Genetic Manipulations. Introduction In order to understand the functioning of a gene/protein we want...
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Transcript of Genetic Manipulations. Introduction In order to understand the functioning of a gene/protein we want...
![Page 1: Genetic Manipulations. Introduction In order to understand the functioning of a gene/protein we want to regulate its concentration/functioning/localization.](https://reader035.fdocuments.us/reader035/viewer/2022062408/56649f125503460f94c26588/html5/thumbnails/1.jpg)
Genetic Manipulations
![Page 2: Genetic Manipulations. Introduction In order to understand the functioning of a gene/protein we want to regulate its concentration/functioning/localization.](https://reader035.fdocuments.us/reader035/viewer/2022062408/56649f125503460f94c26588/html5/thumbnails/2.jpg)
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.
![Page 3: Genetic Manipulations. Introduction In order to understand the functioning of a gene/protein we want to regulate its concentration/functioning/localization.](https://reader035.fdocuments.us/reader035/viewer/2022062408/56649f125503460f94c26588/html5/thumbnails/3.jpg)
Plasmid utilization
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Plasmid utilization
![Page 5: Genetic Manipulations. Introduction In order to understand the functioning of a gene/protein we want to regulate its concentration/functioning/localization.](https://reader035.fdocuments.us/reader035/viewer/2022062408/56649f125503460f94c26588/html5/thumbnails/5.jpg)
Inserting DNA sequences
D:\ANIMATIONS\PLASMIDCLONING.MOV
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Uses of cloned DNA
Producing high levels of proteinsTransfecting cellsTransfecting organisms
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Protein expression systems
![Page 8: Genetic Manipulations. Introduction In order to understand the functioning of a gene/protein we want to regulate its concentration/functioning/localization.](https://reader035.fdocuments.us/reader035/viewer/2022062408/56649f125503460f94c26588/html5/thumbnails/8.jpg)
Uses of cloned DNA
Producing high levels of proteinsTransfecting cellsTransfecting organisms
![Page 9: Genetic Manipulations. Introduction In order to understand the functioning of a gene/protein we want to regulate its concentration/functioning/localization.](https://reader035.fdocuments.us/reader035/viewer/2022062408/56649f125503460f94c26588/html5/thumbnails/9.jpg)
Transfection to mammalian cells
Essentially similar to transformation. Usually the foreign DNA is not incorporated to the host-cell genome.
LiposomesElectroporetionVirusesCalcium phosphate
![Page 10: Genetic Manipulations. Introduction In order to understand the functioning of a gene/protein we want to regulate its concentration/functioning/localization.](https://reader035.fdocuments.us/reader035/viewer/2022062408/56649f125503460f94c26588/html5/thumbnails/10.jpg)
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.
![Page 11: Genetic Manipulations. Introduction In order to understand the functioning of a gene/protein we want to regulate its concentration/functioning/localization.](https://reader035.fdocuments.us/reader035/viewer/2022062408/56649f125503460f94c26588/html5/thumbnails/11.jpg)
Uses of cloned DNA
Producing high levels of proteinsTransfecting cellsTransfecting organisms
![Page 12: Genetic Manipulations. Introduction In order to understand the functioning of a gene/protein we want to regulate its concentration/functioning/localization.](https://reader035.fdocuments.us/reader035/viewer/2022062408/56649f125503460f94c26588/html5/thumbnails/12.jpg)
Viruses
D:\ANIMATIONS\MCB2401N.MOV
Non-enveloped virus Enveloped virus
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Viral Vectors assembly in packaging cells
Kay et al, 2003, Nat Rev Biotech
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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)
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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
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Transgenic / KO mice
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Transgenic mice
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Cre/loxP technology
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Using Cre/loxP in neuroanatomy
Livet et al., Nature 2007
![Page 20: Genetic Manipulations. Introduction In order to understand the functioning of a gene/protein we want to regulate its concentration/functioning/localization.](https://reader035.fdocuments.us/reader035/viewer/2022062408/56649f125503460f94c26588/html5/thumbnails/20.jpg)
Using Cre/loxP in neuroanatomy
Livet et al., Nature 2007