Cloning of protein-coding sequences both in prokaryotic and eukaryotic vectors. Examples of recombinant proteins, mainly in the biomedical field. Fusion proteins and in vitro mutagenesis. Shuttle vectors. Modulation of gene expression in biomedical research: 1) in cell culture (RNA interfering and ectopic expression), 2) in animal models (transgenic animals, classical methods and CRIPR technique. Transgenic plants. Principles of gene therapy. Recombinant vaccines. PCR in diagnostic.
Biologia molecolare (Capranico et al.) – EdiSES
Biologia molecolare della cellula (Alberts) - Zanichelli
Know the general principle and the most recent applications of the recombinant DNA technology, mainly in the biomedical field. Be aware of the impact that these new technologies have on the therapy and diagnostic of several human diseases, on the vaccine prophylaxis and on basic research.
Contents of the "Molecular Biology" course.
Course of the first part of the II semester of the II year.
Type of Assessment
The exam will be an oral colloquium (about 15 minutes long) aimed at verifying the knowledge of the topics presented during the course as well as to assess the ability of the student to organize the new information on the field.
Cloning experiment. The pBR322Vector. The pUC8 vectors and the blue/white selection. Expression vector. Inducible vectors. Cloning and expression of bacterial genes in E.coli. cDNA synthesis and cDNA library construction. Screening with degenerated oligo-deoxy-nucleutides or with specific antibodies. Fusion proteins with signal peptide (growth hormone.GH) or with maltose binding protein (insulin). In vitro mutagenesis and its importance in basic research and for recombinant proteins for therapy use. The examples of insulin, plasminogen activator (tPA) and recombinant pertussis vaccine. Example of a "dominant negative" mutant (TNF). Yeast shuttle vectors and hepatitis B vaccine. Cell cultures. Transfection and stable lines. Vectors for insect cells: baculovirus promoter. Modulation of specific proteins for basic research: over-expression with transfection and down-regulation with RNA interference (siRNA). Monoclonal antibodies. Recombinant chimeric or humanized antibodies and their use in therapy. Transgenic and knock-out mice; their importance in research. The CRISPR technique. Cloned animals. Transgenic plants: gene transfer methods and some applications. Principle of gene therapy and use of viral vectors for gene transfer. The ADA syndrome and cystic fibrosis (CF). Reverse vaccinology: recombinant meningococcal vaccine. DNA-based vaccines. PCR in microbiological, genetic or oncologic diagnostic and in forensic medicine.