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Potential revision Class next
time!!
Alexander Bruce Ph.D.
Aleksandar Mihajlovic M.Sc.
USUAL TIME & PLACE
10 am in lecture theatre
Vasanth Thamodaran M.Sc.
MOLECULAR BIOLOGY – Gene expression analysis
MOLECULAR BIOLOGY TECHNIQUES III.
analysis of gene expression
MOLECULAR BIOLOGY – Gene expression analysis
HYBRIDIZATION
RNA detection
ANTIBODY
protein detection
MOLECULAR BIOLOGY – Gene expression analysis
Where in organism is the gene expressed?
Detection of RNA by in situ hybridization:
Stage 5
Stage 10
Stage 11
Stage 13
MOLECULAR BIOLOGY – Gene expression analysis
DNA
REPORTER
Gen X
Green Fluorescent Protein
Other reporter genes e.g. LacZ
Drosophila – GFP and embryonic development:
http://www.youtube.com/watch?v=MMLVqGEOD58
MOLECULAR BIOLOGY – Gene expression analysis
Mouse embryo histone H2B-GFP reporter
MOLECULAR BIOLOGY – Gene expression analysis
DNA
REPORTER
Gen X
Green Fluorescent Protein
Other reporter genes e.g. LacZ
How?
Enhancer Trap, Knock-in ….
Drosophila – GFP and embryonic development:
http://www.youtube.com/watch?v=MMLVqGEOD58
MOLECULAR BIOLOGY – Gene expression analysis
Various reporter gene vectors for random integration into cell genomes (e.g. ES
cells) leading to production of transgenic animals
Reporter has minimal promoter that
can be activated by a
functioning proximal enhancer
Transposon elements
random integration
Viral intergration elemets
Antibiotic resistence allows
selection of successfully
integrated transgenes
Reporter activity if integration site is proximal to an active gene enhnacer i.e.
cell marked/ highlighted
a) Enhancer trap vectors
MOLECULAR BIOLOGY – Gene expression analysis
Various reporter gene vectors for random integration into cell genomes (e.g. ES
cells) leading to production of transgenic animals
Splice acceptor site/ sequence (no promoter)
random (intronic) integration
Gene fusion ... Simultaneously reports expression and usually inactivates function
(potential cytolocalization analysis in marked cells) - hypomorphic
b) Gene trap vectors
MOLECULAR BIOLOGY – Gene expression analysis
Various reporter gene vectors for random integration into cell genomes (e.g.
ES cells) leading to production of transgenic animals
No splice acceptor site or promoter
random (exon) integration
Active promoter also leads to gene fusion thus reports and usually inactivates gene
function (also potential cytolocalization analysis in marked cells)
c) Promoter trap vectors
MOLECULAR BIOLOGY – Gene expression analysis
Targeting specific genes - BACTERIAL RECOMBINEERING
e.g. creation of C-terminal GFP fusion reporter in mammalian cells
E-coli prom
hygr
GFP
PCR generated
TARGETING construct
Mammalian prom
neor
50bp of homlogous sequence
flanking STOP codon
Bacterial GENOMIC
LIBRARY CLONE
containing the gene
to be targeted
3’UTR
STOP
+ plasmids encoding
RECOMBINASE enzymes
from bacteriophage 
TRANSFORM the targeting construct
into the genomic library bacterial clone
E-coli prom
GFP
Mammalian prom
hygr
neor
RECOMBINASE enzymes recognise
homologous sequences and induce DNA
double strand breaks and strand
CROSSOVER
STOP
3’UTR
MOLECULAR BIOLOGY – Gene expression analysis
Targeting specific genes - BACTERIAL RECOMBINEERING
e.g. creation of C-terminal GFP fusion reporter
hygr
GFP
PCR generated
TARGETING construct
Mammalian prom
E-coli prom
neor
50bp of homlogous sequence
flanking STOP codon
Bacterial GENOMIC
LIBRARY CLONE
containing the gene
to be targeted
3’UTR
STOP
+ plasmids encoding
RECOMBINASE enzymes
from bacteriophage 
TRANSFORM the targeting construct
into the genomic library bacterial clone
Recombinase mediates the successful INSERTION/ INTEGRATION of
the targeting construct into the genomic DNA library DNA creating the
GFP FUSION REPORTER GENE
E-coli prom
GFP
STOP
Mammalian prom
hygr
Successfully recombined bacterial clones can be selected for with antibiotics
neor
3’UTR
MOLECULAR BIOLOGY – Gene expression analysis
Targeting specific genes - BACTERIAL RECOMBINEERING
GFP FUSION REPORTER GENE DNA construct isolated and purified from bacterial DNA
E-coli prom
GFP
Mammalian prom
hygr
neor
3’UTR
TRANSFORM (TRANSFECT) GFP fusion
construct into mammalian cells e.g. ES cells
E-coli prom
GFP
Low frequency mammalian
ENDOGENOUS
RECOMBINATION enzymes
induce strand cross over
Mammalian prom
hygr
neor
3’UTR
Successfully recombination can be selected
for with second antibiotic resistance gene
STOP
3’UTR
The GFP FUSION REPORTER GENE for a specific gene has now replaced the endogenous copies of the
original gene in the mammalian cell genome. These cells can then be used to create TRANSGENEIC animals
The same approaches can be used to disrupt genes by insertion or deletion of DNA sequence to generate
GENETIC KNOCKOUTS to asses specific gene function
MOLECULAR BIOLOGY – Gene expression analysis
NORTHERN BLOT
To what extent is the gene expressed?
RNA
DNA ... Southern blot
MOLECULAR BIOLOGY – Gene expression analysis
PROMOTER
DNA
exon 1
exon 2
exon 3
ATG
TAA AATAAA
TRANSCRIPTION
intron
Pre-mRNA
AUG
intron
UAA AAUAAA
RNA SPLICING
mRNA
AUG
UAA AAUAAA
REVERSE TRANSCRIPTION
cDNA
TAC
ATT TTATTT
HIV
MOLECULAR BIOLOGY – Gene expression analysis
cDNA synthesis
Some reverse
transcriptases
initiate 2nd
stand synthesis
via addition of
hairpins
Ordinary 1st strand synthesis
GENERATION OF A cDNA THAT CAN BE AMPLIFIED USING SPECIFIC PRIMERS BY PCR
MOLECULAR BIOLOGY – Gene expression analysis
Quantitative RT-PCR (reverse transcription)
RP49 control
(house-keeping)
MOLECULAR BIOLOGY – Gene expression analysis
Real-Time PCR
light-cycler
SYBR- green dsDNA
intercalation and
flourescence
MOLECULAR BIOLOGY – Gene expression analysis
Molecular Beacon Probes
Potential for multiplex i.e. more than one probe per sample
MOLECULAR BIOLOGY – Gene expression analysis
Norhern blot, RT-PCR
to study one (couple) gene(s) at once ...
... in genomic era?
MOLECULAR BIOLOGY – Gene expression analysis
Genomic expression analysis using MICROARRAY (CHIP)
MOLECULAR BIOLOGY – Gene expression analysis
MOLECULAR BIOLOGY – Gene expression analysis
Chromatin remodeling
methylase (CG)
+
histone deacetylases (HDACs)
histone acetyltransferases (HATs)
MOLECULAR BIOLOGY – Gene expression analysis
ChIP on chip
Chromatin immunoprecipitation
combined with DNA microarrays (chip)
or
ChIP-seq
Chromatin immunoprecipitation
combined with DNA sequencing
MOLECULAR BIOLOGY – Gene expression analysis
ChIP on chip e.g. of transcription factor binding site discovery
REST
RE1
NEW TARGET GENES
COMBINE WITH HISTONE ChIP on Chip for
extra structure function data
MOLECULAR BIOLOGY – Gene expression analysis
cDNA
more RNA in original sample ...
... more times the particular fragment is sequenced
quantifiable without knowledge of sequence
(microarray needs to know the sequences)
MOLECULAR BIOLOGY – Gene expression analysis
ChIP-seq
Chromatin immunoprecipitation
combined with DNA sequencing
MOLECULAR BIOLOGY – Gene expression analysis
... at the protein level
immunolocalization
ANTIBODY
protein detection
MOLECULAR BIOLOGY – Gene expression analysis
ANTIBODY
MOLECULAR BIOLOGY – Gene expression analysis
MONOCLONAL ANTIBODY
polyclonal serum
MOLECULAR BIOLOGY – Gene expression analysis
POLYACRYLAMIDE-GEL ELECTROPHORESIS
(PAGE)
Figure 8-18a Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Gene expression analysis
RNA
PROTEINS
WESTERN BLOTTING
DNA
MOLECULAR BIOLOGY – Gene expression analysis
MOLECULAR BIOLOGY – Gene expression analysis
1st dimension: Isoelectric focusing
Figure 8-22 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Gene expression analysis
Two-dimensional electrophoresis
MOLECULAR BIOLOGY – Gene expression analysis
Figure 8-23 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Gene expression analysis
PROTEOMICS
What is the difference on B?
identification by
mass spectroscopy
MOLECULAR BIOLOGY – Gene expression analysis
Figure 8-21 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Gene expression analysis
GENOME
TRANSCRIPTOME
PROTEOME
MOLECULAR BIOLOGY – Gene expression analysis
Good Luck
with molecular biology!
RNA
PROTEINS
RT-PCR
Real-Time PCR
Inverse PCR
Nested PCR
DNA
Potential revision Class next
time!!
Alexander Bruce Ph.D.
Aleksandar Mihajlovic M.Sc.
USUAL TIME & PLACE
10 am in lecture theatre
Vasanth Thamodaran M.Sc.