Download 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 nonreplicative 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)
2
Introduction to gene targeting and ends-out recombination
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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)
flanking homologies
(addresses)
the transforming DNA fragment
with selectable marker
selectable marker
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)
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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?
60.0 %
40.0 %
8.9 %
Targeted events
Aberrant genetic events
Molecular analysis of transformants
by Southern blotting (Svetec et al., 2007)
Random integration of the transforming DNA
fragment
10.0 %
Addition of the transforming DNA
fragment next to the homology
21.1 %
Disomic for the chromosome V
*aneuploidy was confirmed by PFGE and FACS
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Observed in all
organisms
analysed so far
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):
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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.
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Acknowledgements:
prof. Ivan-Krešimir Svetec Ph.D.
FUNDING:
Berislav Lisnić Ph.D.
Marina Miklenić M.Sc.
Bojan Žunar M.Sc.
Nataša Tomašević
Dekkera/Brettanomyces
Thank you for your attention
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plasmid isolation
gel purification
&
of the transforming
restriction
fragment
control gel
yeast
electophoresis
transformation
replate transfomants
TO BE OR NOT TO BE
....TRANSFORMED?
yeast genomic DNA
isolation & restriction
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gel electophoresis
Southern blotting
analyse results