The course aims to introduce the advanced Molecular Biology techniques used for isolating eukaryotic genes and studying their expression, and for producing and modifying recombinant proteins in the E.coli bacterium. Also presented are procedures for the expression of exogenous genes in unicellular and multicellular eukaryotic organisms, including those used in the production of transgenic organisms.
Course contents summary
The course will address the following topics:
- Isolation of a specific gene (cDNA, genome region that contains it).
- Transcriptional analysis of the gene whose cDNA has been isolated.
- Analysis of the structure of the genome region containing the gene of interest.
- Expression in bacteria of the protein coded by the gene.
- Mutagenesis of the gene and of the recombinant protein coded by it.
- Chemical synthesis of DNA and creation of artificial genes.
- Gene Cloning in bacteria other than E. coli.
- Expression of heterologous proteins in B.subtilis.
- Expression of heterologous proteins in Streptomyces.
- Gene cloning in S.cerevisiae and other fungi.
- Expression of heterologous proteins in S.cerevisiae.
- Expression of heterologous proteins in P.pastoris.
- Preparation of expression library in yeast.
- Functional complementation in S.cerevisiae.
- Knock-out gene in yeast.
- Scheme of the two hybrids.
- Gene cloning in animal cells.
- Plasmid vector for transient expression (with and without replicons).
- Plasmid vector for the stable expression (replicons and integration).
- Co-transformation and gene amplification.
- Effect of position and remedies.
- Viral transduction.
- Protein Expression system of baculovirus and vaccinia virus.
- Adenovirus, AAV and retroviruses.
- Preparation of transgenic animals.
- Transfection of mouse ES cells.
- Nuclear Transfer and cloning of whole organisms.
- "Gene-targeting" and "knock out" genes in animals.
- Site-specific recombination in animal cells.
- Site-specific mutagenesis in animal cells.
- Chromosomal Engineering.
- Conditional Mutants in mice.
- Knock-out functional (RNA interference, ribozymes).
- Inducible expression systems in animals.
Ingegneria genetica. Principi e tecniche. S.Primrose et al. - Zanichelli
DNA Ricombinante - J.D.Watson et al. – Zanichelli