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Genetic Manipulations
Introduction
In order to understand the functioning of
a gene/protein we want to regulate its
concentration/functioning/localization in
the cell.
 It’s virtually impossible to synthesize
large proteins. Thus, the approach is to
insert encoding DNA to a host-cell that
will alter the encoded protein, or its level.

Plasmid utilization
Plasmid utilization
Inserting DNA sequences
D:\ANIMATIONS\PLASMIDCLONING.MOV
Uses of cloned DNA
Producing high levels of proteins
 Transfecting cells
 Transfecting organisms

Protein expression systems
Uses of cloned DNA
Producing high levels of proteins
 Transfecting cells
 Transfecting organisms

Transfection to mammalian cells
Essentially similar to transformation.
Usually the foreign DNA is not
incorporated to the host-cell genome.
Liposomes
 Electroporetion
 Viruses
 Calcium phosphate

Transfection to mammalian cells

Genes are only transiently expressed
unless incorporated to the cell genome.

Despite so, transient expression is
sometimes enough to study a protein, in
systems as xenopus oocytes, COS-1
etc.
Uses of cloned DNA
Producing high levels of proteins
 Transfecting cells
 Transfecting organisms

Viruses
Non-enveloped virus
Enveloped virus
D:\ANIMATIONS\MCB2401N.MOV
Viral Vectors assembly in packaging
cells
Kay et al, 2003, Nat Rev Biotech
Viral vectors in the CNS
Fate of TGs:
1. non-replicating
extrachromosomal
2. chromosomal
integration
3. extrachromosomal
replication
Gene regulation:
1. Viral promoters
2. Cellular specific
promoter (synapsin,
TH)
Davidson and Breakefield, 2003, Nat Rev Neurosci
Vector delivery to the CNS
1. Different viruses transfect specific cell types
2. Neurotropic viruses are uptaked by terminals and can undergo
retrograde and anterograde transport
3. Tropism can be altered by introducing specific receptor ligands in
capsid proteins.
4. Stereotaxic delivery
Davidson and Breakefield, 2003, Nat Rev Neurosci
Transgenic / KO mice
Transgenic mice
Cre/loxP technology
Using Cre/loxP in neuroanatomy
Livet et al., Nature 2007
Using Cre/loxP in neuroanatomy
Livet et al., Nature 2007