Leonid Stoimenov, Vladan Mihajlovic Faculty of Electronic Engineering, University of Nis
Potential revision Class next time!! Aleksandar Mihajlovic M.Sc. USUAL TIME & PLACE 10 am in lecture...
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Potential revision Class next time!! Aleksandar Mihajlovic M.Sc. USUAL TIME & PLACE 10 am in lecture theatre Alexander Bruce Ph.D. Vasanth Thamodaran M.Sc
Transcript of Potential revision Class next time!! Aleksandar Mihajlovic M.Sc. USUAL TIME & PLACE 10 am in lecture...
Potential revision Class next time!!
Aleksandar Mihajlovic M.Sc.
USUAL TIME & PLACE10 am in lecture theatre
Alexander Bruce Ph.D. Vasanth Thamodaran M.Sc.
MOLECULAR BIOLOGY – Gene expression analysis
MOLECULAR BIOLOGY TECHNIQUES III.
analysis of gene expression
ANTIBODYprotein detection
HYBRIDIZATIONRNA detection
Y
MOLECULAR BIOLOGY – Gene expression analysis
Where in organism is the gene expressed?
Detection of RNA by in situ hybridization:
MOLECULAR BIOLOGY – Gene expression analysis
Stage 5
Stage 10
Stage 11
Stage 13
http://www.youtube.com/watch?v=MMLVqGEOD58
Drosophila – GFP and embryonic development:
Gen X
Green Fluorescent Protein
DNA
MOLECULAR BIOLOGY – Gene expression analysis
REPORTER
Other reporter genes e.g. LacZ
Mouse embryo histone H2B-GFP reporter
MOLECULAR BIOLOGY – Gene expression analysis
http://www.youtube.com/watch?v=MMLVqGEOD58
Drosophila – GFP and embryonic development:
Gen X
Green Fluorescent Protein
DNA
MOLECULAR BIOLOGY – Gene expression analysis
REPORTER
Other reporter genes e.g. LacZ
How? Enhancer Trap, Knock-in ….
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
a) Enhancer trap vectors
Reporter has minimal promoter that can be activated by a functioning proximal enhancer
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
random integrationViral intergration elemets
Transposon elements
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
b) Gene trap vectors
Splice acceptor site/ sequence (no promoter)
Gene fusion ... Simultaneously reports expression and usually inactivates function (potential cytolocalization analysis in marked cells) - hypomorphic
random (intronic) integration
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
c) Promoter trap vectors
random (exon) integration
Active promoter also leads to gene fusion thus reports and usually inactivates gene function (also potential cytolocalization analysis in marked cells)
No splice acceptor site or promoter
MOLECULAR BIOLOGY – Gene expression analysis
Targeting specific genes - BACTERIAL RECOMBINEERING
PCR generated TARGETING construct
GFP hygr neor
E-coli prom Mammalian prom
50bp of homlogous sequence flanking STOP codon
+ plasmids encoding RECOMBINASE enzymes
from bacteriophage TRANSFORM the targeting construct into the genomic library bacterial clone
Bacterial GENOMIC LIBRARY CLONE containing the gene to be targeted
STOP 3’UTR
STOP
GFP hygr neor
E-coli prom Mammalian prom
RECOMBINASE enzymes recognise homologous sequences and induce DNA double strand breaks and strand CROSSOVER
3’UTR
e.g. creation of C-terminal GFP fusion reporter in mammalian cells
MOLECULAR BIOLOGY – Gene expression analysis
Targeting specific genes - BACTERIAL RECOMBINEERING
PCR generated TARGETING construct
GFP hygr neor
E-coli prom Mammalian prom
50bp of homlogous sequence flanking STOP codon
+ plasmids encoding RECOMBINASE enzymes
from bacteriophage TRANSFORM the targeting construct into the genomic library bacterial clone
Bacterial GENOMIC LIBRARY CLONE containing the gene to be targeted
STOP 3’UTR
GFP hygr neor
E-coli prom Mammalian prom
Recombinase mediates the successful INSERTION/ INTEGRATION of the targeting construct into the genomic DNA library DNA creating the GFP FUSION REPORTER GENE
3’UTR
e.g. creation of C-terminal GFP fusion reporter
STOP
Successfully recombined bacterial clones can be selected for with antibiotics
MOLECULAR BIOLOGY – Gene expression analysis
Targeting specific genes - BACTERIAL RECOMBINEERING
GFP hygr neor
E-coli prom Mammalian prom
GFP FUSION REPORTER GENE DNA construct isolated and purified from bacterial DNA
3’UTR
TRANSFORM (TRANSFECT) GFP fusion construct into mammalian cells e.g. ES cells
GFP hygr neor
E-coli prom Mammalian prom
3’UTR
STOP 3’UTR
Low frequency mammalian ENDOGENOUS RECOMBINATION enzymes induce strand cross over
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
Successfully recombination can be selected for with second antibiotic resistance gene
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
To what extent is the gene expressed?N
OR
TH
ER
N B
LO
T
RNA
DNA ... Southern blot
MOLECULAR BIOLOGY – Gene expression analysis
PROMOTER
AATAAA
exon 1 exon 2 exon 3
ATG TAA
AAUAAAAUG UAA
AAUAAAAUG UAA
intron intronTRANSCRIPTION
Pre-mRNA
mRNA
DNA
RNA SPLICING
TTATTTTAC ATTcDNA
REVERSE TRANSCRIPTION
HIV
MOLECULAR BIOLOGY – Gene expression analysis
cDNA synthesis
Ordinary 1st strand synthesis
Some reverse transcriptases
initiate 2nd stand synthesis via addition of
hairpins
GENERATION OF A cDNA THAT CAN BE AMPLIFIED USING SPECIFIC PRIMERS BY PCR
MOLECULAR BIOLOGY – Gene expression analysis
RP49 control(house-keeping)
Quantitative RT-PCR (reverse transcription)
MOLECULAR BIOLOGY – Gene expression analysis
Real-Time PCR
light-cycler
SYBR- green dsDNA intercalation and
flourescence
MOLECULAR BIOLOGY – Gene expression analysis
Potential for multiplex i.e. more than one probe per sample
Molecular Beacon Probes
Norhern blot, RT-PCR
to study one (couple) gene(s) at once ...
... in genomic era?
MOLECULAR BIOLOGY – Gene expression analysis
MOLECULAR BIOLOGY – Gene expression analysis
Genomic expression analysis using MICROARRAY (CHIP)
MOLECULAR BIOLOGY – Gene expression analysis
histone acetyltransferases (HATs)
Chromatin remodeling
methylase (CG)+
histone deacetylases (HDACs)
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
MOLECULAR BIOLOGY – Gene expression analysis
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-seqChromatin immunoprecipitation
combined with DNA sequencing
ANTIBODYprotein detection
Y
MOLECULAR BIOLOGY – Gene expression analysis
immunolocalization
... at the protein level
MOLECULAR BIOLOGY – Gene expression analysis
ANTIBODY
MONOCLONAL ANTIBODY
polyclonal serum
MOLECULAR BIOLOGY – Gene expression analysis
Figure 8-18a Molecular Biology of the Cell (© Garland Science 2008)
POLYACRYLAMIDE-GEL ELECTROPHORESIS
(PAGE)
MOLECULAR BIOLOGY – Gene expression analysis
RNA
DNA
PROTEINS
WESTERN BLOTTING
MOLECULAR BIOLOGY – Gene expression analysis
MOLECULAR BIOLOGY – Gene expression analysis
Figure 8-22 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Gene expression analysis
1st dimension: Isoelectric focusing
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
MOLECULAR BIOLOGY – Gene expression analysis
identification bymass spectroscopy
What is the difference on B?
Figure 8-21 Molecular Biology of the Cell (© Garland Science 2008)
MOLECULAR BIOLOGY – Gene expression analysis
GENOME
TRANSCRIPTOME
PROTEOME
MOLECULAR BIOLOGY – Gene expression analysis
MOLECULAR BIOLOGY – Gene expression analysis
DNA
PROTEINS
RNA
SAGE, CAGE, PAGE ...
GENOMICS
TRANSCRIPTOMICS
PROTEOMICS
RT-PCRReal-Time PCRInverse PCRNested PCR
RFLP
AFLP
Good Luck with molecular biology!
Potential revision Class next time!!
Aleksandar Mihajlovic M.Sc.
USUAL TIME & PLACE10 am in lecture theatre
Alexander Bruce Ph.D. Vasanth Thamodaran M.Sc.
