A GFP-based method facilitates clonal selection of transfected CHO cells.

TLDR: A method based on the co‐expression of enhanced green fluorescent protein (EGFP) that allows clonal selection in standard 96‐well cell culture plates and is an alternative to the identification of high‐producer clones using various cell sorting methods is presented.

Abstract: The identification of highly expressing clones is a crucial step in the development of cell lines for production of recombinant proteins. Here we present a method based on the co-expression of enhanced green fluorescent protein (EGFP) that allows clonal selection in standard 96-well cell culture plates. The genes encoding the EGFP protein and the protein of interest are linked by an internal ribosome entry site and thus are transcribed into the same mRNA but are translated independently. Since both proteins arise from a common mRNA, the EGFP expression level correlates with the expression level of the therapeutic protein for each clone. By expressing recombinant growth factors in CHO cells, we demonstrate the robustness and performance of this technique. The method is an alternative to the identification of high-producer clones using various cell sorting methods, as it can be performed with standard laboratory equipment.

Full text

HAL Id: hal-00514309
https://hal.archives-ouvertes.fr/hal-00514309
Submitted on 2 Sep 2010
HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entic research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.
L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diusion de documents
scientiques de niveau recherche, publiés ou non,
émanant des établissements d’enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.
A GFP-based method facilitates clonal selection of
transfected CHO cells
Denise Freimark, Valérie Jérôme, Ruth Freitag
To cite this version:
Denise Freimark, Valérie Jérôme, Ruth Freitag. A GFP-based method facilitates clonal selec-
tion of transfected CHO cells. Biotechnology Journal, Wiley-VCH Verlag, 2009, 4 (10), pp.24.
�10.1002/biot.200800264�. �hal-00514309�

For Peer Review
A GFP-based method facilitates clonal selection of
transfected CHO cells
Journal:
Biotechnology Journal
Manuscript ID:
BIOT-2008-0264.R2
Wiley - Manuscript type:
Technical Report
Date Submitted by the
Author:
22-Jun-2009
Complete List of Authors:
Freimark, Denise; University of Bayreuth, Chair for Process
Biotechnology
Jérôme, Valérie; University of Bayreuth, Chair for Process
Biotechnology
Freitag, Ruth; Lehrstuhl Bioprozesstechnik, Universität Bayreuth
Keywords:
growth factor, high producer, mammalian cell culture, recombinant
protein production, transfection
Wiley-VCH
Biotechnology Journal

For Peer Review
Page 1 of 19
Technical Report ((4783 words))
A GFP-based method facilitates clonal selection of transfected
CHO cells
Denise Freimark, Valérie Jérôme and Ruth Freitag*
Chair for Process Biotechnology University of Bayreuth, Germany
Key words: growth factor, high producer, mammalian cell culture, recombinant protein
production, transfection
*corresponding author: Chair for Process Biotechnology, University of Bayreuth, 95440
Bayreuth, Germany, phone: 0049 921 55-7371, Fax: 0049 921 55-7375, e-mail:
bioprozesstechnik@uni-bayreuth.de
Abbreviations:
BSA: Bovine serum albumin
CHO: Chinese hamster ovary
EGFP: Enhanced green fluorescent protein
FACS: Fluorescence assisted cell sorting
G418: Geneticin (antibiotic used for screening)
hIGFIA: Human insulin-like growth factor 1A
IRES: Internal ribosome entry site
hVEGFA: Human vascular endothelial growth factor A
Page 1 of 23
Wiley-VCH
Biotechnology Journal
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60

For Peer Review
Page 2 of 19
Abstract
The identification of highly expressing clones is a crucial step in the development of cell lines
for production of recombinant proteins. Here we present a method based on the co-expression
of enhanced GFP that allows clonal selection in standard 96-well cell culture plates. The
genes encoding the EGFP protein and the protein of interest are linked by an internal
ribosome entry site and thus are transcribed into the same mRNA but are translated
independently. Since both proteins arise from a common mRNA, the EGFP expression level
correlates with the expression level of the therapeutic protein for each clone. By expressing
recombinant growth factors in CHO cells, we demonstrate the robustness and performance of
this technique. The method is an alternative to the identification of high-producer clones using
various cell sorting methods, as it can be performed with standard laboratory equipment.
Page 2 of 23
Wiley-VCH
Biotechnology Journal
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60

For Peer Review
Page 3 of 19
Introduction
A key step in the generation of cell lines for the efficient production of recombinant proteins
is the selection of high producer clones following transfection and selection [1]. Most often,
successfully transfected cells pass through a subcloning stage using 96-well plates. A robust
method for screening at this level is valuable, as it helps to focus on good producers early on.
Analysis of the product titre in cell culture supernatant by ELISA is commonly used at this
stage to identify clones secreting high levels of recombinant protein. However, the process of
randomly picking a large number of clones produced, e.g. by limited dilution, and assaying
them individually by ELISA is tedious, time consuming, and reportedly not always very
effective [2,3]. Moreover, this method does not account for differences in either cell density
or media volume between wells. Apart from the already mentioned tediousness, this approach
may therefore not accurately predict the clones with high specific productivity.
High throughput screening methods based, e.g., on fluorescence-activated cell sorting (FACS)
or magnetic bead-based cell separation are faster [4,5], but require, especially in case of
FACS, not only expensive equipment but also highly skilled operators. Hence such methods
tend to be restricted to labs in frequent need of cell sorting. Moreover, unless the target
molecule itself shows fluorescence, labelling or co-expression of a fluorescent or otherwise
detectable molecule is required [6,7]. The enhanced green fluorescent protein (EGFP) has
been suggested in the past as co-expressed indicator protein for applications in FACS-based
cell separation, but also for the screening of cultures in multiwell plates [8-10]. In this
approach the recombinant cells express both the fluorescent and the target protein, the titres
are assumed to be linked. However, mostly the genes for EGFP and the target protein were
co-transfected into the cells via individual plasmids, e.g. [9,11]. Hence no physical connection
between the expression of the two proteins existed.
Page 3 of 23
Wiley-VCH
Biotechnology Journal
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60