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TRANSGENIC MODELS AND MOLECULAR METHODS FOR NEUROSCIENCES
MASSIMO PASQUALETTI
Academic year2023/24
CourseNEUROSCIENCE
Code415EE
Credits6
PeriodSemester 2
LanguageEnglish

ModulesAreaTypeHoursTeacher(s)
TRANSGENIC MODELS AND MOLECULAR METHODS FOR NEUROSCIENCESBIO/06LEZIONI56
MASSIMO PASQUALETTI unimap
Obiettivi di apprendimento
Learning outcomes
Conoscenze

Il corso è erogato in lingua Inglese. Le informazioni richieste sono riportate nel campo dedicato.

Knowledge

By the end of the course, students will have acquired the skills to understand the methodologies used in the generation, characterisation and utilisation of animal models with particular reference to their use in neuroscience.

Modalità di verifica delle conoscenze

Il corso è erogato in lingua Inglese. Le informazioni richieste sono riportate nel campo dedicato.

Assessment criteria of knowledge

For the assessment of the knowledge will be held discussion during classe between the teacher and the students.

Capacità

Il corso è erogato in lingua Inglese. Le informazioni richieste sono riportate nel campo dedicato.

Skills

Acquired skills will enable not only a thorough understanding of a scientific article when reading it but also the theoretical basis for designing a research project involving the generation and use of animal models.

Modalità di verifica delle capacità

Il corso è erogato in lingua Inglese. Le informazioni richieste sono riportate nel campo dedicato.

Assessment criteria of skills

During the lessons, tools will be used to ascertain the students' acquisition of the established objectives. Different didactic strategies will be used in order to favor interactive dynamics between the teacher and the students. Practical activities will be carried out with the aid of multimedia tools and through the design of laboratory and observation activities in tha lab.

Comportamenti

Il corso è erogato in lingua Inglese. Le informazioni richieste sono riportate nel campo dedicato.

Behaviors

Students will be able to acquire and develop sensitivity to issues related to genome manipulation and genaration of animal model of human disease.

Modalità di verifica dei comportamenti

Il corso è erogato in lingua Inglese. Le informazioni richieste sono riportate nel campo dedicato.

Assessment criteria of behaviors

During the lessons, different teaching strategies will be used in order to encourage interactive dynamics between the teacher and students, aimed at verifying the degree of acquisition of the topics covered. During the laboratory sessions, the accuracy and precision of the activities carried out will be evaluated.

Prerequisiti (conoscenze iniziali)

Il corso è erogato in lingua Inglese. Le informazioni richieste sono riportate nel campo dedicato.

Prerequisites

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

Indicazioni metodologiche

Il corso è erogato in lingua Inglese. Le informazioni richieste sono riportate nel campo dedicato.

Teaching methods

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.

Programma (contenuti dell'insegnamento)

Il corso è erogato in lingua Inglese. Le informazioni richieste sono riportate nel campo dedicato.

Syllabus

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.

Bibliografia e materiale didattico

Il corso è erogato in lingua Inglese. Le informazioni richieste sono riportate nel campo dedicato.

Bibliography

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.

Modalità d'esame

Il corso è erogato in lingua Inglese. Le informazioni richieste sono riportate nel campo dedicato.

Assessment methods

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.

Note

Il corso è erogato in lingua Inglese. Le informazioni richieste sono riportate nel campo dedicato.

Notes

Prof Massimo Pasqualetti

Dott.ssa Serena Nazzi (Cultore della materia)

Dott.ssa Sara Migliarini (Cultore della materia)

Supplente:

Dott.ssa Noemi Barsotti

Updated: 19/12/2023 10:58