Educational goals The course's objectives are to provide knowledge of the main biotechnology techniques applied to the animal production. They will therefore address issues related to animal experimentation and possible alternative techniques, basic theoretical knowledge on in vitro cultures of cells and animal tissues and their applications, methodologies related to the production of transgenic animals and the most used technologies in quality and traceability of animal products, in the control of genetic improvement and in the maintenance of animal biodiversity. Methods such as extraction of nucleic acids, Polymerase Chain Reaction, sanger and new generation sequencing, microarrays, will be discussed to analyze topics such as the use of markers useful for diagnostic and selective purposes, gene expression analysis, Polymorphisms associated with genetic improvement through molecular genetic techniques, traceability of animal products.
Expected learning outcomes KNOWLEDGE AND UNDERSTANDING. At the end of the course the student will acquire specific knowledge about the biotechnology aspects of animals, the theoretical bases of animal cell culture techniques and their application as experimental models; Knowledge of the main analytical methods of gene expression in animals, knowledge of methodologies related to the production of transgenic animals, as well as molecular genetics and its applications for animal genetic improvement and traceability. APPLYING KNOWLEDGE AND UNDERSTANDING. Ability to interpret and execute broad spectrum methodological and instrumental procedures for biological and molecular research. JUDGMENT. Acquisition of conscious judgment autonomy with reference to the interpretation of protocols and the evaluation and interpretation of laboratory experimental data. COMMUNICATION SKILLS. Acquiring appropriate skills and tools for communication: data processing and presentation, ability to work in groups.
Animal cell cultures: Background. The laboratory of cell culture: general principles on the maintenance of cell cultures. Description of main equipment of animal cell cultures laboratory. Tools and material to isolate and grow cell cultures. The main types of animal cell cultures: primary cultures and cell lines. Differences between primary and cell lines cultures. Basic techniques for immortalized cell cultures in adhesion and in suspension. Trypsinization, counted and propagation of a cell line. Analysis of cell viability. Techniques of disinfection and sterilization. Material and methods of sterilization. Contamination control. Elimination of microbial contamination in cell cultures. Safety standards in laboratory cell cultures. Techniques of cell preservation: cryopreservation. "In vitro" cytotoxicity tests.
Animal transgenesis: history and application. Overview of gene transfer strategies in animal cells. DNA-mediated transformation. Transformation techniques. Transformation transient and stable transformation. The selectable markers. The reporter genes. Main expression systems in animal cells. Retroviral vectors. Methods for the production/generation of transgenic mice. Microinjection of the pronucleus. Transfection of early embryos. Transfection of ES cells. Random recombination and homologous recombination. "Gene targeting" in ES cells. Introduction of small mutations. Targeting vectors: insertion and replacement. "Gene targeting." The knock out. The knock on. Positive-negative selection of ES cells. Use of transgenic animals in biotechnology and in basic research. Animal cloning.
RNA interference (RNAi). The discovery of gene interference and gene silencing with small RNAs. siRNA and miRNA. The function of miRNAs. The biochemistry of gene silencing mediated by small RNAs. Potential therapeutic applications of RNAi.
Quantitative PCR (Real Time PCR); differences between QPCR endpoint and QPCR in real time; chemical fluorogenic in real time PCR "specific" and "non-specific"; absolute quantification and relative quantification. Examples of real time PCR in livestock farming.
Analysis of gene expression using a micro-array. Molecular basis of microarray technology. Chip cDNA and oligonucleotide chips. Generation of microarray sequences from databases with automatic annotation. experimental design with microarray. elements of analysis. diagnostic applications. microarray applications: SNP detection arrays - to identify single nucleotide polymorphism in the genome of different populations; comparative genomic hybridization (Array CGH) - mRNA or gene expression profiling - to study the expression levels of thousands of genes simultaneously; Chromatin immunoprecipitation (ChIP) studies - to determine the binding of proteins specifc in specific portions of DNA (ChIP-on-chip technology).
DNA sequencing: New DNA sequencing techniques. Technological platforms for next generation sequencing for molecular diagnostics in vitro (Solexa Illumina, Affymetrix gene-chips, Roche 454, Applied Biosystems Solid, Helicos). Applications in the livestock production.
Applications of molecular markers in domestic species. Control of diseases and food safety using DNA for the traceability of animal products, crossings and improved selections, increased efficiency of livestock production, individual identification, sex identification, assessment relationship, identification of genetic markers associated with genes responsible for diseases. SNPs (Single Nucleotide Polymorphisms) as marker for the identification of polymorphisms in candidate genes associated to marker assisted selection.
GMOs in animal feed. What is a GMO. Global area of GM crops. The GM varieties. Legislation on the use of GM plants. Assessment of risks to human and animal health. Toxicity, allergy, antibiotic resistance, presence of transgenic fragments in tissues and / or organs. Analytical methods to identify GMOs in food.
Practical classes: Animal Cell culture: epithelial cells of bovine mammary gland. Trypsinization and cells counting. Cellular protein extraction and quantification by colorimetric assay. RNA extraction from cell culture and quantification using fluorometric assay, reverse transcription of RNA. Quantitative PCR (Real Time PCR) for gene expression evaluation in bovine mammary gland cells.
G.L. Mariottini et al. " Introduzione alle colture cellulari", Ed.Tecniche Nuove, 2010. J. D. Watson, A. A. Caudy, R. M. Myers, J. A. Witkowski “DNA ricombinante«, Zanichelli, 2008. Barcaccia G. e Falcinelli M. «Genetica e genomica» vol. III, Genomica e biotecnologie genetiche, capitolo 21, pag 1041-1123. Liguori editore. - Didactic material provided by the teacher.
Dates of beginning and end of teaching activities
Objectives of the course
Università degli Studi della Tuscia - Rettorato, Via S.M. in Gradi n.4, 01100 Viterbo, ITALY - Tel. 0761.3571