The Piscine SAF-1 Cell Line: Genetic Stability and Labeling
Julia Béjar, Javier Porta, J. José Borrego, M. Carmen Alvarez
Department of Genetics, Faculty of Sciences, University of Málaga, 29071 Málaga, Spain
Received: 14 July 2004 / Accepted: 18 November 2004 / Online publication: 4 June 2005
Abstract
Fish cell lines are increasingly important research
tools. The SAF-1 cell line, fibroblast-like culture
derived from the marine fish gilthead seabream
(Sparus aurata), has proved useful in many applications, especially in viral research. For cell lines intended as in vitro models, characterization of their
properties and authentication are essential for deeper
understanding of their performance and thus more
precise experimental design and applicability. In this
study we characterized the SAF-1 cell line in terms
of genetic stability through time and genetic labeling. Methods for determining stability include telomerase activity, karyotyping, mapping of ribosomal
RNA regions, and DNA content. For genetic labeling
12 microsatellite loci were used. The results indicate
that telomerase has been activated in the course of
SAF-1 development, and the highest levels of telomerase activity correlate with an increase in cell
proliferation, thus supporting a permanent cell line.
This stability is in agreement with the normal situation presented by the cytogenetic traits and DNA
content values, and the genotypic profile allows
SAF-1 authentication at the single individual level.
This study increases the value of SAF-1 as an in vitro
system, which is now one of the few well-characterized cell lines from a marine fish.
Key words: Sparus aurata — fish cell line — genetic
stability — telomerase — microsatellites
Introduction
Fish cell lines are increasingly important as research
resources, both to gain basic knowledge and to obtain
fish products that can be used in aquaculture. Even
though a number of cell lines have been developed,
they still represent an exiguous sample of the large
Correspondence to: Julia Béjar, E-mail: [email protected]
number of existent bony fish species (Bols and Lee,
1991). The marine fish Sparus aurata (gilthead seabream) is one of the most important species in European aquaculture. Because much is known about its
biology, it is a suitable model for the domestication
of other sparid fish. The SAF-1 cell line (Figure 1) was
developed in 1996 from this species. It consists of
fibroblast-like cells derived from fin of an adult
specimen that spontaneously immortalized (Béjar et
al., 1997). It has been largely used as a successful
experimental system, mostly in the study of marine
fish pathogens (Béjar et al., 1997; Garcia-Rosado et
al., 1999; Pérez-prieto et al., 1999; Tafalla et al.,
2004), as well as in fish immunology (Pelegrı́n et al.,
2004). Its interest as a model system is expected to
increase because of the absence of available cell lines
from marine species of economic importance.
Characterization of the properties of cell lines
over time is important as their genetic profile can
change owing to selection of new mutations during
culturing. An initial characterization of SAF-1
showed normal karyotype (Béjar et al., 1997), suggesting genetic stability. For cell lines intended as
model systems, genetic stability is a main attribute;
thus SAF-1 was further characterized after several
years of culture to assess its genetic stability through
time. Consequently, we examined the SAF-1 cell
line in terms of the following traits: telomerase
activity, cytogenetic markers including karyotype,
and major minor ribosomal RNA loci and DNA
content. The results revealed that telomerase had
been activated during the immortalization process,
and that the activity levels have been steadily
maintained through the life span. This stability is
consistent with the normal situation for karyologic
traits. Another important issue for cell lines is to
determine whether there is contamination with
other cell lines, which represents a potential problem (Langdon, 2004). For identification purposes genetic labeling was performed by means of
microsatellites. The genotypic profile obtained supports the species-specific origin and clonal nature of
DOI: 10.1007/s10126-004-4083-0 Volume 7, 389–395 (2005) Springer Science+Business Media, Inc. 2005
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Fig. 1. SAF-1 cells in culture showing typical fibroblastlike morphology. Bar represents 100 lm.
the cells, and allows the identification of SAF-1 at
virtually the single individual level.
Materials and Methods
Cell Cultures. For all tests performed in this study,
SAF-1 cells from different passages were used. Cells
were cultured in L-15 Leibowitz medium, supplemented with 15% fetal bovine serum (FBS), 4 mM
L-glutamine, and antibiotics (penicillin, 100 U/ml;
streptomycin, 100 lg/ml; and amphotericin, 250 ng/
ml) (Béjar et al., 1997). The cultures were seeded in
plastic tissue culture flasks or dishes, incubated at
25C in a normal atmosphere incubator, and routinely subcultured according to the standard trypsinization method.
Fibroblastic primary cultures derived from fin
explants were obtained according to Alvarez et al.,
(1991).
Telomerase Assay. The telomerase activity of
SAF-1 cells at various passages, and of other cellular
groups was analyzed by a modified version of the
telomeric repeat amplification protocol (TRAP) assay (Pyatiszek et al., 1995). The TRAP assay was
performed on lysates prepared from about 105 cells of
3 replicates from each group. The assay was performed with a commercial kit (Boehringer), following the instructions provided by the manufacturer.
Polymerase chain reaction (PCR) products were
analyzed by a photometric enzyme-linked immunosorbent assay (ELISA). Negative controls from
each group consisted of the corresponding cells
treated with RNase (10lg/ll).
Chromosome Preparations, Fluorescence in situ
Hybridization, and DNA Content. Chromosome
metaphases were obtained to assess the karyotype and
the rRNA regions. For chromosome plates exponentially growing cultures from passages 50, 100, and 150
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were treated according to Alvarez et al., (1991). Cells
were incubated in colchicine (final concentration, 10
ng/ml) for 2 hours and dissociated with trypsin. The
cell suspension was centrifuged for 5 minutes at 1200
rpm; the pellet was gently resuspended in 20 mM KCl
and incubated for 30 minutes at room temperature.
After hypotonic treatment the cell suspension was
washed 3 times in fresh cold fixative (3:1 methanol–
acetic acid), and the cells were then dropped onto cold
wet slides. The slides were air-dried overnight at 4C.
A 4% Giemsa staining solution was used for conventional staining. Chromosome counting was based on
54 metaphase plates from passage 50, 56 from passage
100, and 61 from passage 150.
Ribosomal RNA regions were revealed by the
fluorescence in situ hybridization technique (FISH) in
cells from passage 100. The rRNA genes are represented by the major rRNA (5.8S, 18S, and 28S) and the
minor rRNA (5S) families. For major rRNA genes 28S
rDNAs from salmon were labeled by nick translation
with biotin-16-dUTP. The probes for minor rRNA
genes were obtained by PCR from genomic DNA of
Diplodus puntazzo, a species close to S. aurata, by
using 2 conserved primers designed from a 5S rRNA
region of rainbow trout (Komiya and Takemura,
1979). PCR products were labeled by nick translation.
Slides were pretreated with RNAse, and the FISH
protocol was followed (Péndas et al., 1993). Metaphases were visualized under a Zeiss UV microscope
equipped with a CCD photometric camera.
For DNA content determination quantitative
DNA analysis was performed by image cytometry on
cells stained with propidium iodide (Béjar et al.,
1997). The cell line was screened at passages 50, 100,
and 150. Fibroblast-like cells from primary fin tissue
culture were used as control.
Microsatellite
Analysis. For
microsatellite
analysis, DNA was extracted from a SAF-1 cell pellet, following a saline precipitation method (Martı́nez et al., 1988).
A set of 12 highly polymorphic microsatellite loci,
specifically developed for S. aurata and previously
tested in our laboratory for population genetics studies, were selected for SAF-1 labeling purposes. These
were SauK140 (AY173042), SauI47 (AY173041),
SauE97 (AY173036), SauE82 (AY173035), SauD182
(AY173034), and SauAn (AY173032) (Launey et al.,
2003); pSAGT26 (Y17266) (Batargias et al., 1999);
SaI19 (AY322111), SaI15 (AY322110), SaI14
(AY322109),
SaI12
(AY322108),
and
SaI10
(AY322107). The matching probability (Mp), as the
number of individuals that may be surveyed before
finding the same genotype in a randomly selected
sample [15], has been calculated.
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Table 1. Telomerase Activity in Different Cell Groups
a
Absorbance units
Cell group
S
NC
WAC-2 tumoral cell line
Seabream blastocytes
Primary culture
SAFs-1, 50 passages
SAFs-1, 100 passages
SAFs-1, 150 passages
0.924
1.161
0.110
0.317
0.439
0.422
0.088
0.081
0.108
0.052
0.092
0.087
a
Mean values obtained from 3 replicates are shown. Differences
between the replicates were never higher than 15%; S indicates
sample; NC, negative controls of each sample, corresponding to
cells treated with RNase before the telomerase assay.
Each locus was PCR-amplified with its respective pair of primers, with similar conditions used for
all of them: 5 minutes at 94C, followed by 30 cycles
of 30 seconds at 94C, 30 seconds at 58C, and 45
seconds at 72C, with a final extension of 7 minutes
at 72C. The PCR products were run on an ABI310
automated sequencer (Applied Biosystems) to determine the allele sizes. Genotypes were determined
with GENOTYPER Version 3.7 software.
Results
Telomerase Activity. The mean values of telomerase activity obtained from different cellular groups
are presented as absorbance units in Table 1. The
data reveal that SAF-1 cells at the 3 screened passages, 50, 100, and 150 (Table 1), show significantly
lower levels of telomerase activity than those of the 2
groups used as positive controls (human tumor cells
and gilthead seabream embryonic cells) and higher
than those from primary cultures that represent the
type of cells from which SAF-1 has been derived, with
virtually no telomerase activity. However telomerase
activity of SAF-1 cells is slightly lower at passage 50
than those at passages 100 and 150 (Table 1). These
data indicate that telomerase was apparently activated sometime between primary cultures and passage 50 and slightly increased to stable levels over the
course of passages 100 and 150.
Cytogenetic Traits. Changes in the karyotype
affecting chromosome number or morphology have
often been associated with adaptation of cells to
in vitro conditions (Ghosh and Chaudhuri, 1984).
Therefore the first step of a genetic characterization
might consist of checking the diploidy status of the
cells. The results of chromosome counts on SAF-1
cells at 50, 100, and 150 passages (Figure 2, A, B, and
C, respectively) show in the 3 cases a distribution
with a modal peak value at 2n = 48, which corresponds with the diploid number of the species (Sola
Fig. 2. Chromosome number distribution of SAF-1 cells at
50 passages (a), 100 passages (b), and 150 passages (c).
and Cataudella, 1978; Alvarez et al., 1991). In the 3
curves several chromosome numbers appear below
and very few appear above the normal diploid number. The aneuploid values under 48 are regularly
produced by artifacts that are inherent to the
production of chromosome spreads, from either in
vivo or in vitro cellular sources; however, values
over 48 represent aneuploid cells that can occur by
incorrect segregation under culture conditions
(Mathieu et al., 2004). As a whole the 3 distributions
of chromosome numbers fit the standard profile of a
cultured population of euploid cells. Moreover,
392
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Fig. 3. Karyogram obtained from a SAF1 metaphasic cell showing normal
morphology of each chromosome pair.
Bar represents 10 lm.
metaphases with normal diploid number also displayed normal karyotype morphology (Figure 3 and
Alvarez et al., 1991).
The major and minor rRNA regions are known
for high instability (Miller, 1983) due to their DNA
arrangement in the form of tandem repeats. This
instability can result in rearrangements revealed as
chromosome polymorphisms in the rRNA regions,
which have been described in both fish stocks
(Phillips et al., 1998) and fish cell lines (Sánchez
et al., 1993).
In the SAF-1 cells the major rRNA genes appeared as one signal on the telomeres of the short
arms of the largest biarmed chromosomes (Figure 4a,
arrows; Figure 3, pair 1), which coincides with the
results obtained in S. aurata (Garrido-Ramos et al.,
1995). Hybridization with the 5S rDNA probe produced a single signal in an interstitial position of a
medium-sized acrocentric pair (Figure 4B), which
coincides with the authors’’ previous observations in
this species (unpublished results). Altogether these
results indicate that the rRNA regions in SAF-1 have
the standard species-specific pattern, suggesting genetic stability for these regions.
DNA Content. In Figure 5 the DNA contents of
4 cell groups are represented as flow cytometry histograms: a euploid population of primary cultures
from fin clips of S. aurata (A) and SAF-1 cell samples
from passages 50, 100, and 150 (B, C, and D,
respectively). All of them show similar profiles with
2 peaks in the same position of the abscissa. The first
peak corresponds to cells in G1 with DNA content
around the 2c value for this species (Béjar et al.,
1997). The second peak represents the fraction of
cells in G2–M with DNA content around the 4c value. In spite of the similarity in the 4 distributions,
the second peak is lower in primary cultures and
SAF-1 of 50 passages (Figure 5, A and B, respectively), than in SAF-1 from passages 100 and 150 (C
and D, respectively), indicating an increase in the
division rate sometime between passages 50 and 100.
Microsatellite Genotyping. The DNA typing of
the SAF-1 cells with 12 microsatellites is shown in
Table 2. All alleles of SAF-1 are widely represented
in the cultivated stock from which the donor individual came (unpublished results). Interestingly, the
cell line is heterozygous for all loci except the SauAn
locus, which only presents one allele. The power of
discrimination of this set of loci is extremely high
(Mp = 2.13048 · 10)31), thus allowing the identification of SAF-1 at the single individual level.
Discussion
The long-term SAF-1 cell line (Figure 1) was established in 1996 (Béjar et al., 1997), and since then it
Fig. 4. FISH on SAF-1 metaphase
plates hybridized with rDNA
probes. a: Hybridization with 28S
rDNA probes. b: Hybridization
with 5S rDNA probes. Arrows
indicate fluorescent signals. Bar
represents 10 lm.
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393
Fig. 5. Flow cytometric analysis for
DNA content. The x axis represents
the relative amount of DNA per cell.
The y axis represents the relative
number of cells. Primary culture
cells (a), SAF-1 cells at passage 50 (b),
SAF-1 cells at passage 100 (c), and
SAF-1 cells at passage 150 (d).
has been a successful experimental system both in
the study of marine fish pathogens (Béjar et al., 1997;
Garcı́a Rosado et al., 1999; Pérez-Prieto et al., 1999;
Tafalla et al., 2004), and in fish immunology (Pelegrin et al., 2004). Early characterization of SAF-1
showed normal karyotype and DNA content (Béjar
et al., 1997), indicating genetic stability. After nearly
200 passages it seems to be maintained in a steady
state. Owing to the interest in the SAF-1 line as a
fish in vitro model, a survey of stability traits
through time was undertaken to allow a deeper
understanding of its performance and thus more
precise experimental designs and applicability.
When cells from differentiated somatic tissues
are seeded in vitro, they tend to become senescent
after a few passages because of the progressive
shortening of telomeres (Dell’Orco et al., 1973). One
mechanism for maintaining telomere length involves de novo addition of telomeric sequences by
telomerase; thus telomerase activity can be considered a good indicator of stability and proliferation in
cell cultures (Holt et al., 1996). The rare ability of
cell cultures to be maintained indefinitely (immortalization) can either occur spontaneously or be induced by a variety of treatments. Spontaneous
immortalization is unusual in mammalian cells,
with the exception of certain murine lines (Todaro
and Green, 1963). In fish, however, the limited
information available indicates that the majority of
Table 2. Microsatellite Characterization of saf-1 Line with
12 Microsatellitesa
Locus
Genotype
Matching probability
SauK140
Saul47
SauE97
SauE82
SauD182
SauAn
PSAGT26
SaI19
SaI15
SaI14
SaI12
Sal10
133/135
131/133
179/181
162/180
134/136
159/159
218/242
234/250
126/134
243/245
112/132
196/210
0.00284
0.011658
0.002189
0.003442
0.00234
0.0127
0.003274
0.001694
0.000659
0.001213
0.003314
0.001956
a
Genotypes correspond to the allele sizes of SAF-1 cells for each
locus.
cell lines have arisen spontaneously (Bols and Lee,
1991), as it is the case for SAF-1. This variable performance of animal species in immortalization has
been attributed to different mechanisms regulating
proliferative capacity (reviewed in Rhim, 2000).
This study reveals that the permanent SAF-1 line
is positive for telomerase activity, which might be
expected considering that it shows traits of immortality, and telomerase expression represents, in
general, a hallmark of immortality in cultured cells
(Counter et al., 1992). However, this statement is not
applicable to all types of mammalian cells, as human
immortal cells lines with long telomeres but no
detectable telomerase activity have been reported
(Bryan et al., 1995), a situation suggesting the existence of telomerase-independent mechanisms for
telomere maintenance. In the 4 cases of fish cell
lines for which telomerase activity has been analyzed so far (Barker et al., 2000; Béjar et al., 2002;
Ossum et al., 2004), and in the present report, telomerase activity is a common feature suggesting that
cells of piscine origin support the telomerase-related
immortalization model.
Data from Table 1 allow the inference of a temporal pattern of telomerase expression throughout
SAF-1 development. The lack of detectable telomerase activity in fin-derived primary cultures (Table 1),
representing the type of cells from which SAF-1 originated, indicates that telomerase has not been activated within a few days after seeding, as apparently
occurred in a leukocyte line from catfish (Barker et al.,
2000). However, in SAF-1, telomerase activity appeared later, in agreement with what happens in
general in mammalian cells (Counter et al., 1994). The
highest telomerase levels of SAF-1 that remain stable
through passage 150 are, however, significantly lower
than those presented by gilthead seabream blastocytes (Table 1) and the blastocyte-derived gilthead
seabream cell line SaBE-1c, with telomerase levels
similar to the parental cells (Béjar et al., 2000). So far,
the few examples of telomerase studies in fish cell
lines have indicated that levels of healthy telomeres
and strategies to keep them differ between cells types.
In spite of the controversy on the role of telomerase in immortalization, there is a consensus that
telomerase activity is a marker for cell proliferation
394
(Belair et al., 1997). Interestingly, in our study the
highest values of telomerase activity, appearing at
passages 100 and 150, coincide with those showing
the highest peaks of G2–M cells (Figure 5, C and D),
which indicates a higher proliferation rate. This
correlation in SAF-1 supports the idea that sufficient
levels of telomerase activity and the ability of cells
to increase the proliferation rate are requisites for
continual long-term proliferation and normal function of the cell line.
A common feature in the adaptation of cells to
in vitro conditions is the occurrence of changes in the
karyotype, affecting chromosome number or morphology, or both, as result of chromosome instability.
Chromosome rearrangements have been used as distinctive markers for specific cell lines (Sánchez et al.,
1993). This instability can be generated by culture
conditions that damage chromosomes, or defective
telomeres caused by replication-mediated shortening,
or defective telomere-associated proteins (reviewed in
Mathieu et al., 2004). In this way the diploidy status is
a good indicator of chromosome stability that may be
relevant to the proximity of a cellular system to the
original physiologic status. SAF-1 remains euploid, as
have other spontaneously immortalized fish cells
lines (Hong and Schartl, 1996; Barker et al., 2000).
Stability in chromosome number is supported by
normal karyotype (Figure 3), and by normal traits of
the rRNA regions (Figure 4). Additionally, the temporal evaluation of the DNA content, from primary
cultures to passage 150 (Figure 5), reinforces the evidence that no gross DNA additions or deletions have
been produced in SAF-1.
The capacity to authenticate a cell line is very
useful, especially when contamination with other
cell lines is suspected. The labeling of SAF-1 with 12
microsatellites demonstrated the species-specific
origin and the clonal nature of the cells, and allowed
levels of discrimination at the single individual level. For practical purposes and because of the high
heterozygosity presented, only 3 or 4 loci of Table 2
would be sufficient for authentication.
In conclusion, this study documents the genetic
stability of a permanent cell line from gilthead
seabream, SAF-1, that along with the labeling with
genetic markers, confers an added value to the line,
whose interest as an in vitro system is expected to
increase, considering the few available cell lines
from marine fish.
Acknowledgments
The authors are grateful to Dr. Alberto M. Pendás for
the FISH experiments and Teresa Méndez for excellent technical assistance.
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