MODULE OF RECOMBINANT DNA TECHNOLOGY, CLASS LESSONS (5 CFUs, 40 hours)

• DNA as an informational molecule (2 hours)
• Molecular cloning: summary (2 hours)
• DNA ligase-independent cloning strategies(1 hour)
• Second and third generation plasmid-based cloning vectors. Expression vectors (2 hours)
• High-capacity cloning tools: lambda, P1 and cosmid vectors. PAC, Yeast Artificial Chromosomes (YACs) and Bacterial Artificial Chromosomes (BACs). Viral vectors for eucaryotic cells (2 hours)
• Principles of molecular hybridization assays (1 hour)
• Applications of molecular hybridization techniques – 1: Southern, northern and zoo blot. Colony hybridization (2 hours)
• Applications of molecular hybridization techniques – 2: fluorescence labelling of nucleic acids and fluorescence in situ hybridization (FISH), RNA in situ hybridization (RNA ISH), comparative genomic hybridization (CGH), microarray hybridization (2 hours)
• Introduction and applications of PCR: Mutation screening and detection, genomic/cDNA screening, reverse transcription PCR, cloning by PCR, DOP-PCR. (2 hours)
• Real-time and digital PCR : principles and experimental approaches (2 hours)
• Whole genome amplification by PCR: DOP-WGA and Multiple Displacement Amplification (MDA) (2 hours)
• Genomic DNA libraries: introduction and mode of assembly. Library complexity and genome equivalent values. cDNA libraries (2 hours)
• Methods for DNA mutagenesis: (2 hours)
• Gene transfer assays in eucaryotic cells (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), Chromatin conformation Capture (CCC) (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)
• Chromosome engineering. Genome editing by means of and zinc-finger nucleases, TALEN and CRISPR/Cas9 assays(2 hours)
• Genetic maps and molecular markers. (2 hours)
• DNA profiling (1 hours)

MODULE OF RECOMBINANT DNA TECHNOLOGY, LABORATORY LESSONS (1 CFU, 16 hours)
OUTLINE OF THE EXPERIMENT: subcloning of a specific protein coding sequence into a mammalian expression vector, to express it as a fusion with GFP, transfection of the construct in a human cell line and analysis of its expression by fluorescence microscopy and by qPCR.

DETAIL OF THE PROCEDURES:
• analysis of restriction sites using specific programs;
• digestion with restriction endonucleases;
• agarose gel electrophoresis;
• purification of DNA fragments;
• ligation
• Transformation of ligated products into E. coli;
• solid and liquid cultures of bacteria
• Plasmid DNA extraction and purification
• Transfection of the prepared constructs into a human cell line
• Observation of transfected cells at an epifluorescence inverted microscope
• qPCR for relative quantification of the target transcript of interest in cells and calculation of its fold increase using the 2ˆ-DeltaDelta Cq method (Livak 2001).