RECOMBINANT TECHNOLOGIES
- Overview
- Assessment methods
- Learning objectives
- Contents
- Full programme
- Delivery method
- Teaching methods
- Contacts/Info
It is recommended to begin the course with a strong background in Cytology/Histology, Molecular Biology and Genetics. Moreover, a good knowledge of English language will be important to read and understand texts, publications and videos that will be provided to the students as teaching material.
The learning outcomes will be assessed through an oral interview lasting approximately 20-25 minutes, during which several topics selected from the syllabus will be discussed. The final evaluation will include an overall assessment regarding: 1) the level of knowledge about different topics related to the course content and the precision and overall quality of the answers (50% of the final mark); 2) the student’s ability to properly motivate his/her statements and to sustain an adequate discussion (30% of the final mark) and 3) the communication skills shown during the exam session (20% of the final mark). A few questions concerning the laboratory module might be asked as well. The final mark will be calculated on the above mentioned criteria and the exam will be considered passed with a score equal or above the 18/30 threshold mark.
The Course of Recombinant DNA technology is aimed at providing students with the theoretical bases of genetic engineering approaches, coupled to a detailed knowledge of the underlying experimental strategies. This course is organized in two distinct modules, represented by class lessons and a laboratory training section. Class lessons will be focused on a detailed overview of the recombinant DNA methodologies that have been developed in the last four decades, together with a constant reference to the biological problems they were expected to address. Laboratory lessons will provide students the opportunity to autonomously carry out, in a fully equipped molecular biology laboratory, a highly focused experimental plan related to a particular molecular genetics issue. On the basis of the regular and crucial technological support provided by the current recombinant DNA approaches in every facet of modern biotechnologies, this course represents an essential source of knowledge which is in total keeping with the academic purposes of a Bachelor Degree Course in Biotechnology
At the end of the course, attending students are expected to:
• Acquire the knowledge of both theoretical and practical bases of the main methodologies concerning recombinant DNA technologies.
• Demonstrate the ability to properly draw an experimental plan within the context of the current research in the fields of molecular genetics and molecular biology, on the basis of the acquired knowledge.
• Carry out bibliography searches and to synthesize the retrieved informations in oral and/or visual representation.
• Achieve an informed judgment, adequate expertise and communication skills in relation to both the experimental approaches and the main scientific achievements in genome sciences.
• Develop both a critic awareness and ability to analyze and discuss issues related to the course contents and the comprehension skills required to develop and maintain issues related to the acquired knowledge, by means of critical reasoning and problem-solving attitudes.
CLASS LESSONS (5 CFUs, 40 hours)
• DNA as an informational molecule
• Molecular cloning: summary
• Second generation cloning strategies
• Second and third generation plasmid-based cloning vectors. Expression vectors
• High-capacity cloning tools: lambda, P1 and cosmid vectors. PAC, Yeast Artificial Chromosomes (YACs) and Bacterial Artificial Chromosomes (BACs). Viral vectors for eucaryotic cells
• Human artificial chromosomes
• Principles of molecular hybridization assays
• Applications of molecular hybridization techniques – 1: Southern, northern and zoo blot. Colony hybridization
• Applications of molecular hybridization techniques – 2: fluorescence labelling of nucleic acids and fluorescence in situ hybridization (FISH), RNA in situ hybridization (RNA ISH), comparative genomic hybridization (CGH), microarray hybridization
• Introduction and applications of PCR: Mutation screening and detection, genomic/cDNA screening, reverse transcription PCR, cloning by PCR, DOP-PCR.
• Real-time and digital PCR : principles and experimental approaches
• Whole genome amplification by PCR: DOP-WGA and Multiple Displacement Amplification (MDA)
• Genomic DNA libraries: introduction and mode of assembly. Libraries complexity and genome equivalent values. cDNA libraries
• Methods for DNA mutagenesis:
• Gene transfer assays in eucaryotic cells
• Methods for the identification of a gene’s regulatory elements: DNase hypersensitive site’s mapping, gene transfer with reporter vectors, DNA footprinting, Electrophoretic Mobility Shift Assay (EMSA), Chromatin conformation Capture (CCC)
• Introduction to NGS techniques. RNA-Sequencing.
• Introduction to the key model organisms in experimental biology. Key concepts for transgenesis. Methods for producing and analyzing transgenic mice
• Transgenic systems with inducible gene expression
• Gene targeting principles and approaches. Gene “knock-out” e “knock-in” in mouse model systems. Conditional gene knock-out
• Chromosome engineering
• RNA interference assays
• Genome editing by means of and zinc-finger nucleases, TALEN and CRISPR/Cas9 assays
• Genetic maps and molecular markers.
• DNA profiling
LABORATORY LESSONS (1 CFU, 12 hours)
Three practical laboratory training lessons (4 hours each) will be held. The first part of the teaching module is focused on the use of a recombinant DNA molecule previously assembled in the lab session of the Molecular Biology course. By standard cloning techniques, the coding sequence of a human gene has been subcloned into an eucaryotic expression vector. During the Recombinant DNA Technology lab session, the above mentioned recombinant construct will be introduced in cultured human cells by means of transient transfection. Subsequently, the expression of the gene of interest will we evaluated by both fluorescence microscopy on the transfected cells and realtime quantitative PCR. Moreover, a PCR-based gene profile assay on human genomic DNA samples will be carried out in order to simulate a procedure of gene profiling for forensic applications. Students will also be offered a “virtual lab” experience focussed on topics discussed in the classroom by means of 3D viewer devices.
CLASS LESSONS (5 CFUs, 40 hours)
• DNA as an informational molecule
• Molecular cloning: summary
• Second generation cloning strategies
• Second and third generation plasmid-based cloning vectors. Expression vectors
• High-capacity cloning tools: lambda, P1 and cosmid vectors. PAC, Yeast Artificial Chromosomes (YACs) and Bacterial Artificial Chromosomes (BACs). Viral vectors for eucaryotic cells
• Human artificial chromosomes
• Principles of molecular hybridization assays
• Applications of molecular hybridization techniques – 1: Southern, northern and zoo blot. Colony hybridization
• Applications of molecular hybridization techniques – 2: fluorescence labelling of nucleic acids and fluorescence in situ hybridization (FISH), RNA in situ hybridization (RNA ISH), comparative genomic hybridization (CGH), microarray hybridization
• Introduction and applications of PCR: Mutation screening and detection, genomic/cDNA screening, reverse transcription PCR, cloning by PCR, DOP-PCR.
• Real-time and digital PCR : principles and experimental approaches
• Whole genome amplification by PCR: DOP-WGA and Multiple Displacement Amplification (MDA)
• Genomic DNA libraries: introduction and mode of assembly. Libraries complexity and genome equivalent values. cDNA libraries
• Methods for DNA mutagenesis:
• Gene transfer assays in eucaryotic cells
• Methods for the identification of a gene’s regulatory elements: DNase hypersensitive site’s mapping, gene transfer with reporter vectors, DNA footprinting, Electrophoretic Mobility Shift Assay (EMSA), Chromatin conformation Capture (CCC)
• Introduction to NGS techniques. RNA-Sequencing.
• Introduction to the key model organisms in experimental biology. Key concepts for transgenesis. Methods for producing and analyzing transgenic mice
• Transgenic systems with inducible gene expression
• Gene targeting principles and approaches. Gene “knock-out” e “knock-in” in mouse model systems. Conditional gene knock-out
• Chromosome engineering
• RNA interference assays
• Genome editing by means of and zinc-finger nucleases, TALEN and CRISPR/Cas9 assays
• Genetic maps and molecular markers.
• DNA profiling
LABORATORY LESSONS (1 CFU, 12 hours)
Three practical laboratory training lessons (4 hours each) will be held. The first part of the teaching module is focused on the use of a recombinant DNA molecule previously assembled in the lab session of the Molecular Biology course. By standard cloning techniques, the coding sequence of a human gene has been subcloned into an eucaryotic expression vector. During the Recombinant DNA Technology lab session, the above mentioned recombinant construct will be introduced in cultured human cells by means of transient transfection. Subsequently, the expression of the gene of interest will we evaluated by both fluorescence microscopy on the transfected cells and realtime quantitative PCR. Moreover, a PCR-based gene profile assay on human genomic DNA samples will be carried out in order to simulate a procedure of gene profiling for forensic applications. Students will also be offered a “virtual lab” experience focussed on topics discussed in the classroom by means of 3D viewer devices.
The module will be split in standard class lessons (5 CFU) and an experimental laboratory module (1 CFU). Class lessons will be held with the aid of Power Point slide presentation sessions, coupled to projection of didactic movies when required. A set of “brain training” questions will be regularly uploaded in the e-learning platform in order to give the students the opportunity to self-evaluate their level of knowledge and understanding of the topics explained in the classroom. The experimental laboratory module will be held in one of the Experimental Biology Lab at the Department of Biotechnology and Life Sciences, via JH Dunant 3, Varese. Each student will be assigned a workstation endowed will all necessary equipment for the implementation of the proposed experimental plan. A printed tutorial guide will also be distributed to the students. Lab attendance is mandatory for all students, who can skip no more than one lesson. Students are required to attend the laboratory lessons with a personal lab coat. Students with known intolerance or allergy to drugs or chemicals that might be present in a research lab must absolutely inform the teacher before the beginning of the lab module.
The teacher is available under appointment for conversations with the students focused on both topics discussed in the course and organizing issues related to the course, either by phone or e-mail. Students are kindly required not to ask bureaucratic/administrative question unrelated to the course content, if not really urgent.
Telephone: ++39-0332-421512
Email address: francesco.acquati@uninsubria.it
Professors
Borrowers
-
Degree course in: BIOLOGICAL SCIENCES