Elective course in Experimental Systems

Analysing gene expression

“Recent developments in detecting

mRNAs and proteins”

Dr. Ian Simpson

ian.simpson@ed.ac.uk

Centre for Integrative Physiology

&
Institue for Adaptive and Neural Computation
 Recent and emerging technologies

1. Gene expression arrays

2. Protein arrays
3. Next generation sequencing
4. Protein profiling

Ian Simpson, University of Edinburgh

 Measuring gene expression with microarrays

making the chips labelling and hybridisation

Ian Simpson, University of Edinburgh

 An example microarray experiment

peripheral nervous system development (fruitfly) experimental design

Ian Simpson, University of Edinburgh

Cluster genes by their expression profiles

Ian Simpson, University of Edinburgh

 Downstream applications

gene prioritisation

validation

Ian Simpson, University of Edinburgh

Protein microarray

Ian Simpson, University of Edinburgh

 Next-generation sequencing (ngs)

What is ngs ?

Why is it important ?

What can it do ?

An example :-

    The haploid human genome is 3 billion base pairs.

    The human genome project took 13 years and cost $3 billion in the US alone
    
    A single Solexa run takes 4 days, sequences 18billion base pairs and costs about $5000

Ian Simpson, University of Edinburgh

Next-generation sequencing (Solexa)

Ian Simpson, University of Edinburgh

Next-generation sequencing (Solexa)

Ian Simpson, University of Edinburgh

 Applications for next generation sequencing

1. Transcriptome sequencing (RNA-seq)

2. snRNA profiling
3. Mapping gene splicing events
4. DNA methylation profiling
5. Histone modification
6. DNA-protein binding site identification
7. Transcription factor binding site identification (ChIP-seq)
8.  Chromatin structure
9. SNP assays (e.g. genome wide association studies)
10.Personal genomics (patient customised treatment)

and many more..

Ian Simpson, University of Edinburgh

 Proteomics (protein profiling)

2D protein gel

Ian Simpson, University of Edinburgh

 Extra Slides

(i.e. last years genetic

engineering talk)
 The restriction endonuclease EcoRI

5'---G AATTC---3'
3'---CTTAA G---5'

Cut inside double stranded DNA

Over 900 different types cloned.

Vary from very rare cutters NotI, to very common BamHI

Used widely in molecular biology for cloning and mapping

Being replaced in cloning by “recombineering”

Ian Simpson, Centre for Integrative Physiology, University of Edinburgh

 Genetic engineering, cloning DNA molecules

SmaI
Types of restriction enzyme cut, include 5'-CCC|GGG-3'
Blunt end 3'-GGG|CCC-5'
Sticky end (5' recessed or 3' recessed) KpnI
5'-GGTAC|C-3'
3'-C|CATGG-5'

Ian Simpson, Centre for Integrative Physiology, University of Edinburgh

 Anatomy of a DNA cloning vector

MCS

Ian Simpson, Centre for Integrative Physiology, University of Edinburgh

 Cloning DNA fragments

Ian Simpson, Centre for Integrative Physiology, University of Edinburgh

 Blue white selection for insert screening

Ian Simpson, Centre for Integrative Physiology, University of Edinburgh

 Different vector technologies can take different sized inserts

1-4kb – plasmids

5-10kb – bacteriophage

10-50kb – Cosmids

100kb – 10Mb - YACs

Ian Simpson, Centre for Integrative Physiology, University of Edinburgh

 Screening a clone/DNA library

Ian Simpson, Centre for Integrative Physiology, University of Edinburgh

 Transfecting cells to express a coding sequence

polymer based
transfection

mammalian
expression
vector

Ian Simpson, Centre for Integrative Physiology, University of Edinburgh

 DNA sequencing by the Sanger Method

Stats:
• read lengths up to 1,000
bp
• accuracy 99.999%
• costs $0.50 per kilobase
• low throughput

Ian Simpson, Centre for Integrative Physiology, University of Edinburgh

 An Example ABI sequence trace

Ian Simpson, Centre for Integrative Physiology, University of Edinburgh

 Altering gene expression – functional genetics

Over-expression experiments
driving expression from a vector with a strong promoter

Ablation/Depletion experiments
RNAi – RNA interference (miRNA, siRNA)
antisense – drive antisense RNA from expression vector
knockout – traditional or conditional

Ian Simpson, Centre for Integrative Physiology, University of Edinburgh

 Generating a transgenic mouse

Ian Simpson, Centre for Integrative Physiology, University of Edinburgh

 The anatomy of a targeting construct

Ian Simpson, Centre for Integrative Physiology, University of Edinburgh

 Assessing the effects of your manipulation
Immuno-histochemistry for Pax6 before ablation

Ian Simpson, Centre for Integrative Physiology, University of Edinburgh

Describing the overlap between Pax6 and Six3Cre

Ian Simpson, Centre for Integrative Physiology, University of Edinburgh