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

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Uses of cloned DNA

Producing high levels of proteinsTransfecting cellsTransfecting organisms

Protein expression systems

Uses of cloned DNA

Producing high levels of proteinsTransfecting cellsTransfecting organisms

Transfection to mammalian cells

Essentially similar to transformation. Usually the foreign DNA is not incorporated to the host-cell genome.

LiposomesElectroporetionVirusesCalcium 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 proteinsTransfecting cellsTransfecting organisms

Viruses

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Non-enveloped virus Enveloped virus

Viral Vectors assembly in packaging cells

Kay et al, 2003, Nat Rev Biotech

Viral vectors in the CNS

Davidson and Breakefield, 2003, Nat Rev Neurosci

Fate of TGs:

1. non-replicating extrachromosomal

2. chromosomal integration

3. extrachromosomal replication

Gene regulation:

1. Viral promoters2. Cellular specific

promoter (synapsin, TH)

Vector delivery to the CNS

Davidson and Breakefield, 2003, Nat Rev Neurosci

1. Different viruses transfect specific cell types2. Neurotropic viruses are uptaked by terminals and can undergo

retrograde and anterograde transport3. Tropism can be altered by introducing specific receptor ligands in

capsid proteins.4. Stereotaxic delivery

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