Introduction
Somatic cell nuclear transfer (SCNT) in combination with transfection of somatic cells provides a more efficient approach to produce transgenic animal than the traditional pronuclear DNA microinjection method, which has been the major way of transgenic animal production since it was first applied (Churchill et al., 2004). SCNT has shown its tremendous wide-ranging application in high-quality domestic animal reproduction, for the mammary gland bioreactor, for stem-cell techniques and for many other applications. Transgenically cloned large domestic animals have many potential applications, such as the production of therapeutic and nutritional proteins, xenotransplantation and basic research. To date, scientists have successfully produced cloned mammals including sheep (Wilmut et al., Reference Wilmut, Schnieke, McWhir, Kind and Campbell1997), mice (Wakayama et al., Reference Wakayama, Perry, Zuccotti, Johnson and Yanagimachi1998), cattle (Cibelli et al., Reference Cibelli, Stice, Golueke, Kane, Jerry, Blackwell, Ponce de Leon and Robl1998; Kato et al., Reference Kato, Tani, Sotomaru, Kurokawa, Kato, Doguchi, Yasue and Tsunoda1998; Wells et al., Reference Wells, Misica and Tervit1999), goats (Baguisi et al., Reference Baguisi, Behboodi, Melican, Pollock, Destrempes, Cammuso, Williams, Nims, Porter, Midura, Palacios, Ayres, Denniston, Hayes, Ziomek, Meade, Godke, Gavin, Overstrom and Echelard1999), pigs (Polejaeva et al., Reference Polejaeva, Chen, Vaught, Page, Mullins, Ball, Dai, Boone, Walker, Ayares, Colman and Campbell2000), cats (Shin et al., Reference Shin, Kraemer, Pryor, Liu, Rugila, Howe, Buck, Murphy, Lyons and Westhusin2002), rabbits (Chesne et al., Reference Chesne, Adenot, Viglietta, Baratte, Boulanger and Renard2002), mules (Holden, Reference Holden2003), horses (Galli et al., Reference Galli, Lagutina, Crotti, Colleoni, Turini, Ponderato, Duchi and Lazzari2003) and rats (Zhou et al., Reference Zhou, Jouneau, Brochard, Adenot and Renard2001) by SCNT using mammary gland epithelial cells (Wilmut et al., Reference Wilmut, Schnieke, McWhir, Kind and Campbell1997), cumulus oophorus (cumulus ovaricus) cells (Wakayama et al., Reference Wakayama, Perry, Zuccotti, Johnson and Yanagimachi1998), uterine tubal epithelium cells (Kato et al., Reference Kato, Tani, Sotomaru, Kurokawa, Kato, Doguchi, Yasue and Tsunoda1998), fetus fibroblast (Baguisi et al., Reference Baguisi, Behboodi, Melican, Pollock, Destrempes, Cammuso, Williams, Nims, Porter, Midura, Palacios, Ayres, Denniston, Hayes, Ziomek, Meade, Godke, Gavin, Overstrom and Echelard1999), muscle cells (Qi et al., Reference Qi, Kishigami, Nakagawa, Iida and Sokabe2004) and fetal germ cells (Zakhartchenko et al., Reference Zakhartchenko, Durcova-Hills, Schernthaner, Stojkovic, Reichenbach, Mueller, Steinborn, Mueller, Wenigerkind, Prelle, Wolf and Brem1999) at different developmental stages. Nonetheless, the low efficiency of SCNT is a major obstacle to the extensive use of this technology (Renard et al., Reference Renard, Zhou, LeBourhis, Chavatte-Palmer, Hue, Heyman and Vignon2002).
The development of reconstructed embryos following nuclear transfer appears to be dependent on a variety of factors. The most important factor identified thus far is cell-cycle synchrony of donor nuclei with recipient enucleated oocytes (Campbell et al., Reference Campbell, Loi, Otaegui and Wilmut1996; Prather et al., Reference Prather, Boquest and Day1999). For example, Wilmut et al. (Reference Wilmut, Schnieke, McWhir, Kind and Campbell1997) believe strongly that their attempts would not succeed without first inducing donor cells into the quiescent stage. Unfortunately, there is currently no system that provides 100% synchronization of cells at a particular stage of the cell cycle (Baguisi et al., Reference Baguisi, Behboodi, Melican, Pollock, Destrempes, Cammuso, Williams, Nims, Porter, Midura, Palacios, Ayres, Denniston, Hayes, Ziomek, Meade, Godke, Gavin, Overstrom and Echelard1999). The preparation of cell-cycle stage donor cells for SCNT is still a subject for debate (Cibelli et al., Reference Cibelli, Stice, Golueke, Kane, Jerry, Blackwell, Ponce de Leon and Robl1998). Lai et al. (Reference Lai, Park, Cheong, Kuhholzer, Samuel, Bonk, Im, Rieke, Day, Murphy, Carter and Prather2002) and Zhou et al. (Reference Zhou, Jouneau, Brochard, Adenot and Renard2001) stated that serum starvation is not necessary for the success of SCNT. Interestingly, although there are several ways of preparing somatic cells for SCNT, many researchers prefer to use serum starvation to synchronize donor cells when they first attempt to clone a new species (Baguisi et al., Reference Baguisi, Behboodi, Melican, Pollock, Destrempes, Cammuso, Williams, Nims, Porter, Midura, Palacios, Ayres, Denniston, Hayes, Ziomek, Meade, Godke, Gavin, Overstrom and Echelard1999; Chesne et al., Reference Chesne, Adenot, Viglietta, Baratte, Boulanger and Renard2002).
The cell-cycle stage of the donor cells is one of the most important considerations. The importance of the stage of the donor cell-cycle has been noted since the first SCNT mammal – Dolly – was produced (Wilmut et al., Reference Wilmut, Schnieke, McWhir, Kind and Campbell1997; Kato et al., Reference Kato, Tani, Sotomaru, Kurokawa, Kato, Doguchi, Yasue and Tsunoda1998; Baguisi et al., Reference Baguisi, Behboodi, Melican, Pollock, Destrempes, Cammuso, Williams, Nims, Porter, Midura, Palacios, Ayres, Denniston, Hayes, Ziomek, Meade, Godke, Gavin, Overstrom and Echelard1999). The use of donor cells arrested at the G0 stage of the cell cycle always was the first choice in SCNT, although cycling cells have also been used as donor nuclei and in these cases SCNT embryos have developed to full term (Cibelli et al., Reference Cibelli, Stice, Golueke, Kane, Jerry, Blackwell, Ponce de Leon and Robl1998; Kasinathan et al., Reference Kasinathan, Knott, Moreira, Burnside, Jerry and Robl2001; Urakawa et al., Reference Urakawa, Ideta, Sawada and Aoyagi2004).
Yin et al. (2006) reviewed methods of arresting cells in the G0/G1 phase of the cell cycle (Yin et al., Reference Yin, Lee, Kim, Shin, Kim and Kong2007). However, to date, studies on cell-cycle synchronization of cells from transgenic cloned cattle, especially when preparing somatic cells for the reproduction of precious cloned animals by using a re-cloning method, are very limited. Therefore, this study was designed to examine the effects of cell-cycle synchronization protocols, such as confluent inhibition (CI), roscovitine treatment (R) and serum starvation (SS) in ear fibroblast cells from transgenic cloned cattle, in order to seek a more reliable and more efficient method to prepare donor cells for the production of re-cloned embryos from transgenic cattle. Here we provide data on cell-cycle synchronization for the re-cloning of transgenic cloned cattle.
Materials and Methods
Ear tissue was cut from transgenic cloned cattle aseptically and taken to the laboratory. After removal of the cartilage tissue using iris scissors and forceps, the remaining tissues were rinsed twice in Ca2+- and Mg2+-free phosphate-buffered saline (DPBS; GIBCO) and cut into 1 mm3 pieces. The pieces were seeded into T-25 cell culture flask and cultured in an incubator at 37.5 °C, less than 5% CO2 in air and 100% humidity, for 4–6 h until tissue explants were attached. Then, fresh culture medium, [Dulbecco's Modified Essential Medium (DMEM; GIBCO), 10% fetal bovine serum (FBS; GIBCO) and 1% non-essential amino acids plus penicillin and streptomycin], was added. After the cells grew to 70–80% confluency, the spent culture medium was discarded and DPBS was used to wash the cells three times, followed by addition of 0.25% trypsin and 0.02% EDTA to harvest the cells. After centrifugation at 1500 rpm for 5 min, supernatants were removed and one group of pellets was resuspended in fresh cell-culture medium and seeded into new T-25 flasks. A second group of pellets was resuspended in cell cryopreservation medium [DMEM, 10% dimethylsulfoxide (DMSO) and 20% FBS] and aliquoted into cryovials. After freezing at −70 °C overnight, pellets were then stored at −196 °C in liquid nitrogen.
Flow cytometry analysis
DNA content of fetal cells was analysed by ethanol fixation and staining with propidium iodide. Trypsinized cells were resuspended in DMEM with 10% FBS and dispensed into 15 ml tubes, at 5 × 105 cells per tube. Cells were pelleted by centrifugation at 500 g for 10 min and pelleted cells were washed in DPBS. Ethanol (3 ml at 4 °C) was added slowly to each tube with vortexing. After ethanol fixation (at least 12 h at 4 °C), cells were pelleted (1000 g for 5 min), then washed once with PBS + 10% FBS (1000 g for 5 min). Pelleted cells were washed with 2 ml Triton X-100 (Sigma), and the pellet was stained with 1 ml PBS containing 30 mg/ml propidium iodide (Sigma) and 0.3 mg/ml RNase A (Sigma). Staining was carried out at room temperature for a minimum of 1 h. Stained cells were filtered through a 30 mm nylon mesh (Spectrum) immediately prior to flow cytometric analysis.
Preparation of recipient oocytes
Ovaries were collected from a local slaughterhouse and transported to the laboratory in a thermos flask filled with physiological saline at 25–35 °C. Cumulus–oocyte complexes (COCs) were aspirated from follicles (2–8 mm in diameter) and selected based on their morphology. Complexes were washed twice in maturation medium, which was comprised of tissue culture medium (TCM-199; Life Technologies) supplemented with 10% FBS, 0.01 U/ml FSH, 0.01 U/ml LH, 1 mg/ml 17β-estradiol and 1% (v/v) penicillin/streptomycin (Life Technologies). Approximately 50–60 COCs were transferred into 0.5 ml of maturation medium in 4-well dishes, overlaid with paraffin oil and cultured at 38.5 °C in 5% CO2 in air for 18–20 h. After maturation, cumulus cells were removed by vortex in 0.1% (w/v) hyaluronidase for 2–3 min.
Nuclear transfer
Enucleation was achieved by piercing the zona pellucida with a glass needle, followed by removal of the polar body and surrounding cytoplasm. Successful enucleation was confirmed by Hoechst 33342 staining of pushed-out karyoplasts. Donor cells were then transferred individually into the perivitelline space of enucleated recipient oocytes. Reconstructed embryos were fused electrically, 24 h after maturation, in a chamber filled with Zimmerman cell fusion medium, by two stainless steel electrodes. Cytoplast–cell complexes were aligned manually with a fine glass needle, so that the contact surface between cytoplast and donor cell was parallel to the electrodes. Cell fusion was induced with two DC pulses of 2.5 kV/cm for 10 μs, each at 1 s apart, and delivered by a BTX2001 Electro Cell Manipulator (BTX). Activation was induced by incubation in 10 μg/ml cycloheximide and 2.5 μg/ml cytochalasin-D in CR1aa medium supplemented with 0.1% (w/v) bovine serum albumin (BSA) for 1 h and then cycloheximide (10 mg/ml) alone for a further 4 h. After activation, embryos were cultured further in CR1aa medium supplemented with 0.1% (w/v) BSA for 48 h in an atmosphere consisting of 5% O2, 5% CO2 and 90% N2. Cleaved embryos were then selected and cultured for an additional 5 days in CR1aa medium supplemented with 5% (v/v) FBS on cumulus cell monolayers in an atmosphere of 5% CO2 in air. The medium was changed every 2 days throughout the culture period.
Experimental design
In Experiment 1, the cell-cycle stages of ear fibroblasts from transgenic cloned cattle were evaluated. Three experimental groups were designed and cells were passaged four times and cultured near to confluency: (i) cells were cultured either in DMEM + 10% FBS until confluency [confluent inhibition (CI) group]; (ii) in DMEM + 10% FBS + 15 μM roscovitine [roscovitine (R) group]; or (iii) in DMEM + 0.5% FBS [serum starvation (SS) group] for 5 days. Every group sample was evaluated at 24 h, 48 h, 72 h, 96 h and 120 h after treatment.
In Experiment 2, nuclear transferred embryos (NTEs) were reconstructed using the serum-starvation and contact-inhibition treatment for 3 days and ear fibroblasts from transgenic cloned cattle as donors.
Statistical analysis
The data were analysed using the chi-squared test. A value of chi-squared corresponding to p < 0.05 was considered significant.
Results
In the cycling fibroblasts (50–60% confluent), 59.29% cells were allocated in the G0/G1 phases. The efficiency of obtaining G0/G1 phase cells was lower when R was employed to synchronize the cell cycle than for the SS and CI methods (89.7 versus 91.1% and 91.0%, respectively, p < 0.05) (Table 1).
Table 1 Cell-cycle synchronization of transgenic cloned cattle fibroblasts cultured in different conditions
a,b Mean statistical differences p < 0.05.
For the SS group, the proportion of G0/G1 cells was significantly higher when treatment lasted for 3, 4 and 5 days rather than treatment for 1 or 2 days only (91.5, 91.7 and 93.5% versus 90.1 and 88.8%, respectively, p < 0.05). No statistical difference was observed between values for cells treated for 3, 4 or 5 days (Table 2).
Table 2 Distribution of cell cycle stages with the treatment time
a–dMean statistical differences, p < 0.05.
For the CI group, as expected, an increasing number of G0/G1 stage cells was found following 3 days of CI (from 89.4% at day 1 and 90.4% at day 2 to 91.4% at day 3, p < 0.05), the latter being similar to values for day 4 (91.6%) and day 5 of CI treatment (92.1%) (Table 2).
For the R group, the number of cells that had developed to the G0/G1 stage was significantly lower after 1 day of treatment than that after 2 to 5 days (86.51 versus 91.1, 90.1, 89.4 and 91.3%, respectively, p < 0.05), for which similar numbers of cells in G0/G1 phase were observed (Table 2).
Nuclear transferred embryos were reconstructed using SS- or CI-treated ear fibroblasts from transgenic cloned cattle cells as donors. Obvious difference were found in fused couplets and blastocysts (89.88 ± 2.70 versus 87.40 ± 5.13; 44.10 ± 8.62 versus 58.38 ± 13.28, respectively, p < 0.05) (Table 3) while no significant difference was found between cleaved embryos.
Table 3 In vitro developmental competence of cloned embryos
Embryos were examined using serum starvation and contact inhibition treatment transgenic cloned cattle ear fibroblasts cells as donors.
a–dMean statistical differences, p < 0.05.
Discussion
Data from our present study indicate clearly that ear fibroblasts from transgenic cloned cattle could be synchronized effectively at G0/G1 stages using all the three different treatments: CI, R and SS. Most of the cells (>80%) in all groups were arrested at the G0/G1 stage, while in the cycling fibroblasts (50–60% confluent), 59.29 % cells allocated in the G0/G1.
Serum starvation has long been known to withdraw growth factors and other related components from cultured cells (Chesne et al., Reference Chesne, Adenot, Viglietta, Baratte, Boulanger and Renard2002). In other studies, effective synchronization of donor cells in presumptive G0/G1 has been achieved by SS treatment for 4 days of sheep cells (Wilmut et al., Reference Wilmut, Schnieke, McWhir, Kind and Campbell1997), 5–10 days in rabbits cells (Zhou et al., Reference Zhou, Yang, Ding, He, Xie, Hildebrandt, Mitalipov, Tang, Wolf and Ji2006) and 5 days in cattle cells (Kubota et al., Reference Kubota, Yamakuchi, Todoroki, Mizoshita, Tabara, Barber and Yang2000). It takes 5 days of continuous starvation to reach the plateau (93%) found in this study and no statistical difference was observed following 3, 4 or 5 days of treatment. Cho et al. (Reference Cho, Ock, Yoo, Kumar, Choe and Rho2005) reported that they achieved 82.9% cattle fetal fibroblasts cells in G0/G1 phase with SS treatment (Cho et al., Reference Cho, Ock, Yoo, Kumar, Choe and Rho2005). Differences in results may be attributed to the influence of individuals, cell types and treatment methods.
Culture to 100% confluency is another strategy used to move cells into G0 in response to overcrowding (Zetterberg and Auer, Reference Zetterberg and Auer1970). Boquest et al. (Reference Boquest, Day and Prather1999) reported that a larger portion (85%) of confluent porcine foetal fibroblasts cells was in G0/G1 phase when compared with those in cycling cultures. Cho et al. (Reference Cho, Ock, Yoo, Kumar, Choe and Rho2005) reported that 86.9% of confluent cattle fetal fibroblasts cells were in G0/G1 phase. Differences between results might be attributed to the influence of cell types and passage. However, these results show that monitoring the cell-cycle stage distribution for each cell line and for each treatment before their use as donors is of great value.
In this study no significant different was observed in the G0/G1 cells that underwent the SS and CI treatments (91.11 ± 1.77 and 90.97 ± 1.18, p > 0.05), which is similar to results found for rabbit fibroblast (Liu et al., Reference Liu, Yu and Ju2004) and porcine mammary cells (Prather et al., Reference Prather, Boquest and Day1999). We also tested the response of transgenic cloned cattle fibroblasts to the cell cycle inhibitor roscovitine, which has been shown to arrest cultured fetal fibroblast cells from cattle in G0/G1 (Cho et al., Reference Cho, Ock, Yoo, Kumar, Choe and Rho2005). Roscovitine, a potent inhibitor of specific Cdk2 and maturation promoting factor (MPF) effectively arrested human fibroblasts in the G0/G1 phase of the cell cycle and, following its removal, cells arrested in G0/G1 resumed cycling and entered the S phase as expected (Alessi et al., Reference Alessi, Quarta, Savio, Riva, Rossi, Stivala, Scovassi, Meijer and Prosperi1998). Gibbons et al. (Reference Gibbons, Arat, Rzucidlo, Miyoshi, Waltenburg, Respess, Venable and Stice2002) reported that roscovitine-treated adult bovine granulosa cells were more efficiently synchronized in G0/G1 phase of the cell cycle than serum-starved cells (Gibbons et al., Reference Gibbons, Arat, Rzucidlo, Miyoshi, Waltenburg, Respess, Venable and Stice2002). Our results showed that the efficiency of SS and CI treatment was higher than that of R treatment in synchronized cells in G0/G1 in ear fibroblast cells from transgenic cloned cattle. Cho et al. (Reference Cho, Ock, Yoo, Kumar, Choe and Rho2005) reported that 82.8% fetal fibroblasts cells in R were synchronized in G0/G1 phase (Cho et al., Reference Cho, Ock, Yoo, Kumar, Choe and Rho2005). Furthermore, Gibbons (2002) showed that roscovitine culture could synchronize the donor cells and can increase cloning efficiency. Unfortunately, these finding have not been confirmed by other studies to date (Gibbons et al., Reference Gibbons, Arat, Rzucidlo, Miyoshi, Waltenburg, Respess, Venable and Stice2002).
In our current research, the efficiency of SS and CI treatment was higher than that of R treatment, so that nuclear-transferred embryos were reconstructed using ear fibroblasts cells from transgenic cloned cattle and SS or CI treatment as donors. Although several researchers have confirmed that normal non-transgenic offspring could be obtained using SCNT with SS- or CI-treated cells as donors (Wilmut et al., Reference Wilmut, Schnieke, McWhir, Kind and Campbell1997; Cibelli et al., Reference Cibelli, Stice, Golueke, Kane, Jerry, Blackwell, Ponce de Leon and Robl1998; Wells et al., Reference Wells, Misica and Tervit1999), information on effects of different cell cycle synchronization methods and on the subsequent development of re-cloned embryos is very scarce. Here in the present study, we observed obvious differences in the rate of fused couplets and blastocysts (89.88 ± 2.70 versus 87.40 ± 5.13; 44.10 ± 8.62 versus 58.38 ± 13.28, respectively, p < 0.05), while there was no difference in the number of cleaved embryos.
Three different treatments (confluent, roscovitine and serum starvation) could be synchronized effectively at the G0/G1 stages. Cho et al. (Reference Cho, Ock, Yoo, Kumar, Choe and Rho2005) analysed chromosomes from all of the three groups and showed that approximately 70% of cells had normal chromosome sets (58 autosomes and two sex chromosomes) (Cho et al., Reference Cho, Ock, Yoo, Kumar, Choe and Rho2005). These results suggested that these donor treatments could be used for SCNT.
In conclusion, a detailed analysis of cell-cycle stages for ear fibroblasts from transgenic cloned cattle was presented in this study. To our knowledge, few studies have been carried out on the cell-cycle stage of ear fibroblast cells from transgenic cloned cattle. These cells could be synchronized effectively at the G0/G1 stages by all three treatments, confluent, roscovitine and serum starvation. Our data indicate that to harvest adequate G0/G1 stage cells for re-cloning transgenic cattle from fibroblasts and established from ear skin from newborn transgenic cloned calves, 3 days of treatment is sufficient regardless of method. Further research is needed to evaluate the developmental competence of cloned embryos derived from nuclear donors prepared by these treatments. A more efficient synchronization or different control regimes of the cell cycle stages needs to be addressed for ear fibroblasts from transgenic cloned cattle, which would be informative for future SCNT in transgenic cloned cattle re-cloning studies.
Acknowledgements
We kindly thanks Lijing, Liyan at Beijing GenProtein Biotech Company Ltd for help with SCNT. This work was supported by grants from the Hi-tech Research and Development Program of China and the Beijing Natural Scientific Foundation of China.
