1. Genetic Manipulations

2. 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.

3. Plasmid utilization

4. Plasmid utilization

5. Inserting DNA sequences
D:\ANIMATIONS\PLASMIDCLONING.MOV

6. Uses of cloned DNA Producing high levels of proteins
Transfecting cells
Transfecting organisms

7. Protein expression systems

8. Uses of cloned DNA Producing high levels of proteins
Transfecting cells
Transfecting organisms

9. 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

10. 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.

11. Uses of cloned DNA Producing high levels of proteins
Transfecting cells
Transfecting organisms

12. Viruses
Non-enveloped virus
Enveloped virus
D:\ANIMATIONS\MCB2401N.MOV

13. Viral Vectors assembly in packaging cells
Kay et al, 2003, Nat Rev Biotech

14. Viral vectors in the CNS
Gene regulation:
Viral promoters
Cellular specific promoter (synapsin, TH)
Fate of TGs:
non-replicating extrachromosomal
chromosomal integration
extrachromosomal replication
Davidson and Breakefield, 2003, Nat Rev Neurosci

15. Vector delivery to the CNS
Different viruses transfect specific cell types
Neurotropic viruses are uptaked by terminals and can undergo retrograde and anterograde transport
Tropism can be altered by introducing specific receptor ligands in capsid proteins.
Stereotaxic delivery
Davidson and Breakefield, 2003, Nat Rev Neurosci

16. Transgenic / KO mice

17. Transgenic mice

18. Cre/loxP technology

19. Using Cre/loxP in neuroanatomy
Livet et al., Nature 2007

20. Using Cre/loxP in neuroanatomy
Livet et al., Nature 2007