In-depth knowledge of cellular and molecular biology is necessary to approach the course in a proficient manner

Lectures will be held, with the help of slides. Support tools will consist of websites, seminars. Help and completion will be provided by support staff and by colleagues during practicals. The laboratory activities will be organized in groups. The elearning site of the course will be used for enrolling to the course and email will be used for teacher-student communications.

Introduction to the Course and discussion of aspects on animal experimentation. Objection of conscience to animal experimentation. Notes on the rules for the use of animals for scientific purposes: Legislative Decree No. 26 of March 4, 2014, and Directive No. 2010/63/EU. Introduction of the 3Rs principle. Use of mouse as an experimental model; main characteristics: advantages - disadvantages. Strain concept (inbred - outbred). Pre-implantation development; manipulation and in vitro culture of mouse zygotes/blastocysts; pronuclear microinjection. The manipulation of pre-implantation mouse embryos. Recombinant DNA and transgenesis. Concept of transgene; preparation of constructs for transgenesis. Historical background and milestones of transgenesis in the mouse; integration, position effect, regulation and expression characteristics of transgene. Generation of a new transgenic line and importance of transgene expression profile characterization. The example of the transgenic mouse line Pet1/Bgal. Transgenesis in large mammals and use of transgenesis for bioreactor generation.

Introduction to principal animal models used in biomedical research alternative to the mouse: birds, amphibians, fish insects. Introduction to the use of chicken as a model system. Development of quail/chicken transplantation by Nicole Le Douarin, concept of homo- heterotopic/chronic grafts and experimental strategies. "In ovo" electroporation. Strategies of transgenesis in birds. Introduction to the use of Xenopus laevis/tropicalis and zebrafish (Danio rerio) as model systems. Transgenesis in Xenopus and zebrafish. Gain- and loss-of-function experiments via 7-Methylguanosine (m7G) messenger RNA (capped) and morpholino oligo, respectively. Use of zebrafish in drug screening. Introduction to the use of Drosophila as a model system. Transgenesis in Drosophila, transposase and use of P elements. Use of transgenesis in drosophila for the study of gene expression regulation. Gain- and loss-of-function experiments to study gene function: the example of eyless (ey) as a master gene of the eye.

Methodology and experimental strategy for enhancer trap and gene trap in mouse embryonic stem cells: utility, advantages. Examples of its application: the case of Apaf1. Strategies for studying promoters in vivo using transgenesis in the mouse. Identification of time- and tissue-specific promoters: promoter "dissection." Strategies for studying promoters in vivo by using transgenesis in the mouse. Identification of tissue-specific enhancers: bioinformatics analysis, next generation sequencing (NGS), ChIP-seq. Introduction to insulator elements and their use for "positional effect" containment.

Definition, characteristics and use of bacterial artificial chromosomes (BACs). Strategies for the use of BACs in transgenesis: homologous recombination in E. coli cells; examples of the use of BACs in mouse transgenesis: GENESAT.

Gene targeting: gene knock-out/knock-in in mouse; homologous recombination in embryonic stem cells; injection of ES cells into blastocysts; principles of in vitro aggregation and utility of tetraploidization in mouse embryos. Strategies for generations of knock-out mice; examples of knock-out animals: study of gene function. Concept and examples of knock-in (KI). Reporter genes, GFP, betaGal, peroxidase. Generation of animal models for human disease. Employment of targeted gene replacement in mouse and pig. Introduction to new strategies for targeted gene editing: Zinc-Finger Nucleases system, Transcription Activator-Like Effector Nucleases (TALENs) system, and CRISPR/Cas9 system. Advantages and limitations of new gene editing strategies. "Gene Targeting" in rat and other animal systems for "modeling" human diseases.

Binary transgenesis for modulating control of transcriptional activity: UAS/GAL4 system, tet-on and tet-off tetracycline system. Characteristics and use of site-specific recombinases: cre/loxP and flp/FRT system: inversion, excision, translocation. Control of gene expression by somatic recombination. Employment of somatic recombination for fate mapping and intersectional fate mapping studies. Strategies for induction of Cre recombinase activity: time and tissue-specific conditional knock-out. Study of gene function and generation of human disease models based on the use of Cre recombinase-mediated somatic recombination.

Use of transgenesis for the generation of optogenetic models: strategies for the activation and inhibition of neural activity using a light source. Methods of use and applications. Use of transgenesis for the generation of chemogenetic models for neural activation and inhibition by administering molecules: Designed receptors activated exclusively by designed drugs (DREADD). The two systems for remote control of neural activity compared: advantages vs. disadvantages. Introduction to the Double-Floxed Inverted Open reading frame (DIO) or 'Flex' system. New methodologies for monitoring neural activity: Magnetogenetics. Employment of Cre recombinase in strategies for combinatorial activation of reporter genes in the central nervous system: generation of "tools" for the study of connectomics. The example of the "Brainbow" system. Novel Genetic strategies for intersectional Cre-mediated somatic recombination and to access activated neurons: Split-Cre and Targeted Recombination in Active Populations (TRAP).

Study of structural connectivity: the connectome. Anterograde and retrograde marking methods. Lipophilic and hydrophilic tracers: DiI and dextrans. The use of recombinant rabic viruses. Pseudotyping and connectivity study using trans-synaptic tracers. Examples of the use of recombinant and psuedotyped rabic virus for the study of connectomics and neuroanatomical features of monoaminergic neurotransmission systems. Toxicity and limitations to the use of the use of rabic viruses in long-term experiments. Strategies for the generation of new variants of rabic viruses for the promotion of somatic recombination and use in longitudinal long term. General summary and final discussion on aspects covered in the course.

As the topics covered are extremely up-to-date and varied, no textbook is available. Students are strongly advised to follow the lectures in the classroom and the topics covered can be further developed with the help of the ppt presentations shown in class and specific articles/reviews suggested by the lecturer.

The exam consists of an oral test. The oral exam consists of an interview between the candidate and the teacher. The oral examination is not passed if the candidate shows an inability to express him/herself in a clear manner using the correct terminology and if the candidate repeatedly shows the inability to relate parts of the program and notions that he must use jointly to answer a question correctly.

Prof Massimo Pasqualetti

Dott.ssa Serena Nazzi (Cultore della materia)

Dott.ssa Sara Migliarini (Cultore della materia)

Supplente:

Dott.ssa Noemi Barsotti