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)

• 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)

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The 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)

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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

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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

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Thank you for your attention

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plasmid isolation

&

restriction

gel purification

of the transforming

fragment

control gel

electophoresis

yeast

transformationreplate transfomants

yeast genomic DNA

isolation & restriction Southern blotting analyse resultsgel electophoresis

TO BE OR NOT TO

BE ....TRANSFORMED?