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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