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

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)

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) the transforming DNA fragment with selectable marker selectable marker flanking homologies (addresses) genomic allele gene X genomic allele after gene replacement ends-out recombination is used for: inactivation of genes (knock-out mutants) correction of mutations (knock-in mutants = gene therapy)

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) successful ends-out recombination phylamentous fungi (Paietta and Marzluf, 1985) Trypanosoma brucei (Gibson et al., 1996) Physcomitrella patens (Schaefer and Zyrd, 1996) DT40 cell line (Buerstedde and Takeda, 1991)

The proportion of targeted events in ends-out assay? 60.0 % Molecular analysis of transformants by Southern blotting (Svetec et al., 2007) Aberrant genetic events 40.0 % 8.9 % Random integration of the transforming DNA fragment Observed in all organisms analysed so far Addition of the transforming DNA fragment next to the homology 10.0 % 21.1 % Disomic for the chromosome V *aneuploidy was confirmed by PFGE and FACS

*aneuploidy was confirmed Parameters that influence the proportion of targeted events? 1. length of flanking homologies (Bailis and Maines, 1996) 2. systematic investigation of ends-out recombination (Štafa et al., manuscript in preparation): type of gene/genome modification - insertion, replacement, deletion transformation method - lithium acetate transformation, spheroplast transformation and electroporation *aneuploidy was confirmed by PFGE and FACS

Take home message Modifying any region in genome may result in generation of unwanted (aberrant) alterations (disomic transformants and/or direct and dispersed repetas) that could easily go unnoticed. It is necessary to use molecular methods to confirm both the presence of modified allele and the absence of starting (unmodified) allele. The transforming DNA fragments that insert or replace, rather than delete, result in lower percentage of aberrant events.

Acknowledgements: prof. Ivan-Krešimir Svetec Ph.D. FUNDING: Berislav Lisnić Ph.D. Marina Miklenić M.Sc. Bojan Žunar M.Sc. Dekkera/Brettanomyces Nataša Tomašević

Thank you for your attention

isolation & restriction plasmid isolation & restriction gel purification of the transforming fragment control gel electophoresis yeast transformation replate transfomants yeast genomic DNA isolation & restriction Southern blotting analyse results gel electophoresis TO BE OR NOT TO BE ....TRANSFORMED?