KBC/CGI Cloning and Genetic Engineering
Lecturer: Petr Galuszka
Lecture: 2 hours/week + seminar 1 hour/week
Credits: 3
Summer semester
Form of course completion: colloquium
The tools used for gene cloning – restriction endonucleases, DNA polymerases, DNA ligase and reverse transcriptase, plasmid and cloning vectors. PCR techniques: the principle, optimization, types and applications. Mutagenesis in vitro, directed changes in the genetic material, random mutagenesis, mutagenesis using mutated oligonucleotides, site-directed mutagenesis, cassette mutagenesis, suppressor tRNA technique. Preparation and application of cDNA and genomic libraries. Gene chips, DNA microarrays, Affymetrix chips. Cloning strategies: optimization of the expression of the cloned genes, factors influencing the expression in heterologous host, selection markers. Gene cloning in prokaryotes. Gene cloning in yeast and its application for the analysis of eukaryotic genome. Methods of gene delivery into eukaryotic cells (microinjection, electroporation, transfection, vectors, biolistic methods). Gene cloning in plants and its applications. Transformation using Ti-derived plasmids. RNAi gene silencing and gene knock-out. Gene cloning in animal cells. Transfer of genes into embryonic cells of insects, amphibians and mammals. Applications of recombinant DNA technology in agriculture, industry and medicine. Bt maize, DNA vaccines and plantbiotics, biocontrol of the environment, protection against bioterrorism, etc. Transgenic animals: Dolly and Polly, Cumulina, human proteins, human genome cloning. Gene therapy, main strategies in vitro and in vivo. Cloning using stem cells. Preparation of trangenic organisms. Risks connected with the transgenic organisms, safety measures. Ethic problems connected with gene transfer between different species and preparation of transgenic organisms. GMO legislative.
The seminar focuses on learning and discussion of cloning strategies. Each student will prepare own strategy for functional expression of a given gene. Students are instructed how to use software for molecular genetic analysis, sequence editing and comparison, primer design and restriction analysis. They will learn how to use genetic databases available on the Internet.
