Anamarija tafa Ph.D.

lessfrustratedΒιοτεχνολογία

23 Οκτ 2013 (πριν από 3 χρόνια και 9 μήνες)

85 εμφανίσεις

Anamarija
Š
tafa Ph
.
D
.


Laboratory for Biology and Microbial Genetics

Department

of Bi
o
chemical Engineering

Faculty

of Food Technology and Biotechnology

Uni
versity 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
)


s
uitable
for genetic
manipulation

-

first
eukaryotic organism
stabily transformed
with exogenous
non
-
replicative
DNA,
by integration in
to

the
genome,
via homologous recombination
(
Hinnen et al., 1978
)



w
ide application in
biotechnology



production of beer, wine, strong alcohol and dough (classical biotechnology)


p
roduction of insulin, glucagon, somatotropin, interferon and vaccines

(rDNA technology)

3

Introduction to
gene targeting and
ends
-
out recombination




g
ene
targeting


is
a

genetic

technique that uses

homologous recombination

to
modify

an

endogenous

gene


ends
point
away

from each other
(ends
-
out recombination)


t
he
transforming
DNA f
ragment is
supposed to
replace

targeted gene
(gene replacement
)

g
enomic allele after

gene replacement

g
enomic allele

g
ene X


ends
-
out recombination is used for:


inactivation
of genes
(knock
-
out
mutants)


correction
of mutations
(knock
-
in
mutants

=

gene therapy
)

t
he transforming DNA fragment

with selectable marker

s
el ectabl e marker

f
l anki ng homol ogi es

(addresses)

4

Introduction to
gene targeting and
ends
-
out recombination





y
east
Saccharomyces cerevisiae


(
Bailis and Maines,
1996)


p
roteins involved in homologous recombination are evolutionary conserved among eukaryotes
(
Karpenshif and Bernstein, 2012; Krejci et al., 2012;
Aggarwal and Brosh, 2012
)




succes
s
ful 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?


Targeted events

60.0 %


Aberrant genetic events

4
0.0 %

Observed in all

organisms

analysed so far

8.9 %

Random
i
ntegration
of the transforming
DNA

fragment

Addition

of the transforming DNA
fragment
next

to
the homology

10.0 %

21.1 %

Disomic for the chromosome V

*
a
neuploidy

was confirmed by
PFGE

and FACS

Molecular analysis of
transformants


by Southern blotting
(Svetec et al., 2007)

6

Parameters that influence the proportion of
targeted events
?

1. length of flanking homologies
(Bailis and Maines, 1996)


2.
s
ystematic investigation of ends
-
out recombination
(Štafa et al., manuscript in preparation):


type of gene/genome modificatio
n

-

insertion
, replacement, deletion






transformation method


-

lithium
acetate transformation
,

spheroplast transformation and

electroporation

*a
neuploidy

was confirmed

by
PFGE

and FACS

7

Take home message


M
odifying

any

region

in

genome

m
ay

result

in

generation

of

unwante
d

(aberrant)

alterations

(
disomic

transformants


and/or

direct

and

dispersed

repetas)

that

could

easily

go

unnoticed
.


I
t

is

necessary

to

use

molecular

methods

to

confirm

both

the

presence

of

modified

allele

and

the

absence

of

starting

(unmodified)

allele
.



T
he

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
Tomaševi
ć

9

T
hank you for your attention

10

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?