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Genetic side-effects during gene replacement in yeast Saccharomyces cerevisiaeAnamarija tafa Ph.D.

Laboratory for Biology and Microbial GeneticsDepartment of Biochemical EngineeringFaculty of Food Technology and BiotechnologyUniversity of Zagreb

2Svetec groupPalindromes 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 biotechnology production of beer, wine, strong alcohol and dough (classical biotechnology)production of insulin, glucagon, somatotropin, interferon and vaccines (rDNA technology)3Introduction to gene targeting and ends-out recombination gene targeting is agenetictechnique that useshomologous recombinationto modify anendogenousgeneends 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 allelegene Xends-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 markerselectable markerflanking homologies(addresses)4Introduction to gene targeting and ends-out recombinationyeast 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)

5The proportion of targeted events in ends-out assay?Targeted events60.0 % Aberrant genetic events40.0 %Observed in all organisms analysed so far

8.9 %Random integration of the transforming DNA fragment

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

Molecular analysis of transformants by Southern blotting (Svetec et al., 2007)6Parameters that influence the proportion of targeted events?1. length of flanking homologies (Bailis and Maines, 1996)

2. systematic investigation of ends-out recombination (tafa 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 FACS67Take 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.

Acknowledgements:

prof. Ivan-Kreimir Svetec Ph.D. FUNDING: Berislav Lisni Ph.D.Marina Mikleni M.Sc.Bojan unar M.Sc. Dekkera/BrettanomycesNataa Tomaevi

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Thank you for your attention10

plasmid isolation& restrictiongel purification of the transforming fragment control gel electophoresisyeasttransformation

replate transfomants

yeast genomic DNA isolation & restriction Southern blottinganalyse resultsgel electophoresis

TO BE OR NOT TO BE ....TRANSFORMED?