Teacher
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BASIRICO' Loredana
(syllabus)
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.
Nutraceutics of products of animal origin and for animal welfare. Reproductive biotechnology: instrumental insemination, techniques for the production of embryos, MOET and OPU, in vitro fertilization, sexing of semen and embryos. 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. 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. Genetic improvement to. Quantitative genetics b. Genomics Practical 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 candidate genes associated to marker assisted selection.
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.
(reference books)
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. SANDRUCCI A., TREVISI E. (A CURA DI), Produzioni Animali. ED. EDISES, 2022. - Didactic material provided by the teacher. Lecture notes or material posted on the dedicated website (Moodle platform).
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