Single-cell cloning enables the selection of more productive Drosophila melanogaster S2 cells for recombinant protein expression

Abstract

The generation of monoclonal cell lines is an important early process development step for recombinant protein production. Although single-cell cloning is an established method in mammalian cell lines, straightforward protocols are not yet available for insect cells. We describe a new method for the generation of monoclonal insect cells without using fetal bovine serum and/or feeder cells pretreated by irradiation or exposure to mitomycin. Highly productive clones of Drosophila melanogaster S2 cells were prepared in a two-step procedure, comprising the establishment of a polyclonal population and subsequent single cell isolation by limiting dilution. Necessary growth factors were provided by co-cultivation of single transformants with untransfected feeder cells, which were later removed by antibiotic selection. Enhanced expression of EGFP and two target peptides was confirmed by flow cytometry and dot/western blotting. Highly productive clones were stable, showed a uniform expression profile and typically a sixfold to tenfold increase in cell-specific productivity.

Full text

Single-cell
cloning
enables
the
selection
of
more
productive
Drosophila
melanogaster
S2
cells
for
recombinant
protein
expression
Jan
Zitzmann
a
,
Christine
Schreiber
a
,
Joel
Eichmann
a
,
Roberto
Otmar
Bilz
a
,
Denise
Salzig
a
,
Tobias
Weidner
a
,
Peter
Czermak
a,b,c,d,
*
a
Institute
of
Bioprocess
Engineering
and
Pharmaceutical
Technology,
University
of
Applied
Sciences
Mittelhessen,
Giessen,
Germany
b
Department
of
Chemical
Engineering,
Kansas
State
University,
Manhattan
KS,
USA
c
Faculty
of
Biology
and
Chemistry,
Justus-Liebig
University
of
Giessen,
Germany
d
Fraunhofer
Institute
for
Molecular
Biology
and
Applied
Ecology
(IME),
Project
group
Bioresources,
Giessen,
Germany
A
R
T
I
C
L
E
I
N
F
O
Article
history:
Received
24
April
2018
Received
in
revised
form
18
June
2018
Accepted
22
June
2018
Keywords:
Single-cell
cloning
Stably
transformed
D.
melanogaster
S2
cells
Recombinant
protein
expression
Monoclonal
cell
line
Insect
cell
culture
A
B
S
T
R
A
C
T
The
generation
of
monoclonal
cell
lines
is
an
important
early
process
development
step
for
recombinant
protein
production.
Although
single-cell
cloning
is
an
established
method
in
mammalian
cell
lines,
straightforward
protocols
are
not
yet
available
for
insect
cells.
We
describe
a
new
method
for
the
generation
of
monoclonal
insect
cells
without
using
fetal
bovine
serum
and/or
feeder
cells
pretreated
by
irradiation
or
exposure
to
mitomycin.
Highly
productive
clones
of
Drosophila
melanogaster
S2
cells
were
prepared
in
a
two-step
procedure,
comprising
the
establishment
of
a
polyclonal
population
and
subsequent
single
cell
isolation
by
limiting
dilution.
Necessary
growth
factors
were
provided
by
co-
cultivation
of
single
transformants
with
untransfected
feeder
cells,
which
were
later
removed
by
antibiotic
selection.
Enhanced
expression
of
EGFP
and
two
target
peptides
was
confirmed
by
flow
cytometry
and
dot/western
blotting.
Highly
productive
clones
were
stable,
showed
a
uniform
expression
profile
and
typically
a
sixfold
to
tenfold
increase
in
cell-specific
productivity.
©
2018
The
Authors.
Published
by
Elsevier
B.V.
This
is
an
open
access
article
under
the
CC
BY-NC-ND
license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
1.
Introduction
Stably
transformed
Drosophila
melanogaster
S2
cells
(rS2)
have
emerged
as
a
key
platform
for
recombinant
protein
expression,
and
several
related
products
have
already
entered
clinical
trials
[1,2].
Like
other
frequently
used
expression
systems
based
on
mamma-
lian
cell
lines
or
baculovirus
vectors,
rS2
cell
lines
must
undergo
comprehensive
optimization
during
process
development
[2].
This
not
only
includes
the
optimization
of
transfection
conditions
[3,4],
but
also
the
selection
of
highly
productive
subpopulations
[5–7]
or
clonal
derivatives
[8–10].
Although
single-cell
cloning
is
the
state
of
the
art
in
mammalian
cell
lines
[11–13],
the
same
approach
in
stably
transformed
S2
cells
is
controversial,
as
highlighted
by
the
following
statements
in
the
literature:
“[
.
.
.
]
the
additional
effort
for
cloning
does
not
seem
worthwhile,
because
the
levels
of
expression
reported
for
high-expressing
clones
[
.
.
.
]
appear
to
be
similar
to
those
expected
from
a
polyclonal
population
[
.
.
.
]
“[14]
“[
.
.
.
]One
can,
with
a
little
more
effort,
clone
the
selected
cells;
the
resulting
clonal
lines
are
generally
more
nearly
homogeneous,
and
individual
clones
may
differ
sufficiently
in
their
properties
that
the
experimenter
can
choose
a
clone
most
suitable
for
his
purposes.[
.
.
.
]”
[15]
Despite
the
ongoing
discussion,
several
protocols
for
single-cell
cloning
are
available
for
insect
cell
lines
(Table
1).
The
methods
differ
in
terms
of
the
separation
technology
(limiting
dilution
or
plating
in
soft
agar)
and
the
method
used
to
provide
essential
autocrine
growth
factors,
for
example
by
adding
fetal
bovine
serum
(FBS),
conditioned
medium
or
pre-treated
feeder
cells.
The
latter
aspect
is
important
because
S2
cells
cease
proliferation
when
seeded
at
low
density,
which
reflects
their
demand
for
high
concentrations
of
autocrine
growth
factors
[16].
Non-transfected,
Abbreviations:
AMP,
antimicrobial
peptide/protein;
BR021,
Harmonia
axyridis
antimicrobial
peptide
BR021;
BSA,
bovine
serum
albumin;
DMSO,
dimethyl
sulfoxide;
EGFP,
enhanced
green
fluorescent
protein;
FACS,
fluorescence
activated
cell
sorting;
FBS,
fetal
bovine
serum;
GmGlv,
Galleria
mellonella
antimicrobial
peptide
Gloverin;
GMP,
good
manufacturing
practice;
OD
600
,
optical
density
at
600nm;
PBS,
phosphate-buffered
saline;
PCR,
polymerase
chain
reaction;
PVDF,
polyvinylidene
difluoride;
RMCE,
recombinase
mediated
cassette
exchange;
rS2,
recombinant
Drosophila
melanogaster
Schneider
2
cells;
SDS-PAGE,
sodium
dodecylsulfate
polyacrylamide
gel
electrophoresis;
Sf9,
clonal
isolate
of
Spodoptera
frugiperda
Sf21
cells;
SFM,
serum
free
medium.
*
Corresponding
author
at:
Institute
of
Bioprocess
Engineering
and
Pharmaceu-
tical
Technology,
University
of
Applied
Sciences
Mittelhessen,
Giessen,
Germany.
E-mail
address:
peter.czermak@lse.thm.de
(P.
Czermak).
https://doi.org/10.1016/j.btre.2018.e00272
2215-017X/©
2018
The
Authors.
Published
by
Elsevier
B.V.
This
is
an
open
access
article
under
the
CC
BY-NC-ND
license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Biotechnology
Reports
19
(2018)
e00272
Contents
lists
available
at
ScienceDirect
Biotechnology
Reports
journal
homepage:
www.else
vie
r.com/locat
e/btre

irradiated
feeder
cells
have
often
been
used
to
support
the
growth
of
S2
cells
[17]
but
the
necessary
X-ray
or
g
sources
are
not
readily
available
in
every
cell
culture
laboratory
and
irradiation
requires
extensive
empirical
testing
to
ensure
the
complete
inactivation
of
the
feeder
cells
(typically
wild-type
S2
cells)
while
maintaining
their
viability.
The
same
restrictions
apply
when
using
mitomycin
C
to
chemically
block
cell
division.
Although
all
the
methods
shown
in
Table
1
can
successfully
yield
monoclonal
1
rS2
lines,
the
specific
protocols
are
often
cumber-
some
or
inefficient.
We
encountered
difficult
handling
when
using
soft
agar
and
found
only
a
poor
growth
support
when
working
with
conditioned
or
FBS-supplemented
medium
instead
of
feeder
cells.
To
overcome
this
challenge,
we
combined
the
limiting
dilution
of
polyclonal
transformants
and
co-cultivation
with
untreated
feeder
cells
with
a
second
round
of
antibiotic
selection.
This
simple
approach
has
been
used
before,
but
was
only
reported
peripherally
in
the
context
of
other
research
[9,10].
Within
the
previous
works
either
only
a
small
number
of
clones
was
generated
(e.g.
n
=
8
in
[9])
or
a
comparison
between
different
clones
and
the
parental
population
was
missing.
Here
we
provide
a
comprehensive
analysis
of
the
protocol
to
confirm
that
it
offers
a
simple,
robust
and
broadly
available
replacement
for
traditional
single-cell
cloning
methods.
In
contrast
to
most
of
the
older
methods,
the
new
protocol
enabled
the
preparation
of
monoclonal
cell
lines
in
a
completely
serum-free
environment,
which
is
highly
desirable
in
context
of
good
manufacturing
practice
(GMP)-compliant
cell
line
development.
2.
Materials
and
methods
2.1.
Construction
of
expression
plasmids
for
the
generation
of
recombinant
S2
cells
The
recombinant
S2
cells
were
generated
either
by
the
transfection
with
a
single
plasmid
containing
an
expression
cassette
and
a
selection
cassette
or
by
co-transfection
with
two
separate
plasmids
(Fig.
1).
Both
systems
are
reliable
for
the
stable
transformation
of
S2
cells
[17,29]
and
were
used
here
to
produce
different
proteins.
Enhanced
green
fluorescent
protein
(EGFP)
was
used
as
a
fluorescent
reporter
for
the
establishment
and
investigation
of
the
limiting
dilution
assay,
whereas
the
antimi-
crobial
peptides
(AMPs)
Galleria
mellonella
gloverin
(GmGlv)
[8,30]
and
BR021
[31]
were
used
as
representative
target
molecules.
2.1.1.
Plasmid
construction
by
Golden
Gate
assembly
The
Golden
Gate
(GG)
assembly
of
expression
plasmids
for
cell
lines
1,
2
and
4
was
conducted
as
previously
described
[32].
Corresponding
donor
and
acceptor
plasmids
were
synthesized
by
GenScript
(Piscataway,
New
Jersey,
USA)
or
were
already
part
of
an
existing
plasmid
library
[32].
The
reaction
volume
was
20
mL,
comprising
40
fmol
of
each
plasmid,
20
U
T4
DNA
ligase
(Promega,
Mannheim,
Germany),
2
mL
of
the
corresponding
T4
DNA
ligase
buffer
(Promega)
and
10
U
BsaI
(NEB,
Frankfurt
am
Main,
Germany).
The
GG
mix
was
incubated
in
a
PCR
cycler
(PEQLAB,
Erlangen,
Germany)
at
37

C
for
15
min,
followed
by
30
cycles
at
37

C
(2
min)
and
16

C
(5
min).
Subsequently,
the
enzymes
were
heat-inactivated
at
50

C
for
15
min
and
65

C
for
5
min.
Finally,
5
mL
of
the
GG
mix
was
introduced
into
chemically
competent
E.
coli
NEB
10-β
cells
(NEB)
as
described
in
Section
2.1.3.
2.1.2.
Plasmid
construction
by
classical
restriction-ligation
cloning
For
cell
line
3,
we
used
the
commercially
available
DES
1
plasmids
pMT/BiP/V5-His
B
and
pCoBlast
(Thermo
Fisher
Scientific,
Darmstadt,
Germany).
The
BR021
sequence
was
amplified
by
PCR
using
primers
to
introduce
a
C-terminal
thrombin
cleavage
site
as
well
as
BglII
and
XhoI
restriction
sites
(all
primer
sequences
are
provided
in
Supplementary
Material
1).
Following
digestion
with
BglII
and
XhoI
(NEB)
and
agarose
gel
electrophoresis
of
the
backbone,
the
insert
and
the
backbone
were
purified
using
the
GeneJet
Gel
Extraction
Kit
(Thermo
Fisher
Scientific)
according
to
the
manufacturer’s
instructions.
Subsequent
ligation
was
carried
out
with
Instant
Sticky
End
Ligase
Mix
(NEB)
using
100
ng
of
the
backbone
and
23
ng
of
the
insert.
2.1.3.
Plasmid
propagation,
verification
and
purification
Following
the
construction
of
the
plasmids,
5
mL
of
the
resulting
DNA
solution
was
added
to
80
mL
of
lysogeny
broth
(LB)
medium
containing
chemically
competent
E.
coli
NEB
10-β
cells.
The
mixture
was
incubated
for
15
min
on
ice,
before
a
heat
shock
(42

C
for
60
s)
to
facilitate
DNA
uptake.
After
incubation
on
ice
for
a
further
5
min,
we
added
250
mL
LB
medium.
The
cells
were
allowed
to
recover
at
37

C
for
1
h,
while
shaking
at
110 0
rpm
in
a
thermomixer
(Eppendorf,
Hamburg,
Germany).
The
trans-
formants
were
plated
on
selective
LB
agar
containing
either
10
mg/
mL
bleomycin
(GG-plasmids)
or
100
mg/mL
ampicillin
(DES
1
plasmids).
After
incubation
for
1
day
at
37

C,
colonies
were
picked
and
propagated
in
3
mL
selective
LB
medium
to
enable
small-scale
plasmid
isolation
with
the
NucleoSpin
Plasmid
EasyPure
kit
(Macherey–Nagel,
Düren,
Germany).
The
resulting
plasmid
stocks
were
digested
and
sequenced
by
Microsynth
Seqlab
(Göttingen,
Germany)
to
confirm
the
correct
integration
of
the
gene
of
interest.
Raw
material
for
large-scale
plasmid
purification
was
generated
by
culturing
the
transformed
E.
coli
stains
in
200-mL
shake
flask
cultures.
Plasmids
were
recovered
by
alkaline
lysis
followed
by
purification
using
the
NucleoBond
1
Xtra
Midi-Kit
(Macherey–
Nagel)
according
to
the
manufacturer’s
instructions.
The
pure
DNA
was
precipitated
with
isopropanol,
washed
with
70%
ethanol
and
resuspended
in
sterile
water
at
a
final
concentration
of
1
mg/mL,
as
determined
by
absorption
at
260/280
nm
using
a
Cytation
3
spectrophotometer
(BioTek,
Bad
Friedrichshall,
Germany).
Table
1
Summary
of
previously
reported
techniques
for
the
generation
of
monoclonal
insect
cell
lines;
(a)
indicates
the
use
of
fetal
bovine
serum
(FBS)
in
the
medium
during
cloning,
(b)
indicates
the
use
of
serum
free
medium
(SFM).
Method
Source
of
growth
factors
Cell
lines
Ref.
Limiting
dilution
Conditioned
medium
Drosophila
melanogaster
S2,
Spodoptera
frugiperda
Sf9
[18]
(a)
[19]
(a)
Feeder
cells
-
mitomycin
C
D.
melanogaster
S2
[20]
(a)
Feeder
cells
-
irradiated
D.
melanogaster
S2
[21,17,22]
(a,b)
Feeder
cells
-
spatially
separated
D.
melanogaster
imaginal
disc
[23]
(a)
Feeder
cells
-
untreated,
living
D.
melanogaster
S2
[8,9,10,24,25,26]
(a,b)
Soft
agar
Conditioned
medium
D.
melanogaster
S2
[18,27 ]
(a)
Feeder
cells
-
irradiated
D.
melanogaster
S2
[17,28]
(a)
1
In
the
present
work,
the
term
monoclonal
cell
line
designates
a
group
of
identical
cells
derived
from
a
single
parental
cell.
2
J.
Zitzmann
et
al.
/
Biotechnology
Reports
19
(2018)
e00272

2.2.
Maintenance
of
D.
melanogaster
S2
cells
The
D.
melanogaster
S2
cells
were
grown
at
27

C
in
ExCell
420
(Sigma-Aldrich,
Taufkrichen,
Germany)
or
Sf-900
II
serum
free
medium
(Thermo
Fisher
Scientific)
containing
8–10
mM
L-gluta-
mine
(Biochrom,
Berlin,
Germany).
For
strain
maintenance,
the
cells
were
split
every
3–4
days
to
a
density
of
1.5 10
6
cells/mL.
Unless
otherwise
stated,
stable
rS2
cells
were
handled
under
selection
pressure
by
the
addition
of
10–25
mg/mL
blasticidin
S
or
300
mg/mL
hygromycin
B
(both
supplied
by
Invivogen,
Toulouse,
France).
Long-term
preservation
was
achieved
by
freezing
1.5 10
7
cells
in
1
mL
of
a
1:1
mixture
of
spent
and
fresh
medium
with
7.5%
dimethyl
sulfoxide
(DMSO)
and
subsequent
storage
in
liquid
nitrogen.
Clones
were
not
weaned
off
antibiotics
before
being
frozen.
However,
we
omitted
antibiotics
in
the
freezing
medium
and
for
the
first
passage
after
thawing
to
allow
cell
recovery.
DMSO
was
removed
after
thawing
by
pelleting
the
cells
and
resuspending
them
in
DMSO
free
medium.
2.3.
Transfection
of
S2
cells
and
construction
of
polyclonal
cell
lines
Wild-type
S2
cells
were
transfected
using
the
TransitIT
1
-Insect
reagent
according
to
the
manufacturer’s
specifications
(Mirus
Bio
LCC,
Madison,
Wisconsin,
USA).
Briefly,
the
cells
were
seeded
18–
24
h
before
transfection
at
a
density
of
1.5 10
6
cells/mL.
Plasmid
DNA
was
mixed
with
Grace’s
Insect
medium
(Sigma-Aldrich)
and
pre-warmed
TransitIT
1
-Insect
at
the
ratios
indicated
in
Fig.
1.
The
resulting
transfection
mixture
was
incubated
for
30
min
at
room
temperature
to
allow
complex
formation,
before
drop-wise
distribution
throughout
the
culture
vessel.
According
to
the
observed
viability,
the
cells
were
allowed
to
recover
for
3–7
days
without
antibiotics.
Selection
was
then
started
by
adding
blasticidin
S
or
hygromycin
B.
2.4.
Single-cell
cloning
by
limiting
dilution
Once
stable
expression
of
the
polyclonal
population
was
verified
by
His
6
-specific
western
blot
or
flow
cytometry
(3–7
weeks
after
transfection),
single-cell
cloning
was
initiated
with
standard
growth
medium
containing
10–15
transfected
cells/mL
and
510
5
untreated,
living
S2
feeder
cells/mL.
For
limiting
dilution,
100
mL/well
was
transferred
to
10
96-well
suspension
plates
(Eppendorf)
in
order
to
seed
statistically
1–1.5
cells
per
well.
Undisturbed
co-cultivation
for
1–3
days
allowed
the
cells
to
proliferate
before
the
second
round
of
selection
commenced
by
the
addition
of
30
mL
growth
medium
supplemented
with
hygromycin
B
or
blasticidin
S
(final
concentration
300
mg/mL
or
25
mg/mL,
respectively).
Clonal
colonies
formed
on
the
decaying
layer
of
feeder
cells
over
the
next
few
weeks.
Colonies
>
1
mm
in
diameter
(confirmed
by
microscopy)
were
picked
by
pipetting
and
transferred
to
another
96-well
plate
containing
50
mL
fresh
medium.
Clonal
cell
lines
were
subsequently
scaled
up
according
to
the
conditions
shown
in
Table
2,
tested
for
protein
expression
and
finally
preserved
in
liquid
nitrogen.
2.5.
Analysis
of
EGFP
expression
by
flow
cytometry
EGFP
expression
was
analyzed
using
an
easyCyte
HT
flow
cytometer
and
the
corresponding
InCyte
software
(Merck
Milli-
pore,
Darmstadt,
Germany).
During
analysis,
dead
cells
were
excluded
by
counterstaining
with
5
mg/L
propidium
iodide
(Carl
Roth,
Karlsruhe,
Germany).
Fig.
1.
Overview
of
techniques
and
corresponding
plasmids
for
the
generation
of
recombinant
S2
cell
lines
(upper
panel).
Abbreviations:
MT:
Drosophila
melanogaster
metallothionein
promoter,
Ac5:
D.
melanogaster
actin
5C
promoter,
Copia:
promoter
from
D.
melanogaster
copia
LTR-retrotransposon,
BIP:
Bip-secretion
signal,
EGFP:
enhanced
green
fluorescent
protein,
rbG:
rabbit
beta-globulin
polyadenylation
signal,
SV40:
Simian
virus
40
polyadenylation
signal,
Thrombin
C
/Thr
C
:
thrombin
cleavage
site,
His
6
:
polyhistidine
tag,
Hygro
R
:
hygromycin
B
resistance,
Blast
R
:
blasticidin
S
resistance.
Overview
of
corresponding
transfection
conditions
(lower
panel).
J.
Zitzmann
et
al.
/
Biotechnology
Reports
19
(2018)
e00272
3

2.6.
Analysis
of
AMP
expression
by
dot
blot
screening
and
western
blot
analysis
The
expression
of
GmGlv
and
BR021
was
analyzed
after
3
days
of
cultivation
at
the
24-well
scale.
Supernatants
from
clonal
derivatives
of
cell
line
2
were
used
directly,
because
protein
expression
was
driven
by
the
constitutive
Ac5
promoter.
Protein
expression
in
clones
originating
from
cell
lines
3
and
4
was
driven
by
the
metallothionein
promoter,
which
had
to
be
induced
with
CuSO
4
.
Therefore,
75
mL
of
the
cell
suspension
was
mixed
with
75
mL
fresh
medium
to
yield
a
final
concentration
of
600
mM
CuSO
4
.
Induced
cells
were
cultivated
for
3
days
in
a
96-
well
plate,
before
the
supernatants
were
collected.
Before
harvest,
the
optical
density
at
600
nm
(OD
600
)
was
determined
as
a
measure
of
the
current
cell
density
using
a
Cytation
3
spectro-
photometer
(Biotek).
Protein
expression
was
detected
by
vacuum-
assisted
dot
blots
specific
for
the
His
6
tag
using
a
VWR
dot
blot
device
(Darmstadt,
Germany).
We
spotted
50
mL
of
the
collected
supernatants
onto
an
Amersham
Protan
0.2
mm
nitrocellulose
membrane
(GE
Healthcare,
Freiburg,
Germany)
allowing
a
binding
phase
of
30
min.
Membranes
were
blocked
with
phosphate-
buffered
saline
(PBS)
containing
5%
bovine
serum
albumin
(BSA),
stained
overnight
with
a
His
5
-HRP
antibody
conjugate
(Qiagen,
Hilden,
Germany)
diluted
1:5000
in
PBS
containing
0.05%
Tween-
20,
and
washed
three
times
with
PBS
containing
0.1%
Tween-20.
The
target
was
detected
by
enhanced
chemiluminescence
using
the
ChemiDoc
TM
system
and
Clarity
Western
ECL
substrate
(Bio-
Rad,
Munich,
Germany).
For
cell
line
screening,
we
calculated
the
quotient
of
the
dot
blot
intensity
and
OD
600
.
Supernatants
from
promising
clones
were
also
analyzed
by
western
blot
to
confirm
the
correct
expression
of
the
target
protein.
Therefore,
proteins
were
resolved
by
sodium
dodecyl
sulfate
polyacrylamide
gel
electrophoresis
(SDS-PAGE)
under
reducing
conditions
on
Criter-
ionXT
4–20%
polyacrylamide
gradient
gels
and
detected
using
stain-free
technology
(Bio-Rad).
Proteins
were
then
transferred
to
polyvinylidene
difluoride
membranes
(PVDF
Trans-Blot1
Turbo
TM
Biorad,
7
min
at
25
V
and
2.5
A,
Trans-Blot1
Turbo
TM
)
and
detected
according
to
the
dot
blot
staining
protocol.
Finally,
the
relative
cell-specific
productivity,
defined
as
western
blot
intensi-
ty/OD
600
,
was
calculated
to
compare
selected
clones
and
the
parental
cell
line.
2.7.
Analysis
of
genomic
DNA
by
Southern
blot
hybridization
In
order
to
identify
the
plasmid
integration
pattern
of
one
EGFP-
expressing
clone,
we
used
the
North2South
TM
Biotin
Random
Prime
Labeling
Kit
and
the
North2South
TM
Chemiluminescent
Hybridiza-
tion
and
Detection
Kit
(Thermo
Fisher
Scientific).
Based
on
the
EGFP
expression
plasmid
and
a
specific
set
of
primers
(Supple-
mentary
Material
1),
we
used
a
Q5
polymerase
(NEB)
for
PCR
to
construct
a
biotinylated
Southern
blot
probe
specific
for
the
EGFP
sequence.
Genomic
DNA
from
rS2
cells
was
isolated
using
the
PureLink
Genomic
DNA
Mini
Kit
(Thermo
Fisher
Scientific)
according
to
the
manufacturer’s
protocol.
We
digested
9
mg
of
purified
DNA
overnight
at
37

C
using
100
U
of
EcoRI-HF
in
CutSmart
buffer
(NEB)
in
a
total
reaction
volume
of
400
mL.
The
digest
was
precipitated
by
the
addition
of
0.1
volumes
of
3
M
sodium
acetate
and
2.5
volumes
of
ice-cold
ethanol
for
2
h
at
80

C.
The
DNA
was
then
pelleted
by
centrifugation
for
20
min
at
16,10 0
g
and
4

C.
The
resulting
pellet
was
air
dried
and
resuspended
in
15
mL
TE
buffer
(10
mM
Tris,
1
mM
EDTA,
pH
8.0).
The
mixture
was
resolved
by
0.8%
agarose
gel
electrophoresis
at
35
V
for
5.5
h.
In
order
to
fragment
large
DNA
molecules
before
blotting,
the
DNA
in
the
gel
was
depurinated
for
10
min
in
0.25
M
HCl.
For
neutraliza-
tion,
the
gel
was
then
incubated
for
30
min
in
neutralization
buffer
(0.5
M
TrisHCl
pH
7.0,
1.5
M
NaCl).
Single-stranded
DNA
was
produced
by
two
denaturing
steps
(each
15
min,
1.5
M
NaCl,
0.5
M
NaOH).
For
the
capillary
blot,
the
blotting
reservoirs
were
filled
with
transfer
buffer
(20x
SSC:
1.5
M
NaCl,
0.15
M
sodium
citrate,
pH
7.0)
and
the
gel
was
placed
together
with
blotting
paper
below
a
Biodyne
B
Nylon
membrane
(Thermo
Fisher
Scientific).
DNA
was
allowed
to
migrate
overnight,
before
the
membrane
was
collected,
washed
in
5x
SCC
buffer
and
DNA
was
crosslinked
to
the
membrane
by
15
min
exposure
to
UV
light.
After
a
final
drying
step
for
3
h,
we
detected
DNA
fragments
containing
the
EGFP
sequence
by
staining
with
the
biotinylated
Southern
blot
probe
according
to
the
North2South
Chemiluminescent
Hybridization
and
Detection
Kit
(Thermo
Fisher
Scientific).
2.8.
Bioreactor-scale
production
The
best
clones
were
cultivated
at
the
1-L
bioreactor
scale
(Labfors
5,
Infors
HT,
Bottmingen,
Switzerland)
as
previously
described
[8].
For
batch
cultivation,
1.5 10
6
cells/mL
were
seeded
and
cultivated
without
antibiotics
at
27

C,
pH
6.4
and
dO
2set
40%.
If
necessary,
cells
were
induced
at
the
mid-exponential
growth
phase
using
600
mM
CuSO
4
.
Harvest
commenced
at
the
onset
of
the
stationary
phase
and
was
timed
using
a
capacitive
biomass
sensor
(Incyte
Arc
View
system,
Hamilton
Bonaduz,
Switzerland).
The
target
products
were
quantified
by
SDS-PAGE
against
purified
protein
standards.
3.
Results
3.1.
Setup
of
the
cloning
protocol
In
the
course
of
establishing
the
transfection
and
cloning
protocol,
we
generated
dose-response
curves
for
the
selection
antibiotics
(Supplementary
Material
2),
which
revealed
different
kinetics
of
feeder
cell
death
for
each
antibiotic.
Blasticidin
S
builds
up
a
higher
selection
pressure
compared
to
hygromycin
B,
achieving
the
faster
removal
of
untransfected
feeder
cells.
However,
despite
the
differences
in
selection
speed,
both
anti-
biotics
induced
the
growth
of
clonal
colonies
and
led
to
growth
arrest
and
decay
of
the
wild-type
feeder
cells.
Microscopically,
the
first
colonies
were
visible
within
the
first
2
weeks
of
selection.
After
3–4
weeks,
well
growing
colonies
had
a
diameter
of
about
1
mm,
yielding
enough
cells
for
further
scale
up
(Fig.
2).
Table
2
Scale
up
during
single-cell
isolation.
Note
that
recommended
volumes
are
plate-dependent
and
may
be
adjusted
according
to
the
equipment
used.
Desiccation
was
prevented
by
ensuring
a
humidified
environment
and
subsequent
re-feeding.
†
The
lower
ratio
between
V
cells
and
V
medium
was
chosen
to
ensure
proper
surface
oxygenation.
Culture
vessel
Culture
method
V
cells
from
previous
step
[
m
L]
V
medium
[
m
L]
V
total
[
m
L]
96-well
plate
static
100
50
†
150
48-well
plate
static
150
150
300
24-well
plate
static
300
300
600
12-well
plate
static
600
600
1200
6-well
plate
static
1200
1200
2400
T25
flask
dynamic
2400
600–2600
3000–5000
4
J.
Zitzmann
et
al.
/
Biotechnology
Reports
19
(2018)
e00272

3.2.
Intracellular
EGFP
expression
in
monoclonal
rS2
cells
The
generation
of
polyclonal
cell
line
1
resulted
in
clearly
detectable
but
very
heterogeneous
EGFP
expression
as
determined
by
flow
cytometry
(Fig.
3a,
small
panel).
The
shape
of
the
corresponding
histogram
can
be
interpreted
as
the
superimposi-
tion
of
multiple
expression
patterns
resulting
from
different
subpopulations
with
varying
copy
numbers
or
integration
loci.
Consequently,
subsequent
single-cell
cloning
led
to
the
isolation
of
19
clones
with
considerably
varying
EGFP
expression
and
the
low,
moderate
and
high
producers
were
clearly
distinguishable
(Fig.
3b).
A
detailed
analysis
of
the
related
expression
profiles
revealed
that
the
clones
showed
more
homogeneous
fluorescence
and
a
sharper
fluorescence
distribution
than
the
original
parental
culture
(Fig.
3a,
main
panel),
indicating
the
successful
segregation
of
the
different
genotypes.
The
fluorescence
signal
in
the
high
producers
was
up
to
tenfold
higher
than
the
polyclonal
population.
To
verify
stable
EGFP
expression
in
the
single-cell
clones
even
without
the
sustained
addition
of
hygromycin
B,
one
of
the
monoclonal
lines
was
cultured
in
the
presence
and
in
the
absence
of
antibiotics
for
3
weeks.
Under
selection
pressure,
almost
all
of
the
cells
remained
EGFP-positive,
but
even
if
the
antibiotic
was
omitted,
the
proportion
of
EGFP-positive
cells
remained
well
above
95%,
indicating
stable
integration
(Supplementary
Material
S3).
Because
the
integration
of
long
tandem
arrays
of
the
donor
transgene
is
a
known
reason
for
high-level
expression
in
Drosophila
S2
cells
[17],
we
analyzed
the
highly
productive
cell
line
19
(Fig.
3b)
by
Southern
blot
hybridization.
To
gain
more
insight
into
the
nature
of
transgene
integration,
isolated
cellular
DNA
was
digested
with
EcoRI,
a
single
cutter
in
the
expression
cassette,
allowing
the
characterization
of
the
integration
pattern
(Fig.
4a
and
b).
The
prominent
band
at
8.4
kb
revealed
that
multiple
copies
of
the
expression
plasmid
were
integrated
in
a
head-to
tail
fashion
(Fig.
4c).
3.3.
Expression
of
antimicrobial
peptides
in
monoclonal
rS2
cells
Following
the
general
setup
of
the
cloning
procedure,
we
analyzed
its
impact
on
the
co-expression
of
EGFP
and
GmGlv
(cell
line
4).
For
the
polyclonal
population,
even
70
days
of
permanent
selection
was
not
sufficient
to
achieve
a
homogeneous
EGFP
expression
profile
and
nonproductive
cells
were
still
present.
In
contrast,
monoclonal
lines
isolated
within
the
same
time
interval
showed
a
unimodal
EGFP
distribution
(Fig.
5).
However,
although
they
were
EGFP
positive,
the
polyclonal
and
most
of
the
monoclonal
lines
produced
almost
undetectable
levels
of
GmGlv
(Fig.
6c).
This
may
reflect
the
silencing
of
the
GmGlv
expression
cassette
or
that
integration
of
the
selection
plasmid
is
favored
over
integration
of
the
expression
plasmid.
Because
of
the
missing
correlation
between
EGFP
and
GmGlv,
screening
for
the
target
peptide
by
dot
blot
was
necessary.
After
cloning
and
dot
blot
screening,
a
highly
productive
clone
was
identified,
which,
in
contrast
to
the
parental
polyclonal
population,
was
suitable
for
further
scale
up
(Table
3).
Fig.
3.
Analysis
of
EGFP-expressing
cell
lines
by
flow
cytometry.
(a)
EGFP
expression
profile
of
wild-type
and
polyclonal
S2
cells
(small
panel)
and
three
representative
monoclonal
cell
lines
representing
low,
moderate
and
high
producers
(indicated
by
different
colors).
(b)
Comparison
of
EGFP
expression
(mean

SD)
in
19
different
monoclonal
cell
lines
and
the
parental
polyclonal
culture.
Fig.
2.
The
process
of
single-cell
colony
growth,
illustrated
with
representative
microscopy
pictures
for
the
different
process
stages.
J.
Zitzmann
et
al.
/
Biotechnology
Reports
19
(2018)
e00272
5

To
further
demonstrate
the
benefit
of
single-cell
cloning,
two
additional
monoclonal
cell
lines
expressing
another
peptide
(BR021)
were
generated
(cell
lines
2
and
3).
Because
cell
line
4
showed
that
the
expression
level
of
the
fluorescent
reporter
protein
did
not
predict
the
expression
level
of
the
target
peptide,
no
reporter
was
used
in
those
two
cell
lines.
Compared
to
the
GmGlv-producing
cell
line
the
number
of
high
producers
was
significantly
higher
and
10–20%
of
the
picked
clones
proved
suitable
for
scale
up
(Tabl e
3).
In
comparison
with
the
corresponding
polyclonal
lines,
the
best
clones
showed
a
sixfold
to
sevenfold
increase
in
cell-specific
productivity,
as
assessed
by
western
blot
analysis
(Fig.
6,
right
panel).
During
subsequent
production
at
the
bioreactor
scale,
we
recovered
17–26
mg/L
of
the
corresponding
peptide.
The
supernatants
were
used
as
basis
for
BR021/GmGlv
purification
and
an
activity
assay
(Supplementary
Material
4).
Fig.
4.
Southern
blot
analysis
of
the
EGFP-expressing
monoclonal
cell
line
19.
Digestion
with
the
single-cutter
EcoRI
should
yield
(a)
8.4-kb
fragments
if
there
are
tandem
head-to-tail
repeats
of
the
integrated
transgene
(EcoRI
P
)
but
(b)
a
single
fragment
whose
size
depends
on
the
position
of
the
next
genomic
EcoRI
site
(EcoRI
G
)
if
there
is
a
single-copy
transgene.
(c)
The
presence
of
an
8.4-kb
band
in
the
Southern
blot
confirms
the
integration
of
multiple
cassettes
in
tandem.
Fig.
5.
Co-expression
of
EGFP
and
the
antimicrobial
protein
GmGlv.
(a)
The
establishment
of
a
polyclonal
population
and
subsequent
strain
maintenance
under
selection
pressure
did
not
lead
to
a
uniform
expression
pattern.
(b)
Single-cell
clones
can
easily
be
distinguished
from
the
decaying
feeder
cells
due
to
the
intracellular
EGFP
fluorescence.
Isolated
monoclonal
cell
lines
show
a
uniform
EGFP
expression
pattern.
6
J.
Zitzmann
et
al.
/
Biotechnology
Reports
19
(2018)
e00272

4.
Discussion
4.1.
General
considerations
on
the
heterogeneity
of
the
S2
cell
line
The
need
for
single-cell
cloning
can
be
justified
by
considering
the
origin
of
the
S2
cell
line
and
the
nature
of
transgene
insertion
into
the
host
cell
genome.
The
wild-type
S2
line
is
heterogeneous
because
it
was
established
from
100
to
300
late-stage
D.
melanogaster
embryos
before
hatching
[33].
According
to
the
German
Collection
of
Microorganisms
and
Cell
Cultures
(DSMZ,
ACC
130),
S2
cells
were
originally
diploid
with
5–
10%
having
an
XY
karyotype,
but
become
now
60–80%
tetraploid
and
exclusively
XX.
This
diversity
is
also
reflected
in
the
fact
that
three
different
isolates
of
the
original
S2
line
showed
vastly
differing
behavior
in
transcriptomic
studies,
demonstrating
the
substantial
divergence
caused
by
long-term
subculture
in
different
laboratories
[15,34].
Plasmid-based
transformation
also
introduces
heterogeneity
because
it
results
in
the
integration
of
multiple
copies
of
the
transgene
at
a
random
genomic
location
[17]
(see
also
Appendix
A).
High-copy-number
transgenic
loci
at
transcriptionally
active
sites
are
generally
beneficial
for
maximizing
protein
expression
[7].
However,
this
causes
an
additional
metabolic
burden,
which
may
inhibit
cell
growth
and
proliferation.
Consequently,
the
long-term
cultivation
of
polyclonal
populations
can
reduce
protein
yields
because
highly-productive
cells
become
over-
populated
by
faster-growing
but
less
productive
neighbor
cells
[2,35].
Especially
in
continuous
bioprocesses,
where
homoge-
neous
cell
populations
are
needed
to
maintain
a
stable
and
controllable
operation
[36],
single-cell
cloning
is
necessary
to
reduce
the
adverse
effects
of
polyclonality.
However,
it
should
be
stressed
that
a
complete
genetic
homogeneity
cannot
be
achieved
even
with
clonal
cell
lines.
This
is
because
of
probable
recombination
events
within
the
multiple
head-to-tail
array
[15].
Furthermore,
it
is
also
conceivable
that
the
long,
repeated
sequences
can
form
heterochromatic
chromatin
structures,
which
interfere
with
protein
expression
[15].
Despite
the
lack
of
complete
homogeneity,
clonal
cell
lines
still
provide
a
better
starting
point
for
process
development
as
they
were
derived
from
a
single
cell,
with
certain
desirable
properties
in
terms
of
growth
and
protein
expression.
Fig.
6.
Expression
analysis
during
the
preparation
of
monoclonal
cell
lines
for
the
production
of
(a)
His
6
-Thr
C
-BR021,
(b)
BR021-Thrc
C
-V5/His
6
and
(c)
His-Thr
C
-Gloverin.
Dot
blot
screening
of
selected
clones
(analyzed
after
reaching
the
24-well
scale)
together
with
a
polyclonal
(poly)
and
a
wild-type
(wt)
control
(left
and
middle
panel).
Western
blot
analysis
to
compare
the
identified
high
producers
with
the
polyclonal
population
(right
panel).
Table
3
Summary
of
the
single-cell
cloning
experiments
for
cell
lines
2–4.
Cell
line
Expressed
protein
Picked
monoclonal
cell
lines
Putative
high
producers
(dot
blot)
Verified
high
producers
(western
blot)
Max.
protein
titer
during
batch
culture
Growth
inhibition
of
E.
coli
at
10
m
M
2
His
6
-Thr
C
-BR021
58
9
(15.5%)
6
(10.3%)
19
mg/L
Not
tested
3
BR021-Thr
C
-V5/His
6
90
24
(26%)
19
(21%)
17
mg/L
Yes
4
His
6
-Thr
C
-Gloverin
42
3
(7.1%)
1
(2.4%)
26
mg/L
Yes
J.
Zitzmann
et
al.
/
Biotechnology
Reports
19
(2018)
e00272
7

4.2.
Single-cell
cloning
overcomes
cell
line
heterogeneity
and
improves
product
yield
Consistent
with
the
statements
above,
our
polyclonal
cell
lines
showed
variable
cell-specific
expression
levels.
This
is
in
agreement
with
previous
reports
concerning
other
polyclonal
rS2
cells
expressing
EGFP
[37]
as
well
as
polyclonal
Sf9
cells
expressing
GFP-tagged
virus-like
particles
[38].
To
overcome
the
limitations
of
polyclonality,
we
isolated
monoclonal
lines
by
limiting
dilution,
which
not
only
unified
the
expression
profile
but
also
achieved
a
multifold
increase
in
cell-specific
productivity.
High-level
expression
in
D.
melanogaster
and
Aedes
spp.
cell
lines
is
usually
associated
with
the
integration
of
multi-copy
head-to-tail
transgene
arrays
[17,18,39,40].
We
also
observed
such
arrays
in
our
monoclonal
line
producing
high
levels
of
EGFP,
increasing
the
fluorescence
signal
by
tenfold
compared
to
the
polyclonal
line.
The
sixfold
to
sevenfold
increase
in
BR021
levels
we
observed,
agreed
with
an
earlier
study
in
which
a
comparable
limiting
dilution
protocol
achieved
a
fivefold
increase
in
productivity
for
the
expression
of
an
antibody
[10].
In
our
GmGlv-producing
cell
line,
an
even
greater
increase
in
productivi-
ty
was
observed;
indeed,
protein
production
was
only
possible
using
the
monoclonal
cell
line,
whereas
polyclonality
resulted
in
negligible
expression
levels
and
no
product
recovery.
The
overall
proportion
of
highly
productive
clones
varied
between
2.4%
and
21%
in
our
assay,
which
is
comparable
to
the
7%
reported
for
a
protocol
based
on
mitomycin
C
[20].
All
of
our
clones
that
were
scaled
up
to
production
level
were
exceptionally
stable
in
terms
of
their
expression
profile,
even
in
the
absence
of
selection
pressure
(e.g.
during
the
5–7
days
of
expression
at
the
bioreactor
scale).
This
behavior
can
be
attributed
to
the
inherent
homogeneity
of
the
monoclonal
population.
However,
as
reported
earlier,
not
all
clones
with
high
productivity
perform
well
during
scale
up
and
it
is
important
to
screen
for
the
best
combination
of
growth
properties,
robustness
and
productivity
[10].
The
AMP
titers
of
17–26
mg/L
we
achieved
in
our
batch
culture
are
competitive
with
those
from
other
expression
systems
used
for
the
production
of
insect
derived
AMPs,
which
typically
generate
titers
of
0.5–68
mg/L
[41,42].
Further
process
intensification
should
be
possible
by
changing
from
batch
to
continuous
perfusion
culture,
which
has
already
enabled
the
production
of
hundreds
to
thousands
of
milligrams
of
target
protein
using
comparable
rS2
cells
[10,43].
Our
study
strongly
supports
arguments
in
favor
of
single-cell
cloning
for
insect
cell
lines
and
confirms
that
the
proposed
limiting
dilution
approach
with
untransfected
S2
feeder
cells
can
be
used
as
a
routine
method.
Highly
productive
monoclonal
cell
lines
were
successfully
prepared
regardless
of
whether
one
or
two
plasmids
were
used
in
the
initial
transfection
mixture.
The
likelihood
of
isolating
superior
clones
is
usually
low,
so
an
appropriate
number
of
clones
must
be
screened.
Future
automation
of
the
process
will
therefore
be
advantageous
because
this
allows
a
considerable
increase
of
the
number
of
clones
that
can
be
examined.
4.3.
Limiting
dilution
compared
to
other
techniques
for
population
enrichment
Although
limiting
dilution
is
broadly
applicable,
the
limita-
tions
of
the
method
include
the
time
consuming
work
and
the
statistical
nature
of
cell
separation,
which
requires
additional
microscopic
validation
of
monoclonality
after
seeding
[44].
Therefore,
other
groups
have
evaluated
less
cumbersome
methods
to
obtain
homogeneous
and
highly-productive
rS2
cells.
The
simplest
method
involves
the
repeated
treatment
of
a
polyclonal
population
with
high
concentrations
of
the
selection
antibiotic
in
order
to
enrich
for
a
high-producer
subpopulation
[6].
Cell
lines
expressing
surface
proteins
can
also
be
isolated
using
immuno-magnetic
selection
[5,45,46].
However,
in
both
cases,
the
corresponding
subpopulations
remain
polyclonal
and
are
therefore
still
genetically
inhomogeneous,
possibly
reducing
the
stability
of
protein
expression
over
time
due
to
differences
in
growth
kinetics.
Fluorescence
activated
cell
sorting
(FACS)
is
an
alternative
method
for
the
controlled
separation
of
single
cells
based
on
expression-related
fluorescence
intensity.
FACS
can
be
used
for
the
simple
enrichment
of
productive
subpopulations
or
even
for
the
verified
isolation
of
single
cells,
and
has
already
been
successfully
applied
to
Sf9
and
rS2
cells
[46,47].
After
separation
by
FACS,
single
rS2
cells
are
expanded
using
essentially
the
same
protocol
as
for
limiting
dilution
[48].
FACS
is
useful,
especially
in
the
context
of
cell
line
generation
by
targeted
gene
insertion
using
recombinase
mediated
cassette
exchange
(RMCE),
because
here
the
fluorescent
tagging
cassette
is
later
exchanged
for
a
targeting
cassette
and
consequently
there
is
a
direct
relationship
between
the
preliminary
fluorescence
signal
and
the
final
product
expression
level
[38,48,49].
However,
unless
the
reporter
is
exchanged
by
RMCE
or
directly
fused
to
the
target
protein,
the
use
of
fluorescence
for
selection
is
problematic.
A
general
drawback
of
antibiotic
or
fluorescence-driven
enrichment
is
that
it
does
not
necessarily
address
the
expression
of
the
actual
target
protein,
as
it
only
selects
for
cells
with
high
antibiotic
resistance
or
reporter
fluorescence.
Furthermore,
the
reporter
usually
does
not
resem-
ble
the
target
protein
in
terms
of
size,
stability
and
other
physicochemical
properties.
In
this
context,
cell
line
4
showed
that
the
expression
level
of
the
target
peptide
did
not
match
that
of
the
co-expressed
EGFP,
and
only
limiting
dilution
combined
with
direct
screening
by
dot
blot
allowed
the
identification
of
a
high
producer.
In
conclusion,
limiting
dilution
is
a
universally
applicable
and
cost-efficient
method
for
the
generation
of
monoclonal
rS2
cell
lines.
Using
the
protocol
described
herein,
limiting
dilution
does
not
require
expensive
equipment
(unlike
FACS),
involves
no
radiation,
gives
reproducible
results
and
is
easy
to
automate.
As
a
future
perspective,
the
combination
of
limiting
dilution
with
upstream
enrichment
techniques
may
achieve
an
increase
in
the
proportion
of
highly
productive
clones.
Furthermore,
the
main
aspects
of
our
protocol
are
also
compatible
with
microfluidic
single-cell
printing,
which
offers
a
more
controlled
single-cell
isolation
and
ensures
the
presence
of
single
cells
in
each
cavity
of
the
microtiter
plate
[44].
5.
Author
contributions
Jan
Zitzmann
conceived,
designed
and
performed
all
experi-
ments
with
cell
lines
2,
3
and
4.
He
wrote
the
manuscript
and
coordinated
the
creation
of
the
paper.
Christine
Schreiber
designed
and
performed
all
steps
for
the
generation
of
cell
line
1
and
the
corresponding
experiments.
Additionally,
she
prepared
the
plasmid
set
for
cell
line
3
and
revised
the
manuscript.
Joel
Eichmann
helped
to
set
up
the
Golden
Gate
assembly
and
revised
the
manuscript.
Roberto
Otmar
Bilz
performed
the
purification
and
activity
assay.
Tobias
Weidner
and
Denise
Salzig
helped
to
draft
and
revise
the
manuscript.
Peter
Czermak
helped
to
draft
and
revise
the
manuscript,
and
supervised
the
research.
All
authors
have
given
their
approval
for
this
final
version
of
the
manuscript.
Conflicts
of
interest
The
authors
declare
no
conflict
of
interest.
The
funding
sponsors
had
no
role
in
the
design
of
the
study,
in
the
collection,
analysis,
or
interpretation
of
data,
in
the
writing
of
the
manuscript,
and
in
the
decision
to
publish
the
results.
8
J.
Zitzmann
et
al.
/
Biotechnology
Reports
19
(2018)
e00272

Acknowledgments
We
would
like
to
thank
the
Hessen
State
Ministry
of
Higher
Education,
Research
and
the
Arts
for
the
financial
support
within
the
Hessen
initiative
for
scientific
and
economic
excellence
(LOEWE-Program).
The
authors
acknowledge
Dr.
Richard
M
Twyman
for
revising
the
paper.
Appendix
A.
Linearizing
the
expression
plasmids
prior
to
transfection.
An
additional
tool
to
improve
homogeneity?
Transfection
is
usually
performed
with
non-linearized
plasmids.
However,
during
cell
line
establishment
the
transfected
circular
plasmid
is
randomly
linearized
within
the
cell,
leading
to
heterogeneity
and
possibly
destroying
important
elements
such
as
the
resistance
gene
or
the
gene
of
interest
[50].
Linearization
of
the
plasmid
prior
to
transfection
by
a
restriction
enzyme
with
a
single
recognition
site
in
a
non-coding
region
preserves
the
integrity
of
all
sensitive
parts
and
may
be
therefore
beneficial.
Although
linear
DNA
providesa well-definedand putativelysuperior
starting
point
forcell
line
generation,
there
are
some
issues
that
need
to
be
addressed
when
using
this
method.
As
an
example,
the
Lipofectamine-
and
PEI-
mediated
transfection
of
HeLa-cells
with
linearized
plasmid-DNA
led
to
decreased
protein
yields
and
enhanced
cytotoxicity
compared
to
standard
circular
plasmids
[51].
The
same
was
observed
for
the
liposome-mediated
transfection
of
Vero
cells
[52].
Both
studies
indicate
changes
in
the
morphology
of
the
transfection
complex
as
a
reason
for
the
deterioration
in
transfection
efficiency.
While
circular
DNA
led
to
compacted
and
roughly
spherical
shaped
complexes,
linear
DNA
results
in
a
worm
like
and
apparently
cytotoxic
structure
[51,52].
This
cytotoxicity
has
to
be
prevented
by
augmenting
the
transfection
mixture
with
cationic
amphiphiles
[52].
Despite
these
issues,
linearization
of
the
plasmidscan be superior,
asdemonstrated
for
the
transfection
of
mammalian
neuronal
cell
lines,
where
it
yielded
a
three-fold
increase
in
the
number
of
stable
colonies
[50].
However,
the
efficiency
of
stable
clone
generation
and
gene
expression
was
highly
dependent
on
the
restriction
site
selected
for
linearization
[50].
For
D.
melanogaster
S2
cells
and
their
associated
transfection
reagents
(e.g.
TransitIT
1
-Insect),
no
comprehensive
investigation
on
the
influence
of
plasmid
linearization
is
available
yet.
As
a
future
perspective
the
regarding
interdependencies
should
be
examined
in
a
structured
way,
ideally
through
statistically
designed
experi-
ments.
Appendix
B.
Supplementary
data
Supplementary
material
related
to
this
article
can
be
found,
in
the
online
version,
at
doi:https://doi.org/10.1016/j.btre.2018.
e00272.
References
[1]
W.A.
de
Jongh,
S.
Salgueiro,
C.
Dyring,
The
use
of
Drosophila
S2
cells
in
R&D
and
bioprocessing,
Pharm.
Bioprocess.
1
(2013)
197–213,
doi:http://dx.doi.org/
10.4155/pbp.13.18.
[2]
J.
Zitzmann,
G.
Sprick,
T.
Weidner,
C.
Schreiber,
P.
Czermak,
Process
optimization
for
recombinant
protein
expression
in
insect
cells,
in:
S.J.T.
Gowder
(Ed.),
New
Insights
Cell
Cult.
Technol.,
1st
ed.,
InTech
Open,
Rijeka,
2017,
pp.
43–98,
doi:http://dx.doi.org/10.5772/67849.
[3]
J.H.
Park,
H.Y.
Kim,
K.H.
Han,
I.S.
Chung,
Optimization
of
transfection
conditions
for
expression
of
green
fluorescent
protein
in
Drosophila
melanogaster
S2
cells,
Enzyme
Microb.
Technol.
25
(1999)
558–563,
doi:
http://dx.doi.org/10.1016/S0141-0229(99)00096-4.
[4]
H.S.
Shin,
H.J.
Cha,
Facile
and
statistical
optimization
of
transfection
conditions
for
secretion
of
foreign
proteins
from
insect
Drosophila
S2
cells
using
green
fluorescent
protein
reporter,
Biotechnol.
Prog.18
(2002)
1187–1194,
doi:http://
dx.doi.org/10.1021/bp025533l.
[5]
N.G.L.
Santos,
M.P.
Rocca,
C.A.
Pereira,
D.C.
Ventini,
A.L.P.
Puglia,
S.A.C.
Jorge,
M.
A.N.
Lemos,
R.M.
Astray,
Impact
of
recombinant
Drosophila
S2
cell
population
enrichment
on
expression
of
rabies
virus
glycoprotein,
Cytotechnology
68
(2016)
2605–2611,
doi:http://dx.doi.org/10.1007/s10616-016-9984-z.
[6]
M.A.N.
Lemos,
A.S.
dos
Santos,
R.M.
Astray,
C.A.
Pereira,
S.A.C.
Jorge,
Rabies
virus
glycoprotein
expression
in
Drosophila
S2
cells.
I:
design
of
expression/
selection
vectors,
subpopulations
selection
and
influence
of
sodium
butyrate
and
culture
medium
on
protein
expression,
J.
Biotechnol.
143
(2009)
103–110,
doi:http://dx.doi.org/10.1016/j.jbiotec.2009.07.003.
[7]
S.A.C.
Jorge,
A.S.
Santos,
Â.
Spina,
C.A.
Pereira,
Expression
of
the
hepatitis
B
virus
surface
antigen
in
Drosophila
S2
cells,
Cytotechnology
57
(2008)
51–59,
doi:http://dx.doi.org/10.1007/s10616-008-9154-z.
[8]
J.
Zitzmann,
T.
Weidner,
P.
Czermak,
Optimized
expression
of
the
antimicrobial
protein
gloverin
from
galleria
mellonella
using
stably
transformed
drosophila
melanogaster
S2
cells,
Cytotechnology
69
(2017)
371–389,
doi:http://dx.doi.
org/10.1007/s10616-017-0068-5.
[9]
E.
Uribe,
M.
Venkatesan,
D.R.
Rose,
K.V.
Ewart,
Expression
of
recombinant
Atlantic
salmon
serum
C-type
lectin
in
Drosophila
melanogaster
Schneider
2
cells,
Cytotechnology
65
(2013)
513–521,
doi:http://dx.doi.org/10.1007/
s10616-012-9505-7.
[10]
L.
Wang,
H.
Hu,
J.
Yang,
F.
Wang,
C.
Kaisermayer,
P.
Zhou,
High
yield
of
human
monoclonal
antibody
produced
by
stably
transfected
Drosophila
schneider
2
cells
in
perfusion
culture
using
wave
bioreactor,
Mol.
Biotechnol.
52
(2012)
170 –179,
doi:http://dx.doi.org/10.1007/s12033-011-9484-5.
[11]
Chapter
8
selection
and
cloning,
in:
R.H.
Burdon,
P.H.
van
Knippenberg
(Eds.),
Lab.
Tech.
Biochem.
Mol.
Biol.,
Elsevier,
1984,
pp.
151–165,
doi:http://dx.doi.
org/10.1016/S0075-7535(08)70700-9.
[12]
S.A.
Fuller,
M.
Takahashi,
J.G.
Hurrell,
Cloning
of
hybridoma
cell
lines
by
limiting
dilution,
Curr.
Protoc.
Mol.
Biol.
(Chapter
11)
(2001),
doi:http://dx.doi.
org/10.1002/0471142727.mb1108s01
Unit11.8.
[13]
W.M.
Yokoyama,
M.
Christensen,
G.
Dos
Santos,
D.
Miller,
J.
Ho,
T.
Wu,
M.
Dziegelewski,
F.A.
Neethling,
Production
of
monoclonal
antibodies,
Curr.
Protoc.
Immunol.
102
(2013),
doi:http://dx.doi.org/10.1002/0471142735.
im0205s102
Unit
2.5..
[14]
J.A.
Schetz,
E.P.N.
Shankar,
Protein
expression
in
the
Drosophila
Schneider
2
cell
system,
Curr.
Protoc.
Neurosci.,
John
Wiley
&
Sons,
2004,
doi:http://dx.doi.
org/10.1002/0471142301.ns0416s27/abstract
(accessed
August
15,
2014).
[15]
L.
Cherbas,
L.
Gong,
Cell
lines,
Methods
San
Diego
Calif.
68
(2014)
74–81,
doi:
http://dx.doi.org/10.1016/j.ymeth.2014.01.006.
[16]
Â.M.
Moraes,
S.A.C.
Jorge,
R.M.
Astray,
C.A.T.
Suazo,
C.E.
Calderón
Riquelme,
E.F.
P.
Augusto,
A.
Tonso,
M.M.
Pamboukian,
R.A.M.
Piccoli,
M.F.
Barral,
C.A.
Pereira,
Drosophila
melanogaster
S2
cells
for
expression
of
heterologous
genes:
From
gene
cloning
to
bioprocess
development,
Biotechnol.
Adv.
30
(2012)
613–628,
doi:http://dx.doi.org/10.1016/j.biotechadv.2011.10.009.
[17]
L.
Cherbas,
P.
Cherbas,
Transformation
of
Drosophila
cell
lines,
in:
D.
Murhammer
(Ed.),
Baculovirus
Insect
Cell
Expr.
Protoc.,
Humana
Press,
Totowa,
N.J,
2007,
pp.
317–340,
doi:http://dx.doi.org/10.1007/978-1-59745-457-5_16.
[18]
S.
Bärtsch,
F.E.
Würgler,
C.
Sengstag,
A
genetic
system
to
detect
mitotic
recombination
between
repeated
chromosomal
sequences
in
Drosophila
Schneider
line
2
cells,
Mutat.
Res.
Toxicol.
Environ.
Mutagen.
395
(1997)
9–27,
doi:http://dx.doi.org/10.1016/S1383-5718(97)00138-1.
[19]
F.
Fernandes,
J.
Vidigal,
M.M.
Dias,
K.L.J.
Prather,
A.S.
Coroadinha,
A.P.
Teixeira,
P.M.
Alves,
Flipase-mediated
cassette
exchange
in
Sf9
insect
cells
for
stable
gene
expression,
Biotechnol.
Bioeng.
109
(2012)
2836–2844,
doi:http://dx.doi.
org/10.1002/bit.24542.
[20]
S.L.
Nilsen,
F.J.
Castellino,
Expression
of
human
plasminogen
in
Drosophila
Schneider
S2
cells,
Protein
Expr.
Purif.
16
(1999)
136–143,
doi:http://dx.doi.
org/10.1006/prep.1999.1045.
[21]
W.
Liu,
V.
Vigdorovich,
C.
Zhan,
Y.
Patskovsky,
J.B.
Bonanno,
S.G.
Nathenson,
S.C.
Almo,
Increased
heterologous
protein
expression
in
Drosophila
S2
cells
for
massive
production
of
immune
Ligands/Receptors
and
structural
analysis
of
human
HVEM,
Mol.
Biotechnol.
57
(2015)
914–922,
doi:http://dx.doi.org/
10.1007/s12033-015-9881-2.
[22]
C.
Rabu,
A.
Quéméner,
Y.
Jacques,
K.
Echasserieau,
P.
Vusio,
F.
Lang,
Production
of
recombinant
human
trimeric
CD137L
(4-1BBL)
cross-linking
is
essential
to
its
t
cell
co-stimulation
activity,
J.
Biol.
Chem.
280
(2005)
41472–414 81,
doi:
http://dx.doi.org/10.1074/jbc.M506881200.
[23]
D.J.
Peel,
M.J.
Milner,
The
diversity
of
cell
morphology
in
cloned
cell
lines
derived
from
Drosophila
imaginal
discs,
Rouxs
Arch.
Dev.
Biol.
198
(1990)
479–
482,
doi:http://dx.doi.org/10.1007/BF00399059.
[24]
L.
Yang,
Y.
Song,
X.
Li,
X.
Huang,
J.
Liu,
H.
Ding,
P.
Zhu,
P.
Zhou,
HIV-1
virus-like
particles
produced
by
stably
transfected
Drosophila
S2
cells:
a
desirable
vaccine
component,
J.
Virol.
86
(2012)
7662 –7676,
doi:http://dx.doi.org/
10.1128/JVI.07164-11.
[25]
A.J.
Scotter,
D.A.
Kuntz,
M.
Saul,
L.A.
Graham,
P.L.
Davies,
D.R.
Rose,
Expression
and
purification
of
sea
raven
type
II
antifreeze
protein
from
Drosophila
melanogaster
S2
cells,
Protein
Expr.
Purif.
47
(2006)
374–383,
doi:http://dx.
doi.org/10.1016/j.pep.2005.10.028.
[26]
M.P.
Belmares,
J.D.
Rabinowitz,
W.
Liu,
E.D.
Mellins,
H.M.
McConnell,
pH
stability
of
HLA-DR4
complexes
with
antigenic
peptides,
Biochemistry
39
(2000)
14558–14566,
doi:http://dx.doi.org/10.1021/bi001544g.
[27]
N.S.
Millar,
H.A.
Baylis,
C.
Reaper,
R.
Bunting,
W.T.
Mason,
D.B.
Sattelle,
Functional
expression
of
a
cloned
Drosophila
muscarinic
acetylcholine
receptor
in
a
stable
Drosophila
cell
line,
J.
Exp.
Biol.
198
(1995)
1843–1850.
J.
Zitzmann
et
al.
/
Biotechnology
Reports
19
(2018)
e00272
9

[28]
J.P.
Incardona,
T.L.
Rosenberry,
Construction
and
characterization
of
secreted
and
chimeric
transmembrane
forms
of
Drosophila
acetylcholinesterase:
a
large
truncation
of
the
C-terminal
signal
peptide
does
not
eliminate
glycoinositol
phospholipid
anchoring,
Mol.
Biol.
Cell
7
(1996)
595–611.
[29]
L.
Cherbas,
R.
Moss,
P.
Cherbas,
Transformation
techniques
for
Drosophila
cell
lines,
in:
L.S.B.G,
E.A.
Fyrberg
(Eds.),
Methods
Cell
Biol.,
Academic
Press,
San
Diego,
New
York,
Boston,
London,
Sydney,
Tokyo,
Toronto,
1994,
pp.
161–179.
(Accessed
May
18,
2016)
http://www.sciencedirect.com/science/article/pii/
S0091679X08609127.
[30]
C.
Kollewe,
Production
of
recombinant
proteins
in
insect
cells,
Am.
J.
Biochem.
Biotechnol.
9
(2013)
255–271,
doi:http://dx.doi.org/10.3844/
ajbbsp.2013.255.271.
[31]
A.
Vilcinskas,
K.
Mukherjee,
H.
Vogel,
Expansion
of
the
antimicrobial
peptide
repertoire
in
the
invasive
ladybird
Harmonia
axyridis,
Proc.
R.
Soc.
B
Biol.
Sci.
280
(2013),
doi:http://dx.doi.org/10.1098/rspb.2012.2113.
[32]
C.
Schreiber,
H.
Müller,
O.
Birrenbach,
M.
Klein,
D.
Heerd,
T.
Weidner,
D.
Salzig,
P.
Czermak,
A
high-throughput
expression
screening
platform
to
optimize
the
production
of
antimicrobial
peptides,
Microb.
Cell
Factories
16
(2017)
29,
doi:
http://dx.doi.org/10.1186/s12934-017-0637-5.
[33]
I.
Schneider,
Cell
lines
derived
from
late
embryonic
stages
of
Drosophila
melanogaster,
J.
Embryol.
Exp.
Morphol.
27
(1972)
353–365.
[34]
L.
Cherbas,
A.
Willingham,
D.
Zhang,
L.
Yang,
Y.
Zou,
B.D.
Eads,
J.W.
Carlson,
J.M.
Landolin,
P.
Kapranov,
J.
Dumais,
A.
Samsonova,
J.-H.
Choi,
J.
Roberts,
C.A.
Davis,
H.
Tang,
M.J.
van
Baren,
S.
Ghosh,
A.
Dobin,
K.
Bell,
W.
Lin,
L.
Langton,
M.O.
Duff,
A.E.
Tenney,
C.
Zaleski,
M.R.
Brent,
R.A.
Hoskins,
T.C.
Kaufman,
J.
Andrews,
B.R.
Graveley,
N.
Perrimon,
S.E.
Celniker,
T.R.
Gingeras,
P.
Cherbas,
The
transcriptional
diversity
of
25
Drosophila
cell
lines,
Genome
Res.
21
(2011)
301–314,
doi:http://dx.doi.org/10.1101/gr.112961.110.
[35]
B.
Baum,
L.
Cherbas,
Drosophila
cell
lines
as
model
systems
and
as
an
experimental
tool,
Methods
Mol.
Biol.
Clifton
NJ.
420
(2008)
391–424,
doi:
http://dx.doi.org/10.1007/978-1-59745-583-1_25.
[36]
R.G.
Werner,
F.
Walz,
W.
Noé,
A.
Konrad,
Safety
and
economic
aspects
of
continuous
mammalian
cell
culture,
J.
Biotechnol.
22
(1992)
51–68,
doi:http://
dx.doi.org/10.1016/0168-1656(92)90132-S.
[37]
M.G.
Santos,
S.A.C.
Jorge,
K.
Brillet,
C.A.
Pereira,
Improving
heterologous
protein
expression
in
transfected
Drosophila
S2
cells
as
assessed
by
EGFP
expression,
Cytotechnology
54
(2007)
15–24,
doi:http://dx.doi.org/10.1007/
s10616-007-9060-9.
[38]
J.
Vidigal,
B.
Fernandes,
M.M.
Dias,
M.
Patrone,
A.
Roldão,
M.J.T.
Carrondo,
P.M .
Alves,
A.P.
Teixeira,
RMCE-based
insect
cell
platform
to
produce
membrane
proteins
captured
on
HIV-1
gag
virus-like
particles,
Appl.
Microbiol.
Biotechnol.
102
(2018)
655–666,
doi:http://dx.doi.org/10.1007/s00253-017-
8628-3.
[39]
M.
Bourouis,
B.
Jarry,
Vectors
containing
a
prokaryotic
dihydrofolate
reductase
gene
transform
Drosophila
cells
to
methotrexate-resistance,
EMBO
J.
2
(1983)
1099–1104 .
[40]
T.J.
Monroe,
M.C.
Muhlmann-Diaz,
M.J.
Kovach,
J.O.
Carlson,
J.S.
Bedford,
B.J.
Beaty,
Stable
transformation
of
a
mosquito
cell
line
results
in
extraordinarily
high
copy
numbers
of
the
plasmid,
Proc.
Natl.
Acad.
Sci.
U.
S.
A.
89
(1992)
5725–
5729.
[41]
H.
Müller,
D.
Salzig,
P.
Czermak,
Considerations
for
the
process
development
of
insect-derived
antimicrobial
peptide
production,
Biotechnol.
Prog.
31
(2015)
1–11,
doi:http://dx.doi.org/10.1002/btpr.2002.
[42]
N.S.
Parachin,
K.C.
Mulder,
A.A.B.
Viana,
S.C.
Dias,
O.L.
Franco,
Expression
systems
for
heterologous
production
of
antimicrobial
peptides,
Peptides
38
(2012)
446–456,
doi:http://dx.doi.org/10.1016/j.peptides.2012.09.020.
[43]
J.
Zitzmann,
T.
Weidner,
G.
Eichner,
D.
Salzig,
P.
Czermak,
Dielectric
spectroscopy
and
optical
density
measurement
for
the
online
monitoring
and
control
of
recombinant
protein
production
in
stably
transformed
Drosophila
melanogaster
S2
cells,
Sensors
18
(2018)
900,
doi:http://dx.doi.org/
10.3390/s18030900.
[44]
A.
Gross,
J.
Schoendube,
S.
Zimmermann,
M.
Steeb,
R.
Zengerle,
P.
Koltay,
Technologies
for
single-cell
isolation,
Int.
J.
Mol.
Sci.
16
(2015)
16897–16919,
doi:http://dx.doi.org/10.3390/ijms160816897.
[45]
K.
Brillet,
B.G.
Perret,
V.
Klein,
F.
Pattus,
R.
Wagner,
Using
EGFP
fusions
to
monitor
the
functional
expression
of
GPCRs
in
the
Drosophila
Schneider
2
cells,
Cytotechnology
57
(2008)
101–109,
doi:http://dx.doi.org/10.1007/
s10616-008-9125-4.
[46]
M.E.
March,
C.C.
Gross,
E.O.
Long,
Use
of
transfected
Drosophila
S2
cells
to
study
NK
sell
activation,
in:
K.
Campbell
(Ed.),
Natural
killer
kell
krotocols.
Methods
in
Molecular
Biology
(Methods
and
Protocols),
612,
Humana
Press,
2010,
doi:
http://dx.doi.org/10.1007/978-1-60761-362-6_6.
[47]
S.
Cumberledge,
M.A.
Krasnow,
Preparation
and
analysis
of
pure
cell
populations
from
Drosophila,
Methods
Cell
Biol.
44
(1994)
143–159.
[48]
L.
Cherbas,
J.
Hackney,
L.
Gong,
C.
Salzer,
E.
Mauser,
D.
Zhang,
P.
Cherbas,
Tools
for
targeted
genome
engineering
of
established
Drosophila
cell
lines,
Genetics
201
(2015)
1307–1318,
doi:http://dx.doi.org/10.1534/genetics.115.181610.
[49]
J.
Vidigal,
M.M.
Dias,
F.
Fernandes,
M.
Patrone,
C.
Bispo,
C.
Andrade,
R.
Gardner,
M.J.T.
Carrondo,
P.M .
Alves,
A.P.
Teixeira,
A
cell
sorting
protocol
for
selecting
high-producing
sub-populations
of
Sf9
and
High
Five
TM
cells,
J.
Biotechnol.168
(2013)
436–439,
doi:http://dx.doi.org/10.1016/j.jbiotec.2013.10.020.
[50]
G.
Stuchbury,
G.
Münch,
Optimizing
the
generation
of
stable
neuronal
cell
lines
via
pre-transfection
restriction
enzyme
digestion
of
plasmid
DNA,
Cytotechnology
62
(2010)
189–194,
doi:http://dx.doi.org/10.1007/s10616-
010-9273-1.
[51]
R.
Lehner,
X.
Wang,
P.
Hunziker,
Plasmid
linearization
changes
shape
and
efficiency
of
transfection
complexes,
Eur.
J.
Nanomed.
5
(2013),
doi:http://dx.
doi.org/10.1515/ejnm-2013-0028.
[52]
A.
vonGroll,
Y.
Levin,
M.C.
Barbosa,
A.P.
Ravazzolo,
Linear
DNA
Low
efficiency
transfection
by
liposome
can
be
improved
by
the
use
of
cationic
lipid
as
charge
neutralizer,
Biotechnol.
Prog.
22
(2006)
1220–1224,
doi:http://dx.doi.org/
10.1021/bp060029s.
10
J.
Zitzmann
et
al.
/
Biotechnology
Reports
19
(2018)
e00272