Attempt at Conserving the Genetic Resources of Tan Sheep by Conserving its Fibroblast Line
Tan sheep is one of the most distinctive local sheep breeds in China, while investigations concerning its cell biology and molecular genetics are still scarce. To preserve the valuable genetic resources of Tan sheep, we established a Tan sheep fibroblast line and identified its biological characteristics, including cell morphology, growth curve, karyotype, isoenzyme polymorphism and expression of exogenous fluorescent genes. The results showed that the viability of the fibroblasts before freezing was 96.75±3.24 and 93.42±2.87% after thawing. The growth curve of Tan sheep ear marginal fibroblasts displayed an obvious S shape and there was no microbial contamination and cross-contamination; all somatic chromosomes were acrocentric autosomes and only the two sex chromosomes were submetacentric; the transfection efficiencies of the three fluorescent protein genes were 20.6-36.2%. It could be concluded that the this study has not only provided the biological characteristics of Tan sheep at cellular level but also made a valuable contribution to the preservation of the genetic resources of the Tan sheep.
Received: April 14, 2013;
Accepted: August 17, 2013;
Published: September 25, 2013
Biodiversity is facing unprecedented challenges worldwide and local breeds
are threatened by the introduction of foreign species, massive destruction of
their natural habitats and industrial pollution. The genetic significance of
local breeds as a reservoir of genetic variations and major genes has been realized
(Horst, 1989) and variation needs to be known and how
it can be conserved and exploited effectively (Karp et
al., 1998). Traditionally, in vitro conservation involves materials
like semen, embryos and oocytes, however, a number of reasons why these techniques
cannot be used for endangered breeds globally. Instead, storage of somatic cells
constitutes an optimal choice, each somatic cell contains the full genetic code
of the whole animal which can be readily collected.
Tan sheep is one of the most distinctive local sheep breeds in China and was
listed among the 138 nationally protected domestic animal breeds by the Chinese
government in (2006) (http://www.agri.gov.cn/blgg/t20060609_626418.htm).
To date, efforts have been focused on analyzing various quantitative traits
of the Tan sheep. While investigations concerning its cell biology and molecular
genetics are still scarce. In this study, Tan sheep fibroblasts from the ear
marginal tissue of adult sheep (20 males and 23 females) were isolated and cultured
and their biological characteristics were subsequently analyzed.
MATERIALS AND METHODS
Preparation of fibroblasts: During all the experiments, all procedures
and protocols were in compliance with the Statement on Animal Care and Usage
in Research and Teaching. The ear marginal tissue samples of Tan sheep were
chopped into 1 mm3 pieces using ophthalmic scissors. Then the tissue
pieces were plated on the bottom of culture flasks with DMEM containing 10%
Fetal Bovine Serum (FBS) and cultured at 37°C, 5% CO2 and saturated
humidity until near confluence.
When they reached 85% confluence, the cells were subcultured with 0.25% trypsin
(m/v) at a ratio of approximate 1:3. Cells were cultured in fresh medium 24
h prior to freezing to make sure the nutrition was sufficiently absorbed. For
freezing, cells were trypsinized and suspended in freezing media (DMEM containing
30% FBS and 10% DMSO) at the concentration of 1.5x106 cells mL-1.
The suspension was aliquoted in cryovials and stored at -70°C for 24 h before
being transferred to liquid nitrogen. For recovery the cryovials were thawed
in 37°C water bath and the cells were then transferred to culture flasks
with medium containing 10% FBS.
Viability and growth kinetics: Cell viability before freezing and after
recovery was determined using Butlers dye exclusion method (Butler,
1999). Growth curve of the cells was plotted following the method of Gu
et al. (2006).
Tests for microbial contamination: The procedure used for detection
of bacteria, fungi and yeast was as described in Doyle et
al. (1990). The cells were stained with Hoechst 33258 according to the
DNA fluorescent staining protocol to detect mycoplasmas which is also recommended
by American type culture collection.
Chromosomal analysis: Karyotypes were prepared following the protocol
described in the Reading Conference report (Ford et
al., 1980). Diploid percentage was determined by analyzing 100 spreads.
Isoenzyme polymorphism: The electrophoretic mobilities of Lactate Dehydrogenase
(LDH) and Malate Dehydrogenase (MDH) were determined using the polyacrylamide
gel electrophoresis protocol described by Macy (1979)
of American type culture collection.
Expression of fluorescent genes in Tan sheep fibroblasts: The fluorescent
protein genes pEGFP-C1, pEGFP-N3, pEYFP-N1 and pDsRed1-N1 were transfected into
cells with Lipofectamine 2000 (Invitrogen Corp., Carlsbad, California) in serum-free
medium. The cells were observed at 24, 48 and 72 h after transfection using
laser confocal microscope (Nikon TE-2000-E, Japan).
In primary culture, fibroblast-like or epithelial-like cells could be seen
migrating from the tissue pieces 5-12 days after explanting (Fig.
1a). Fibroblasts grew rapidly and gradually replaced the epithelial cells
in the entire population. Cells were well spread on the flask bottom, forming
characteristically multipolar or bipolar shapes (Fig. 1b).
The viability (formulated as mean±SD) was 96.75±3.24% before freezing
(Fig. 1c) and 93.42±2.87% after thawing (Fig.
1d), the difference between which is non-statistically significant (p>0.05)
and assumed an obvious S shape (Fig. 2). The population
doubling time was approximately 24 h.
||Morphology of Tan sheep fibroblasts cultured in vitro,
(a) 6 days after explanting, (b) Near confluence (c) Before cryopreservation
and (d) 24 h after recovery
|| Growth curve of Tan sheep fibroblasts
A pure cell culture can easily become contaminated with microorganism. Upon
analysis, the culture medium tested did not become turbid or display other visible
abnormalities whereas the positive control was visibly turbid with precipitation.
No viruses were indicated by the cytopathogenic evidence or by the hemadsorption
test. The nuclei appeared as clear blue ellipses stained with Hoechst 33258,
disproving the existence of mycoplasmas. The results indicated there was no
microbial contamination in the Tan sheep cell line.
Detailed information for karyotype preparation is available in the Reading
Conference report (Ford et al., 1980). In Tan
sheep the diploid chromosome number is 54, consisting of 52 autosomes and two
sex chromosomes, XY or XX. The chromosomal properties were shown in Table
1, the chromosome numbers per spread were counted for 100 spreads of the
1-3 passages and the diploid frequency were 94.5-97.8%. Aberrations in chromosome
numbers tended to increase with increasing numbers of passages, indicating that
in vitro culture affected the heritage of cells slightly but supporting
the inference that the cell line was reproducibly diploid. These results showed
that all autosomes were acrocentric and the two sex chromosomes (XY) were submetacentric
The LDH bands of Tan sheep were compared with those of Songliao Black pig and
five bands (LDH-1,-2,-3,-4,-5) were observed in all samples (Fig.
4a). The MDH bands were compared with those of Suffolk sheep and two bands
(s-MDH, m-MDH) were observed (Fig. 4b). The isoenzyme patterns
of LDH and MDH of Tan sheep fibroblasts were clearly distinguishable from those
of other cell lines in our laboratory.
|| Chromosome parameters of Tan sheep fibroblasts (male)
|| Karyotype of Tan sheep fibroblasts (male)
||Isoenzyme patterns of (a) LDH Lanes1 and 2: Songliao Black
pig; Lane 3 and 4: Tan sheep (b) MDH, Lanes 1 and 2: Tan sheep; Lane 3:
Using the method of Tsuchiya et al. (2002),
the fluorescent protein genes pEGFP-N3, pDsRed1-N1 and pEYFP-N1 were transfected
into cells with Lipofectamine 2000 (Invitrogen Corp., Carlsbad, California)
in serum-free medium. The cells were observed at 24, 48 and 72 h after transfection
using laser confocal microscope (Nikon TE-2000-E, Japan). The results indicated
that the transfection efficiencies of the three fluorescent protein genes were
between 20.6 and 36.2%. And for all the three genes the strongest fluorescence
intensities and the highest transfection efficiencies appeared at 48 h after
transfection (Fig. 5).
Traditionally in vitro conservation involves materials like semen, embryos
and perhaps oocytes. Placement in liquid nitrogen allows indefinite storage,
while recovery is relatively straightforward. There are, however, a number of
reasons why these techniques cannot be used for endangered breeds globally.
Deep freezing of semen and embryos can only be performed on numerable species
and furthermore requires species specific techniques (Groeneveld
et al., 2006). It requires substantial infrastructure which is not
generally available in all countries, not to mention considerable costs, an
issue also pointed out by Woolliams and Wilmut (1999).
Instead, storage of somatic cells constitutes an optimal choice as illustrated
by Corley-Smith and Brandhorst (1999). The principle
rests on the fact that each somatic cell contains the full genetic code of the
whole animal which can be readily collected, for instance, from ear notching,
making the sampling process cheap and fast and thus also applicable in countries
with poor facilities. In addition somatic cells can be collected from every
animal easily, for instance, from ear margin, making the collection of samples
cheap and fast.
Morphology, one of the most important qualitative criteria of epidermal tissue
reconstitution, is commonly evaluated with light and electron microscopy.
In this study the cells exhibited fibrous appearances with turgor vitalis
cytoplasm and during growth they showed typically fibroblast-like morphology
with radiating, flame-like or whirlpool-like migrating patterns.
||Tan sheep fibroblasts cultured for 48 h after transfected
by pEGFP-N3 (a, d, g), pDsRed-N1 (b, e, h) and pEYFP-N1 (c, f, i), (a),
(b), (c) Merged pictures of phase contrast and fluorescence; (d, e, f, g,
h), I pictures in fluorescence; a, b, c, d, e, f (100x); G, H, I (400x)
The genetic stability of cell line is critical to preserve the genetic resources,
prescribing that the fibroblasts must maintain the same diploid character as
those in vivo. As most mutants cultured in vitro still possessed
the division capability, after successive cell divisions a significant differentiation
would appear in the cell lines it which cannot be subsequently used in breed
conservation. Chromosome analysis can identify the gender of the animal from
which a cell line was derived and also distinguish between normal and malignant
cells, due to the instability of chromosome number in the neoplastic ones (Freshney,
2000). Chromosome number per spread was counted from 100 spreads of cells
from passages 1, 2 and 3. The normal number of chromosomes (2n = 54) was observed
in 96.56±2.89% of the cells, further validating the stability of these
cells. Enzyme polymorphism, evidenced by the existence of isoenzymes, occurs
among species and races, as well as tissues within an organism (OBrien
et al., 1977). Isoenzymes can be separated chromatographically or
electrophoretically, displaying characteristic distribution patterns. Biochemical
analysis of isoenzyme polymorphism is currently considered as a standard method
for quality control of cell line and is routinely used by leading biological
resource centers (Parodi et al., 2002).
Apart from the need to determine the tissue of origin of a culture, it is also
important to avoid cross-contamination from other cell lines. Isoenzyme analysis
is an extremely reliable and straightforward technique which provides a rapid
and reliable identification of species of origin. While the species of origin
of a cell line can usually be determined with only two isoenzyme tests (lactate
dehydrogenase and glucose-6-phosphate dehydrogenase), specific identification
of a cell line would require a larger battery of tests (Halton
et al., 1983). This procedure retained the advantage of rapid testing
while also provided a useful level of specificity for identification purposes.
As demonstrated above, biochemical analysis of isoenzyme polymorphism is currently
considered to be a standard method for cell line quality control and identification
and detection of interspecies contamination and is routinely used by the leading
biological resource centers around the world. In this study, the isoenzyme bands
of LDH and MDH of Tan sheep fibroblasts were clear, indicating that the cultures
were free of contamination by other cell types.
The researches about fluorescent protein are mainly focused on tumor cell,
nerve cell and stem cell (Jung et al., 2001).
We could see that the transferring cells at the state of reduplication and different
dividing phase and the growth and reduplication of transfected cell were no
transparent difference with control group. The result showed that the transfected
cells were not been effected by fluorescein under certain range.
In this study, the quality of the Tan sheep fibroblast cell line was evaluated
in the aspects of morphology, viability, microorganism detection, chromosome
analysis, isoenzyme analysis and exogenous gene transfection. The viability
of the fibroblasts before freezing was 96.75±3.24 and 93.42±2.87%
after thawing. The growth curve of Tan sheep ear marginal fibroblasts displayed
an obvious S
shape and there was no microbial contamination and cross-contamination; all
somatic chromosomes were acrocentric autosomes and only the two sex chromosomes
were submetacentric; the transfection efficiencies of the three fluorescent
protein genes were 20.6-36.2%. It could be concluded that the this study has
not only provided the biological characteristics of Tan sheep at cellular level
but also made a valuable contribution to the preservation of the genetic resources
of the Tan sheep.
This research was funded by the Ministry of Agriculture of China for Transgenic
Research Program (2011ZX08009-003-006, 2011ZX08012-002-06), the project National
Infrastructure of Animal Germplasm Resources (2013) and China Agriculture Research
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