Presentation on theme: "Anamarija Štafa Ph.D. Laboratory for Biology and Microbial Genetics"— Presentation transcript:
1 Genetic side-effects during gene replacement in yeast Saccharomyces cerevisiae Anamarija Štafa Ph.D.Laboratory for Biology and Microbial GeneticsDepartment of Biochemical EngineeringFaculty of Food Technology and BiotechnologyUniversity of Zagreb
2 Svetec group”Palindromes in genomes and mechanisms of gene targeting in yeast”Yeast Saccharomyces cerevisiaefirst 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 biotechnologyproduction of beer, wine, strong alcohol and dough (classical biotechnology)production of insulin, glucagon, somatotropin, interferon and vaccines (rDNA technology)
3 Introduction to gene targeting and ends-out recombination gene targeting is a genetic technique that uses homologous recombination to modify an endogenous geneends point away from each other (ends-out recombination)the transforming DNA fragment is supposed to replace targeted gene (gene replacement)the transforming DNA fragmentwith selectable markerselectable markerflanking homologies(addresses)genomic allelegene Xgenomic allele aftergene replacementends-out recombination is used for:inactivation of genes (knock-out mutants)correction of mutations (knock-in mutants = gene therapy)
4 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 recombinationphylamentous fungi (Paietta and Marzluf, 1985)Trypanosoma brucei (Gibson et al., 1996)Physcomitrella patens (Schaefer and Zyrd, 1996)DT40 cell line (Buerstedde and Takeda, 1991)
5 The proportion of targeted events in ends-out assay? 60.0 %Molecular analysis of transformantsby Southern blotting (Svetec et al., 2007)Aberrant genetic events40.0 %8.9 %Random integration of the transforming DNA fragmentObserved in allorganismsanalysed so farAddition of the transforming DNA fragment next to the homology10.0 %21.1 %Disomic for the chromosome V*aneuploidy was confirmed by PFGE and FACS
6 *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, deletiontransformation method- lithium acetate transformation, spheroplast transformation and electroporation*aneuploidy was confirmedby PFGE and FACS
7 Take home messageModifying 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.
10 isolation & restriction plasmid isolation&restrictiongel purificationof the transformingfragmentcontrol gelelectophoresisyeasttransformationreplate transfomantsyeast genomic DNAisolation & restrictionSouthern blottinganalyse resultsgel electophoresisTO BE OR NOT TO BE ....TRANSFORMED?