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 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
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
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