No formal prerequisites or propedeuticity are required. However, a solid knowledge of cell biology, molecular biology and genetics is strongly recommended in order to be able to follow the course fully understanding the different topics

Synthetic program:

Genetic manipulation of eukaryotic cells. Methods for expressing or silencing wt and mutant genes in cultured animal cells. Cellular and molecular biotechnologies to identify protein-protein interactions: the two-hybrid system and its variants, the phage display technology. Biology of embryonic, foetal and tissue stem cells. Applications of stem cells. Regenerative medicine. Induced pluripotent stem cells. Gene therapy: vectors, methodologies and scopes. Cloning and therapeutical cloning.

Program of pratical laboratory classes:

Transfection of mammalian cells. Use of GFP to monitor transfection efficiency. Transfection of GFP-tagged proteins to establish their intracellular localization in mammalian cells. Immunofluorescence analysis. Differentiation of cultured mammalian cells.

The course aims to provide knowledge and skills to work professionally with roles of responsibility in the areas of medical biotechnology which make use of eukaryotic cells (wt or genetically modified).

Learning outcomes

Knowledge to be attained:
• Up to date technologies for genetic manipulation of cultured eucaryotic cells for gene function and therapeutical applications.

• Cellular and molecular biotechnologies to study and unravel molecular interactions

• Biology of embryonic and adult stem cell biology  and of their therapeutical applications.

• Gene therapy approaches

Abilities to be attained:
• Gene expression in mammalian cells
• Gene silencing in mammalian cells
• Use of reporter genes to monitor transfection efficiency, protein intracellular localization and protein activity.

Knowledge and understanding of technologies for the genetic manipulation of mammalian and human cells to investigate gene functions and for therapeutic applications. Knowledge and understanding of cellular and molecular biotechnologies for the discovery and study of molecular interactions. Knowledge and understanding of stem cell biology and its therapeutic applications, particularly in regenerative medicine. Knowledge and understanding of the different gene therapy approaches based on viral and non-viral vectors for the treatment of inherited and acquired diseases.

Ability to apply knowledge and understanding to address open questions in the field of cellular biotechnologies and their applications. Therefore, students will be required to apply the acquired knowledge to identify unresolved issues in research on cellular biotechnology applications, formulate scientific hypotheses and research questions, propose experimental strategies to address these issues, and critically evaluate the results obtained.

Autonomous judgment in the analysis and critical interpretation of the results of scientific experiments and therapeutic protocols related to the applications of cellular biotechnologies. Students will, therefore, be able to read and understand experimental data, interpret results, critically assess the validity of experiments, and identify their strengths and limitations.

Communication skills. Students will be able to clearly and systematically present the main concepts and topics covered in the course and use appropriate scientific terminology when describing experiments and results accurately. Furthermore, they will be able to effectively communicate scientific information related to the topics addressed.

Learning skills aimed at acquiring and developing the competencies required to analyze different experimental and therapeutic approaches through the retrieval of relevant information to address open research questions and unresolved problems. Students will be able to independently acquire new knowledge on innovative cellular biotechnologies, particularly on their therapeutic applications, interpret their molecular mechanisms, and use this knowledge to solve scientific and professional problems.

The achievement of these learning outcomes will be assessed through an oral examination.

Formal lectures (8 cfu= 64 hours) making use of slides and hypertext links to specific Web sites and pratical laboratory classes (1 cfu = 10 hours). Outside these activities, the students are expected to read assigned papers from scientific literature.

The evaluation of the oral examination will be based on the level of the theoretical knowledge and practical abilities acquired through the description of topics and methodologies (60%), on the autonomous judgement (10%), on the critical and problem solving abilities and (20%) and on communication skills (10%).

Methods for expressing or silencing wt and mutant genes in cultured animal cells. Genetic manipulation of eukaryotic cells: principles and scopes. Stable and transient transfection. Plasmid vector for mammalian expression: characteristics. Viral vectors for mammalian expression: vectors based on vaccinia virus, BacMam systems, retrovirus, lentiviral vectors. Expression in COS cells. Constitutive and regulated expression. The Tet-ON and  Tet-OFF systems. Reporter genes and selection markers. Silencing genes using oligonucleotides, PNAs, antisense RNA. RNA interference: mechanism and applications. siRNA and miRNA: physiological roles and applications.  Other kinds of small RNAs to regulate gene expression. shRNA for stable silencing. Methods to introduce molecules in cells: Microinjection, calcium phosphate transfection, DEAE dextran transfection, lipofection, magnet-assisted transfection, dendrimers, nanorods, cell penetrating peptides (CPPs), receptor mediated transfection, etc.

Cellular and molecular biotechnologies to identify protein-protein interactions. The two-hybrid system: principles and applications. Vectors for the two-hybrid system. False positives and false negatives in the two-hybrid system. The two-hybrid system variants: the single hybrid, the three-hybrid, the RNA-based hybrid, the split-hybrid, the Ras-SOS system, etc. Two-hybrid system in bacterial and mammalian cells.  The phage/virion-display: principles and applications. Vectors for phage/virion-display: M13 and baculovirus.

Biology of stem cells. Definition of stem cells. Embryonic, foetal and tissue (adult) stem cells. Potency and plasticity of stem cells. Adult stem cells: hematopoietic stem cells, neural stem cells, epithelial stem cells and mesenchymal stem cells. Homing and engraftment of stem cells. Applications of stem cells. Induced pluripotent stem cells. Regenerative medicine and tissue engineering based on stem cells. Bone marrow transplant, corneal epithelial stem cell transplant, skin production and transplant, bone production and transplant, etc. The theory and the facts on cancer stem cells: implications for therapy.

Gene therapy. Principles of gene therapy. Gene therapy for recessive monogenic diseases. Principles of gene therapy for acquired diseases.  Gene therapy strategies. Modulating the immune system using gene therapy. Examples of gene therapy: successes and problems with ADA-SCID, X-linked SCID, chronic granulomatosis and epidermolysis bullosa gene therapy. Gene therapy for  tumors and infectious diseases. Oncolytic vectors. In vivo and ex vivo gene therapy: advantages and disadvantages.

Animal cloning. The definition of cloning. Cloning methodology for mammals. Therapeutical cloning: technical and ethical problems in humans.

-BIOLOGIA CELLULARE E GENETICA: Parte Prima - Biologia Cellulare a cura di Fantoni, Bozzaro, Del Sal, Ferrari - Casa Editrice PICCIN.

-MOLECULAR BIOLOGY OF THE CELL: Alberts et al., VII edition, Garland Science.