensitive but in live embryos
• mutated GFP CFP (cyan), RFP (red), YFP (yellow)
• DsRed is a red fluorescent protein

enhancer
prom.
GFP
enhancer
prom.
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II. Experimental expression of gene products
Introducing extra gene product

• Fast
• Less reliable

Introducing an extra gene
(Transgenics)

• Slower, more difficult
• more reliable

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II. Experimental expression of gene products
Introducing extra gene product

• Introducing extra protein?
• doesnt usually work, not enough protein can be
  delivered, because there is no amplification
  step.
• Introducing extra RNA
• usually works well, because one mRNA molecule
  produces many proteins. Good for early embryonic
  stages, since proteins are immediately produced.
• Introducing DNA gene constructs
• Un-integrated DNA constructs cause mosaic
  expression, but can be useful if ubiquitous
  promoters are used to express proteins that
  function cell-non-autonomously (signals).

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II. Experimental expression of gene products
Delivering extra gene product
(into cells)

• Microinjection (RNA and DNA)
• works well with large cells, i.e. early Xenopus
  and Zebrafish embryos
• Electroporation (DNA)
• works fairly well with Chick neural tissue.
• Lipofection
• Lipofection reagent facilitates passing of DNA
  through hydrophobic cell membranes

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II. Experimental expression of gene products
Transgenics
(introducing an artificial gene into the genome)

• Transgenic mouse (elaborate procedure, mammalian
  embryo)
• Transgenic Xenopus (efficient procedure,
  vertebrate embryo)
• Transgenic Drosophila (P-element transformation)
• Plasmid 1
• Plasmid 2
• TRANSPOSASE integrates plasmid 1 into the
  Drosophila genome

white gene
promoterPROTEIN
P
P
Transposase gene
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III. Experimental inhibition of gene function
Inhibiting gene products and genes

• Inhibiting protein function good
• Disrupting RNA or inhibiting RNA function better
• Disrupting the DNA of the gene best

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III. Experimental inhibition of gene function
Inhibiting protein function
(good)

• Pharmacology
• small inhibitory molecules, delivery often easy,
  specificity often difficult to assess.
• Dominant-negative protein constructs
• mutated proteins that perturb normal function of
  the endogenous gene product. Fairly advanced
  knowledge of protein function required.
  Specificity often difficult to assess, because
  they inhibit may related gene products from
  several different genes.
• Depletion of endogenous protein using antibodies?
• works well for extract biochemistry, but mostly
  unsuccessful or misleading if used in in vivo
  experimental systems.

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III. Experimental inhibition of gene function
knock down
Inhibiting RNA
(desrupting RNA)
(better)
(inhibiting function)

• double stranded RNA, dsRNA Disrupts target RNA.
• RNAi (long dsRNA)
• Works well in Drosophila and C.elegans, but
  doesnt work well in neural tissue in C.elegans.
• siRNA (21nt small dsRNA)
• Works in mammalian tissues.
• Morpholino antisense oligonucleotides (MO)
• Inhibits RNA function (interferes with
  translation initiation or RNA splicing) Xenopus
  (microinjection), Zebrafish (microinjection),
  Chicken (delivery difficult).

silencing
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III. Experimental inhibition of gene function
knock out
Disrupting the gene
(best)

• Targeted knock out (reverse genetics)
• requires homologous recombination
• Mouse knock out technology
• Integrational mutagenesis
• Drosophila P-element mediated integration of
  exogenous DNA into genome to disrupt endogenous
  genes. Cloning of affected gene easy because
  exogenous DNA can be used as a marker.
• Classic genetics (forward genetics)
• start with a mutant phenotype, map mutation
  (difficult), identify mutated gene

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Molecular Methods Tool Box
I. Analysis II. Overexpression III. Inhibition
Protein Western Blot Proteomics immuno-histochemisty protein chimera (adding proteins to in vitro reactions) pharmacological inhibitors, dominant-negative proteins, protein depletion using antibodies
RNA Northern Blot RNase protection assays microarrays RT-PCR RNA in-situ microinjection RNAi siRNA Morpholinos
DNA (gene) Reporter genes (microinjection) Electroporation, lipofection, transgenics Knock-out Integrational mutagenesis Classic genetics
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Question for Tool box for studying gene function
in neural development lecture in PY4302 course
(2004).
Question Several powerful molecular tools (or
methods) are available to the developmental
neurobiologist for studying differential RNA
expression in the developing central nervous
system of vertebrate embryos. Describe and
compare the methods of Northern Analysis
(Northern Blot) and RNA in situ hybridisation and
their application. Minimal Model
Answer Northern Analysis (alternative correct
term Northern Blot) involves the isolation and
preparation of RNA from tissue (RNA from tissue
taken from different parts of the embryo or from
different embryonic stages can be loaded into
different lanes and compared with each other),
separation of RNA molecules on a gel in an
electric field according to differences in
mobility (according to size or mass is considered
correct), transfer from the gel to a membrane
(blotting paper considered correct) and detection
of a particular RNA fragment with the help of a
labelled (radioactive or epitope-labelled
considered correct) antisense probe that
hybridises specifically to the particular RNA
fragment, followed by detection and imaging (e.g.
film) of the resulting bands in columns
representing the different lanes of the original
gel. RNA in situ hybridisation involves the
fixing of embryos or of embryonic tissue under
conditions that preserve RNA, incubation with an
epitope-labelled (radioactive not taught but also
correct if applied to tissue sections) antisense
RNA probe that will specifically hybridise to a
particular RNA in the tissue, incubation with an
enzyme conjugated antibody that recognises and
binds the epitope label on the antisense RNA
probe, incubation with a colourless substrate
that is converted into a coloured precipitate
only where the enzyme-conjugated antibody bound
to the antisense RNA probe reveals the presence
of the particular RNA studied and imaging of the
resulting embryo or tissue. More advanced
answer Advantages of Northern Analysis include
good resolution for detecting different size gene
products (different RNA products from the same
gene created by alternative promoters and/or
alternative splicing) and of stage-specific
differences of expression (depending on the
accuracy of the original tissue isolation).
Disadvantages include little quantitative
information on levels of gene expression and
limited resolution for detecting spatial (not
cell to cell) differences of expression
(depending on accuracy of original tissue
isolation). Advantages of RNA in situ
hybridisation include good resolution for
detecting spatial differences of expression (at
least cell to cell, if not on the subcellular
level), relatively good information on the
temporal differences of expression (depending on
the accuracy of the staging). Disadvantages
include little quantitative information on
different levels of gene expression and usually
limited specific information on expression of
alternative gene products (depending on the
particular probe used).