Genetic side-effects during gene replacement in yeast Saccharomyces cerevisiae Anamarija Štafa Ph.D. Laboratory for Biology and Microbial Genetics Department of Biochemical Engineering Faculty of Food Technology and Biotechnology University of Zagreb
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Anamarija Štafa Ph.D. Laboratory for Biology and Microbial Genetics Department of Biochemical Engineering Faculty of Food Technology and Biotechnology.
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Genetic side-effects during gene replacement in
yeast Saccharomyces cerevisiae
Anamarija Štafa Ph.D.
Laboratory for Biology and Microbial GeneticsDepartment of Biochemical Engineering
Faculty of Food Technology and BiotechnologyUniversity of Zagreb
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Svetec group
”Palindromes in genomes and mechanisms of gene targeting in yeast”
Yeast Saccharomyces cerevisiae• first eukaryotic organism sequenced (Goffeau et al., 1996)
• suitable for genetic manipulation - first eukaryotic organism stabily transformed with exogenous non-replicative DNA, by integration into the genome, via homologous recombination (Hinnen et al., 1978)
• wide application in biotechnology
• production of beer, wine, strong alcohol and dough (classical biotechnology)
• production of insulin, glucagon, somatotropin, interferon and vaccines (rDNA technology)
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Introduction to gene targeting and ends-out recombination
• gene targeting is a genetic technique that uses homologous recombination to modify an endogenous gene
• ends point away from each other (ends-out recombination)
• the transforming DNA fragment is supposed to replace targeted gene (gene replacement)
genomic allele after gene replacement
genomic allele gene X
• ends-out recombination is used for:• inactivation of genes (knock-out mutants)• correction of mutations (knock-in mutants = gene therapy)
the transforming DNA fragment with selectable marker
selectable marker
flanking homologies(addresses)
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Introduction to gene targeting and ends-out recombination
• yeast Saccharomyces cerevisiae (Bailis and Maines, 1996)
• proteins involved in homologous recombination are evolutionary conserved among eukaryotes (Karpenshif and Bernstein, 2012; Krejci et al., 2012; Aggarwal and Brosh, 2012)