1. 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

2. 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)

3. 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)

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 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)

5. 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

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, deletion
transformation method
- lithium acetate transformation, spheroplast transformation and electroporation
*aneuploidy was confirmed
by PFGE and FACS

7. 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.

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

9. Thank you for your attention

10. 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?