Castilho Leda R., Moraes Angela Maria (Ed.) Animal Cell Technology: From Biopharmaceuticals to Gene Therapy

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previously described, plasmids employed for stable expression contain a

selection cassette with a gene that will provide a new property to the

transfected cell. Both the sequence encoding the product and the selection

gene can be contained in the same plasmid or in individual vectors, and

this has no inﬂuence on the expression levels of the recombinant protein.

When present in the same vector they can be expressed from a polycis-

tronic mRNA (see Section 3.4.3). One strategy to increase the chances of

obtaining high producer cell lines consists of driving the expression of the

resistance gene by a weak promoter. Although this approach signiﬁcantly

reduces the efﬁciency of the transfection, the cells that survive the selective

pressure show higher yields of recombinant protein (Wurm, 2004). The

resistance to a drug can be recessive or dominant. Those genes providing

recessive resistance need a particular host, which must be deﬁcient in the

activity by which it will be selected, whereas the genes confering dominant

resistance can be used independently of the biochemical background of

the cell (Keown et al., 1990). Use of the TK gene is one example of

selection employing a recessive biochemical marker. Mammalian cells

display an absolute dependency on the TK enzyme when they are

cultivated in the presence of aminopterin, which blocks the de novo

synthesis of thymidine. The selection medium HAT, containing hypox-

anthine, aminopterin, and thymidine, is used to rescue cells that express

TK. Obviously, this selection system requires cells that lack TK, also

identiﬁed as TK

–

. In this respect, TK

–

cell lines are available from human

and murine origin (Bacchetti and Graham, 1977; Maitland and McDougall,

1977; Wigler et al., 1977).

The selection systems based on a dominant marker are based on

resistance to antibiotics. A large number of drugs are used for this purpose:

neomycin, hygromycin, puromycin, bleomycin, and zeocin, among others.

However, two aminoglycoside phosphotransferases are most routinely

used, namely neomycin-phosphotransferase and hygromicin-B-phospho-

transferase. Both confer resistance by phosphorylation-mediated inactiva-

tion of the corresponding antibiotics. The toxic metabolites added to the

culture medium are G418 (analog to gentamicin and also known as

geneticin) and hygromycin B, both of which inhibit protein synthesis

(Gonza´lez et al., 1978; Colbere-Garapin et al., 1981).

Another group of biochemical markers is characterized by promoting

the phenomenon of ‘‘gene ampliﬁcation’’ and, hence, known as ampliﬁca-

tion markers. Various features have been shown for the gene ampliﬁcation

process: (i) the ampliﬁcation is obtained after treatment with increasing

concentrations of the selection agent; (ii) the ampliﬁed DNA shows

certain instability; (iii) the size and structure of the ampliﬁed unit is

variable and temporary (Keown et al., 1990). Within the ampliﬁcation

markers, the most commonly used are the enzymes dihydrofolate reduc-

tase (DHFR), involved in the metabolism of nucleotides (Figure 3.4), and

glutamine synthetase (GS). For DHFR, selection takes place in the absence

of hypoxanthine and thymidine, whereas for GS selection takes place in

the absence of glutamine. Although most of the ‘‘ampliﬁed’’ cells express

signiﬁcant amounts of the recombinant protein, the increase in speciﬁc

productivity (usually 10–20-fold) varies from clone to clone (Wirth et al.,

1988). In eukaryotic vectors, the DHFR gene is of prokaryote (E. coli)or

62 Animal Cell Technology

murine origin, the latter producing a protein with higher resistance against

MTX (Simonsen and Levinson, 1983). The extensive use of this system lies

in the availability of CHO cells lacking the DHFR enzyme (Urlaub and

Chasin, 1980). The commonly used clones DUKX-B11 and DG44 can be

obtained from Dr Lawrence Chasin (Columbia University, NY).

In the case of the GS, the enzyme catalyzes the synthesis of glutamine

from glutamate and ammonium. Ammonia is an undesirable byproduct of

cellular metabolism that is released into culture medium. Thus, this system

presents a dual effect: on one hand, it reduces the ammonia levels in the

culture medium and, on the other hand, it provides cells with a continuous

source of glutamine, which is an unstable amino acid. GS is speciﬁcally

and irreversibly inhibited by methionine sulfoximine (MSX), an analog of

glutamine. Therefore, cells overexpressing the enzyme are resistant to high

doses of MSX (Sanders and Wilson, 1984). Similarly to the DHFR system,

the GS/MSX system has proved to be effective for gene ampliﬁcation. A

typical example of this system is based on NS0 cells transfected with the

GS gene and treated with 10–100 M MSX, which allowed the isolation of

resistant clones containing several copies of the GS and the heterologous

gene (Bebbington et al., 1992).

folic acid

DHFR

dihydrofolate (FH )

tetrahydrofolate (FH )

MTX

serine ⫹

glycine

TMP

dUMP

Figure 3.4

Gene ampliﬁcation. (A) The DHFR enzyme catalyzes the conversion of folate to

tetrahydrofolate (FH

)viatheintermediateproductdihydrofolate(FH

). FH

essential for the biosynthesis of purine, glycine, and thymidine monophosphate

(TMP) from deoxyuridine monophosphate (dUMP). (B) In the DHFR system, the

expression of recombinant protein is increased by exposing the cells to high

methotrexate (MTX) concentrations. MTX is an analog of folic acid that binds to

DHFR, inhibiting the enzyme and causing cell death. Increasing concentrations of

MTX in the culture medium induce a selection pressure for cells with high levels of

DHFR and, concomitantly, the gene of interest is ampliﬁed. Highly resistant clones

commonly contain hundreds to t housands of copies of the plasmid integrated in

their chromosomes.

Cloning and expression of heterologous proteins in animal cells 63

Finally, it is worth emphasizing that the optimization of the selection

method must precede any attempt to obtain recombinant cell lines.

Performing a dose-response assay is highly recommended to assess the

lowest concentration of the antibiotic that kills the wild-type cells after a

given period of time.

3.8.3 Reporter markers

The advent of reporter markers has greatly contributed to a better under-

standing of gene expression in eukaryotic cells. A reporter gene encodes

for a protein that can be monitored by simple and sensitive methods.

Reporter genes are commonly used to: (i) optimize and monitor DNA

delivery methods; (ii) study a variety of promoters and regulatory ele-

ments, since the expression levels of the reporter protein will reﬂect the

transcription activity; (iii) analyze the trafﬁc and/or the cellular compart-

mentalization of the studied protein; (iv) characterize transcription factors

and associated proteins, such as co-activators; (v) identify protein–protein

interactions; and (vi) develop high-throughput screening of compounds

with potential therapeutic effects. An ideal reporter gene should meet the

following characteristics: (i) to be absent or expressed in very low levels in

the cell; (ii) to be detected by a simple, sensitive, and reliable method; (iii)

not to induce pleiotropic effects in the host cell. A signiﬁcant number of

reporter genes are suitable for use in mammalian cells, whose expression is

evidenced by colorimetric, ﬂuorescence, or chemiluminescence methods.

Some examples are described below.

Chloramphenicol acetyl transferase (CAT). This bacterial enzyme was

the ﬁrst reporter protein used for studying the transcriptional activity of

eukaryotic regulatory sequences (Gorman et al., 1982). CAT inactivates

chloramphenicol, an inhibitor of prokaryotic protein synthesis, by con-

verting it to the mono- or di-acetylated species. Measurement of CAT

activity requires a

C-radiolabeled chloramphenicol or acetyl-CoA and,

therefore, an additional step is neccessary to separate the radio-labeled

reactant from the product. Novel detection methods based on ﬂuorescent

substrates or ELISA assays, which do not use radiolabeled reagents, have

been described more recently (Bullock and Gorman, 2000).

Beta-galactosidase (-gal). The -gal enzyme from E. coli is the most

frequently used reporter marker in science, because it is functionally active

and well tolerated in a variety of cell lines. -gal activity can be monitored

both qualitatively, by in situ staining, and quantitatively, in cell lysates,

without the need for sophisticated laboratory equipment. The enzyme

hydrolyzes many natural or synthetic -D-galactopyranosides, which

allows the use of different detection methods (Serebriiskii and Golemis,

2000). Because mammalian cells have endogenous -gal activity, this

reporter gene should be used only in cells displaying a low endogenous

activity. In situ detection of -gal requires cell ﬁxation and addition of the

colorless substrate 5-bromo-4-chloro-3-indolyl--D-galactoside (X-Gal),

which is converted into a blue precipitate by the enzyme action. On the

other hand, determination of -gal activity in cellular extracts is performed

by a simple and fast assay based on the conversion of the colorless ortho-

nitrophenyl--D-galactopyranoside (ONPG) into galactose and ortho-

64 Animal Cell Technology

nitrophenol, which is detected at 420 nm (Miller, 1972). Alternatively,

-gal activity can be assessed in vivo using the substrate FdG (ﬂuorescein

di--D-galactopyranoside). Hydrolysis of this compound releases ﬂuor-

escein, which is retained intracellularly and can be measured by ﬂuores-

cent activated cell sorting or FACS (Rossi et al., 2000). Different -gal

detection protocols are reviewed by Alam and Cook (2003).

Luciferase. Luciferase and luciferin are a non-speciﬁc enzyme and its

substrate, respectively, that upon reaction in the presence of ATP, Mg

2þ

and O

generate bioluminiscence. Different genes or cDNAs encoding

luciferases have been isolated from different organisms, such as unicellular

seaweed, marine bacteria, and ﬁsh. Due to the speciﬁcity of the reaction,

many luciferases have been used as reporter proteins either in prokaryotic

or eukaryotic cells (Greer and Szalay, 2002). Fireﬂy luciferase (LUC) is

one of the most widely used. The oxidation reaction produces oxylucifer-

in, CO

, and a photon. The intensity of light emitted during the reaction is

measured with a luminometer. Luciferase-based techniques are sensitive,

simple, rapid, inexpensive, safe (organic solvent or radiolabeled com-

pounds are not required for the test), and applicable to a wide range of cell

lines. One of the most recent advances constitutes the development of

highly sensitive techniques that make the detection of luciferase activity in

living cells and organisms feasible (Greer and Szalay, 2002).

Green ﬂuorescent protein (GFP). GFP was discovered during the

investigation of marine bioluminescent organisms. In the 1960s, it was

demonstrated that Aequoria jellyﬁsh emitted light after the addition of

2þ

, with no need for other cofactors. The gene encoding the Aequoria

GFP was cloned in 1991, and is used as a reporter in cells, where upon

excitation at 395 nm a green ﬂuorescence is emitted at 509 nm. The GFP is

exceptionally stable with respect to pH, temperature, and proteolysis, and

can be detected in living cells and organisms (Yang et al., 1996). Due to the

prolonged time required between the post-translational maturation and

the appearence of ﬂuorescence (3–5 hours) in the wild-type GFP (Brand,

1995), different mutants exhibiting an increased ﬂuorescence intensity

have been generated (Welsh and Kay, 1997). The enhanced GFP (EGFP)

is a variant with two amino acid substitutions (Phe64Leu and Ser65Thr),

whose ﬂuorescence is shifted to the red spectrum. The advantages that

EGFP presents over the wild-type species are: (i) an increased ﬂuorescence

intensity; (ii) a shorter time period between protein synthesis and ﬂuores-

cence; and (iii) higher expression levels in several animal cells. Currently,

there are also yellow (EYFP) and cyan (ECFP) variants, which are excited

at 514 and 434 nm, with a maximal emission at 477 and 527 nm, respec-

tively.

Alkaline phosphatase (AP). APs refer to a class of hydrolase enzymes

that catalyze the hydrolysis of a broad range of monophosphates and have

an optimal activity at alkaline pH (10–10.5). APs are found in all organ-

isms. In earlier times, the use of APs as reporter marker was hampered

because most animal cells exhibit high endogenous activity. This limitation

was overcome by Berger et al. (1988), who generated a secreted form of

the human placental AP, known as SEAP. The enzymatic properties of the

SEAP are equivalent to the wild-type AP. AP activity derived from SEAP

is distinguished from the endogenous AP by assaying the activity in an

Cloning and expression of heterologous proteins in animal cells 65

aliquot of culture supernatant. In general, SEAP offers certain beneﬁts

when compared with other intracellular reporter enzymes: (i) there is no

need for cell lysis; (ii) the kinetics of gene expression is easily monitored

by harvesting and assaying the supernatant periodically; (iii) living trans-

fected cells remain available for additional phenotypic characterization,

such as cell growth, speciﬁc mRNA expression, and other enzymatic

activities; and (iv) the test presents good sensitivity due to the use of

chemiluminiscence substrates (Alam and Cook, 2003)

3.9 Screening, quantitation, and bioassay methods

Once the selection process is completed, many resistant clones may be

obtained, which express the heterologous gene at different levels. Accord-

ing to experienced researchers, the expression levels of ‘‘overproducer’’

clones should be 5–30-fold higher than the average of the pool of clones.

These variations in the expression levels are independent of the cell line

and the transfection method used (Wirth and Hauser, 1993). For that

reason, it is necessary to have screening and monitoring methods that are

sensitive and speciﬁc enough to detect and quantify the protein expressed

by high producer clones in early stages. In general, protein detection can

be carried out by assaying its biological activity (in the case of enzymes,

coagulation factors, cytokines, growth factors, hormones, etc.) or by using

an immunochemical assay (ELISA, Western blot, RIA, dot blot, etc.). The

ﬁrst provides a precise measurement of the protein functionality, while the

second is only useful to identify the molecule without an indication of

activity. In general, the immunochemical methods are technically simple,

fast, and less costly than those based on the assesment of biological

activity.

In the case of recombinant products expressed with a tag or fusion

protein, the immunoassay can be directed towards the detection of the tag,

for example, in the case of an antibody that reacts against myc-tag (see

Section 3.4.3). Another example is the use of bicistronic vectors where,

apart from the gene of interest, a reporter gene is coexpressed and its

properties are exploited for the selection and isolation of recombinant

clones (i.e. GFP-coexpressing cells sorted by ﬂow cytometry). Alterna-

tively, high speed cell sorting can be used for the selection and separation

of cells when the reporter gene is expressed as a surface protein, for

example, a receptor. In this example, the separation is based on the use of a

ﬂuorescent-labeled antibody that reacts against the surface protein.

3.10 Optimizing the initial stage of an animal cell-based

bioprocess

In summary, it can be concluded that the optimization of the expression of

heterologous genes in animal cells deserves careful design. Firstly, the

biological properties of the ﬁnal product and the amount required for its

further application (e.g. diagnostics or therapy) have to be considered.

These two factors determine the strategy and expression system that best

suit the needs of the recombinant product. Both the vector and the target

66 Animal Cell Technology

sequence can be genetically manipulated, adding or removing elements

that can improve the rate of transcription and translation of the gene of

interest. The selected host for the production of the recombinant protein

must be able to introduce the post-translational modiﬁcations required for

the biological activity of the product. It is essential that the host cell is

cultivated under optimal conditions to warrant higher transfection efﬁ-

ciencies, and to facilitate the selection, cloning and growth of the future

recombinant cell lines. Finally, selecting the most suitable transfection

method and choosing the selection marker and screening technique are

pivotal issues that can substantially increase the probability of isolating

recombinant cell lines with high speciﬁc productivities.

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