Transcript Tool box for studying gene function in neural development

Molecular tool box for studying gene
function in neural development
For Eye Development Course
Studying gene function in neural development
Differential Gene Expression
(The Paradigm of Developmental Biology)
approx. 30’000 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.
Studying gene function in neural development
Gene Expression
DNA
GENE
transcription
(The Central Dogma of Molecular Biology)
RNA
translation
GENE PRODUCTS
PROTEIN
GENE
FUNCTION
Studying gene function in neural development
Experimental Design
I.
(“gene A functions in process B”)
Investigation of gene expression: gene A is expressed in the
appropriate tissue and at the appropriate stage to mediate process B.
II. Gain-of-function experiment: gene product of gene A is
sufficient for mediating process B.
III. Loss-of-function experiment:gene A or gene product of gene A
is normally required for process B.
IV. Analysing of experiment by investigating the resulting
phenotype or marker gene expression.
Studying gene function in neural development
Methods tool box
I.
Methods for analysing gene expression (Protein,
RNA, Reporter gene (DNA))
(IV. Analyse experiments with tissue-specific molecular markers.)
II. Methods for expressing gene products (ectopic
expression, over-expression, expression rescue of Protein,
RNA or DNA)
III. Methods for inhibiting gene function (inhibiting
Protein function, disrupting or inhibiting RNA, disrupting
gene (DNA))
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.
Western Blot
Protein A
linked to the enzyme Alkaline Phosphatase (AP) or HRP
(chemiluminescent)
Protein extraction
Protein gel
Blot
Film
Protein A is expressed from stages 10 to 30 during Xenopus development
Immunohistochemistry
endogenous protein in fixed embryonic tissue
Protein A
linked to the enzyme Alkaline Phosphatase (AP)
or linked to fluorescent molecule
coloured precipitate
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)
Northern Blot
endogenous mRNA of gene A:
AAAAAAAAAAAA
radioactively labelled DNA probe
RNA extraction
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
RT-PCR
RNA extraction
endogenous mRNA of gene A:
AAAAAAAAAAAA
reverse transcription
AAAAAAAAAAAA
TTTTTTTTT
cDNA of gene A:
PCR with radioactive nucleotides
TTTTTTTTT
radioactive DNA PCR product:
DNA gel
gene-specific PCR primers
Film
Gene A is expressed from stages 20 onward during Xenopus development
RNA in situ hybridisation
I. Methods for analysing gene expression
Reporter gene DNA constructs
• cis-regulatory DNA elements drive expression of reporter
protein.
– Lac Z:
enhancer
prom.
ß-galactosidase
• very sensitive but only in fixed dead tissue
– Green Fluorescent Protein (GFP):
enhancer prom.
• less sensitive but in live embryos
• mutated GFP: CFP (cyan), RFP (red), YFP (yellow)
• DsRed is a red fluorescent protein
GFP
II. Experimental expression of gene products
Introducing extra gene product
• Fast
• Less reliable
Introducing an extra gene
• Slower, more difficult
• more reliable
(Transgenics)
II. Experimental expression of gene products
Introducing extra gene product
• Introducing extra protein?
doesn’t 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-nonautonomously (signals).
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
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:
P
white+ gene
promoter::PROTEIN
Transposase gene
– TRANSPOSASE integrates plasmid 1 into the Drosophila genome
P
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
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.
III. Experimental inhibition of gene function
Inhibiting RNA
(desrupting RNA)
(inhibiting function)
“knock down”
(better)
• double stranded RNA, dsRNA: Disrupts target RNA.
– RNAi: (long dsRNA)
Works well in Drosophila and C.elegans, but doesn’t work well in neural tissue
in C.elegans.
– siRNA: (21nt small dsRNA)
“silencing”
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).
III. Experimental inhibition of gene function
Disrupting the gene
“knock out”
(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
Molecular Methods Tool Box
Protein
RNA
DNA
(gene)
I. Analysis
II. Overexpression
III. Inhibition
•Western Blot
•Proteomics
•immunohistochemisty
•protein chimera
•(adding proteins to in
vitro reactions)
•Northern Blot
•RNase protection
assays
•microarrays
•RT-PCR
•RNA in-situ
•Reporter genes
•microinjection
•pharmacological
inhibitors,
•dominant-negative
proteins,
• protein depletion using
antibodies
•RNAi &
•siRNA
•Morpholinos
•(microinjection)
•Electroporation,
•lipofection,
•transgenics
•Knock-out
•Integrational mutagenesis
•Classic genetics
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).