RECOMBINANT DNA TECHNOLOGY - CLASS LESSONS (5 CFUs, 40 hours)
• DNA as an informational molecule (2 hours)
• Molecular cloning: history and key concepts (2 hours)
• From natural plasmids to cloning vectors (1 hour)
• Restriction enzymes (1 hour)
• The four cloning steps (2 hours)
• Preparation and analysis of recombinant DNA (0,5 hours)
• Second and third generation plasmid-based cloning vectors. (2 hours)
• High-capacity cloning vectors: lambda, P1 and cosmid vectors. PAC, YACs and BACs). Viral vectors for eucaryotic cells (2 hours)
• Principles of molecular hybridization assays (1 hour)
• Applications hybridization -1: Southern, northern and zoo blot. Colony hybridization (2 hours)
• Applications hybridization -2: FISH assays, RNA ISH, comparative genomic hybridization (CGH), microarray hybridization (2 hours)
• Applications of PCR: Mutation screening and detection, genomic/cDNA screening, RT-PCR, cloning by PCR, DOP-PCR, in situ PCR. Outline on digital PCR (3 hours)
• Real-time PCR and digital : principles and applications (2 hours)
• Genomic DNA libraries: introduction and mode of assembly. Library complexity. cDNA libraries (1,5 hours)
• Methods for DNA mutagenesis (2 hours)
• Gene transfer assays in eucaryotic cells: calcium phosphate transfection, lipofection, electro-poration and viral vectors infections(1 hour)
• Methods for the identification of a gene’s regulatory elements: DNase hypersensitive site’s mapping, gene transfer with reporter vectors, DNA footprinting, Electrophoretic Mobility Shift Assay (EMSA) (3 hours)
• Introduction to the key model organisms in experimental biology. Key concepts for transgenesis. Methods for producing and analyzing transgenic mice (2 hours)
• Transgenic systems with inducible gene expression(2 hours)
• Gene targeting principles and approaches. Gene “knock-out” e “knock-in” in mouse model systems. Conditional gene knock-out (3 hours)
• Use of chromosome engineering and zinc-finger nucleases, TALEN and CRISPR assays for gene targeting in vivo (1 hour)
• Genetic maps and molecular markers. (1 hour)
• DNA profiling (1 hours)

Laboratory lessons (1 CFU, 16 hours)
Four practical laboratory training lessons (4 hours each) will be held. The teaching module is focused on the planning and fulfillment of a molecular cloning experiment, under the following schedule:
• Day 1: Planning of the experimental strategy. Digestion of DNA templates with restriction enzymes, dephosphorilation of the plasmid vector, preparative gel electrophoresis and DNA gel extraction
• Day 2: quantitation of the extracted DNA samples. Planning and execution of the ligation reaction. Bacterial transformation.
• Day 3: analysis of the results. Colony PCR to discriminate recombinant and nonrecombinant clones. Inoculation of PCR-positive clones in liquid medium.
• Day 4: Mini-prep of plasmid DNA. Electrophoretic analysis of the recombinant DNA molecole. Final discussion of the whole data.