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Embryo |
a Department of Gene Expression and Development, The Roslin Institute, Roslin, Midlothian EH25 9PS, Scotland, United Kingdom
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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developmental biology, embryo, oocyte development
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To determine whether the abnormal phenotypes of cloned animals are a consequence of the nuclear transfer technique itself or, instead, of the inherent properties of the donor cell nucleus, inbred and hybrid ES cells were used for nuclear transfer and tetraploid blastocyst injection [10]. Only F1-derived ES cell clones survived to adulthood, whereas inbred ES cell clones died within several minutes of delivery by cesarean section. Because pups derived by blastocyst injection showed a similar pattern of mortality, the death of inbred ES cell clones at term seemed to be caused by the limited developmental potential of inbred ES cells. Previous reports of ES cell cloning have shown similar results with one exception, when one cloned pup from the inbred ES cell line E14 survived [8, 9]. By contrast, pups cloned from both hybrid and inbred somatic cells can survive to adulthood [16]. It is important to determine whether this difference is caused by the inbred ES cells or by the different inbred ES cell lines used.
In the present study, we examined the effect of two factors on the developmental potential of embryos reconstructed from ES cells. First, we examined the effect of the degree of confluence on development and found marked increases in development if the cells were at a high level of confluence. Second, we compared an inbred ES cell line, HM-1, and an intercrossed ES cell line, R1, as donor nuclei for nuclear transfer. Our results show that clones from both inbred and intercrossed ES cell lines can survive to adulthood. Our data indicate that the death of inbred ES clones is not universal but, rather, reflects the developmental potential of the different inbred ES cell lines used.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All inorganic and organic compounds were purchased from Sigma Chemical Company (St. Louis, MO) unless otherwise stated.
ES Cell Lines and Culture Conditions
The hypoxanthine phosphoribosyltransferase-deficient ES cell line HM-1 [17] was derived from an inbred mouse strain 129/Ola and employed for nuclear transfer at passage 19. The developmental potential of these cells was confirmed by production of a high proportion of chimeras with germline transmission after injection into blastocysts [18]. The ES cell line R1 [19], derived from 129/Sv x 129/Sv-CP, was provided by Dr. Lesley Forrester at passage 14 and grown for another 5–11 passages at the Roslin Institute. Thus, the R1 cells used in the present experiments ranged from passage 19 to 25. The HM-1 ES cells were cultured in Glasgow minimum essential medium (Gibco BRL [Life Sciences Ltd.], Paisly, Scotland, U.K.) supplemented with 15% heat-inactivated fetal calf serum, 1000 U/ml of leukemia inhibitory factor, and the following reagents: 2 mM L-glutamine, 1% minimum essential medium nonessential amino acid solution (Gibco BRL), and 1% ß-mercaptoethanol. The R1 ES cells were cultured in Dulbecco modified Eagle medium (Gibco BRL) containing the same reagents as used for the HM-1 cells. One night before the experiment, the serum concentration was reduced to 5%.
Nuclear Transfer
All experiments described in this study were conducted following approval by the Roslin Institute's Animal Ethics Committee and within a project license issued under the Animal (Scientific Procedures) Act of 1986.
Reconstruction of embryos by nuclear transfer was performed according to the method reported by Wakayama et al. [3] with a slight modification. Female B6D2F1 mice (age, 8–10 wk) were used to provide oocytes. Oocytes were collected 13–14 h after hCG injection and precultured in CZB (Chatot, Ziomet, Bavister) [20] medium before enucleation was performed using a Nikon inverted microscope (TE300; Nikon UK Ltd., Kingston upon Thames, Surrey, U.K.) equipped with Eppendorf TransferMan NK micromanipulator (Eppendorf AG, Hamburg, Germany). Oocytes were placed in a drop of Hepes-buffered CZB medium containing 5 µg/ml of cytochalasin B for 3–5 min and then enucleated with a 6- to 8-µm pipette by aspirating the metaphase II spindle with a minimum of cytoplasm. The enucleation pipette was attached to a Piezo Micromanipulator Controller PMM150 (Prime Tech Ltd., Ibaraki, Japan). After enucleation, the oocytes were collected and kept in CZB medium in the incubator before injection. For nuclear transfer, several drops of ES cell suspensions were mixed well with 10% polyvinylpyrrolidone (360KD; ICN Biomedicals Inc., Aurora, OH). A small-sized (<10 µm) ES cell was drawn in and out of the injection pipette (inner diameter, 5 µm) until the cell membrane was broken and separated from the nucleus. Each ES cell nucleus was drawn deep into the pipette before the same pipette was used to pick up other cell nuclei until four to six nuclei were lined up inside the pipette within a few minutes. These nuclei were injected one by one into the enucleated oocytes by using piezo microinjection. The reconstructed oocytes were cultured in CZB medium for 1–3 h before activation.
Embryo Culture and Transfer
Reconstructed oocytes were activated for 5–6 h in calcium-free CZB medium containing 10 mM strontium and 5 µg/ml of cytochalasin B to inhibit polar body extrusion as previously reported [3, 10]. The activated oocytes with pseudopronuclei were collected and cultured in CZB or M16 medium for 72 h. Morula/blastocyst-stage embryos were transferred into the uteri of pseudopregnant surrogate mothers that had been mated with vasectomized male mice 2.5 days earlier. Five to ten embryos were transferred into each uterine horn. Recipient mothers were killed at 19.5 days postcoitum, and the pups were quickly removed from the uteri. After cleaning fluid away from their air passages, the pups were kept in a warm box supplied with oxygen. Surviving pups were raised by lactating mothers.
Immunocytochemistry Staining of Reconstructed Oocytes and Cloned Blastocysts
At different time points following injection of HM-1 ES cells and activation, reconstructed oocytes were processed for immunocytochemistry staining to observe cytoskeletal organization and DNA configuration as previously described [21]. Oocytes were fixed and extracted for 30 min at 37°C in a microtubule stabilization buffer (0.1 M PIPES [pH 6.9], 5 mM MgCl2·6H2O, and 2.5 mM EGTA) containing 2% formaldehyde, 0.5% Triton X-100, 50% deuterium oxide, and 1 mM dithiothreitol. After washing three times in a blocking solution of PBS containing 10% normal goat serum (NGS), 0.1% Triton X-100, and 0.02% sodium azide, the oocytes were stored at 4°C until processed. To evaluate microtubule and microfilament dynamics and chromatin configuration, multiple fluorescence labeling using triple-stain analysis was performed. Oocytes were incubated with fluorescein isothiocyanate-conjugated anti-
-tubulin antibody (final dilution, 1:500; Sigma) and rhodamine phalloidin (final dilution, 1:4000; Molecular Probes, Eugene, OR) in a blocking solution of PBS containing 5% NGS in the dark at 37°C for 1 h. After washing three times in the blocking solution, the oocytes were mounted in Vectashield (Vector Laboratories, Burlingame, CA) containing 5 µg/ml of 4',6-diamidino-2-phenylindole (DAPI). Labeled oocytes were viewed using a Zeiss Axiovert S 100 photomicroscope equipped with fluorescein (Zeiss 487709), Texas Red (Zeiss 487714), and Hoechst (Zeiss 487702) (all from Imaging Associates Ltd., Thame, U.K.) selective filter sets and a 50-W mercury arc lamp using a 40x Neofluar objective (Imaging Associates Ltd.). Images were acquired using the Kinetic Imaging System (Imaging Associates Ltd.). Cloned blastocysts were treated with 0.9% sodium citrate medium for 30 min at room temperature and then fixed in ethanol:acetic acid:water (3:2:1) solution for less than 1 min. The blastocysts were transferred to a slide and allowed to spread, stained with DAPI, and viewed by the same microscope.
Flow Cytometric Cell Cycle Analysis
Cells were collected by trypsin treatment, washed once with cold PBS, and processed for cell cycle analysis as previously described [22]. Briefly, centrifuged cells were resuspended in one volume of cold "saline GM"(6.1 mM glucose, 137 mM NaCl, 5.4 mM KCl, 1.5 mM Na2HPO4·H2O, 0.9 mM KH2PO4, and 0.5 mM EDTA) and fixed by adding three volumes of 95% ethanol (-20°C). After overnight fixation at 4°C, cells were pelleted, washed once with PBS/EDTA (5 mM), and stained for 2 h at room temperature with 50 µg/ml of propidium iodide and 100 µg/ml of RNase A (Sigma) in PBS containing 0.1% sodium azide and 0.1% BSA. Samples were analyzed using a FAC Scan cytometer (Becton Dickinson, San Jose, CA) equipped with an argon laser (excitation wavelength, 488 nM) and a 610-nM long-pass filter. For each sample, 104 events were stored for analysis. The doublet discrimination module was applied to exclude doublets or higher-order aggregates from DNA analysis. The percentage of cells existing in the various stages of the cell cycle were calculated using the CELLQuest program (Becton Dickinson) to visualize a histogram plot of red fluorescence (DNA) and by gating the cell populations with 2N (G0/G1 phase), 2N/4N (S phase), and 4N (G2/M phase) content of DNA.
Genomic DNA Analysis of Cloned Mice
Polymerase chain reaction amplification of the microsatellite markers D4Mit204 and D7Mit22 was performed as previously reported [8]. The DNA was extracted from tail tips of the first two surviving clone mice, oocyte donor mouse, surrogate mother, and HM-1 ES cell pellets. Reactions (20 µl) were subjected to 34 cycles of 30 sec at 94°C, 1 min at 60°C, and 2 min at 72°C, and products were separated on a 4% agarose gel and visualized after staining with ethidium bromide.
Statistical Analysis
Results were evaluated using the chi-square test or ANOVA, with a P value of less than 0.05 considered to be statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Microtubule patterns and nucleus changes in reconstructed embryos after nuclear transfer and activation are shown in Figure 1. The HM-1 ES cell nucleus remained intact 5 min after injection, and no spindle started to organize (Fig. 1A). A bipolar spindle that resembled a metaphase II oocyte spindle formed 1 h after nuclear transfer, the nucleus condensed into chromosomes, and these chromosomes aligned on the metaphase equator (Fig. 1B). In all embryos examined, two groups of chromosomes segregated to their respective poles 1 h after the onset of activation, at which time the reconstructed oocyte was at the telophase stage (Fig. 1C). Two groups of chromosomes decondensed into two pseudopronuclei 5 h after activation, and several nucleoli were visible in both pronuclei (Fig. 1D).
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Cloned Mice from Inbred HM-1 ES Cells
As shown in Table 1, more than 95% of oocytes survived piezo microinjection, and 98% of reconstructed oocytes activated after exposure to strontium. An effect of degree of confluence was found on subsequent development of cloned embryos with both donor cell populations. For 80–90% cell confluence (Fig. 2), 84.7% of cloned embryos cleaved, and 49% developed to morulae/blastocysts (Fig. 3, A and B). Some cloned embryos were cultured to 84 h after activation, and Figure 3C shows one blastocyst with more than 50 cells after staining. Twenty-seven of 197 (13.7%) transferred embryos developed to term, 18 pups were alive at term, and 5 pups developed into healthy adults. Most of the remaining 13 live pups died within 1 h following cesarean section because of respiratory failure (Table 2). The five surviving cloned pups were all males with chinchilla coats and pink eyes (Fig. 4A), as expected for the ES cell line HM-1, which is an XY line derived from the 129/Ola strain. Results of microsatellite analysis confirmed that the first two clones were from HM-1 ES cells (Fig. 5). The fertility of the surviving adults has been proved through mating with 129/Ola or B6D2F1 female mice.
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In contrast to 80–90% cell confluence, with 60–70% cell confluence, only 59.3% of activated oocytes cleaved and 22.2% of embryos developed to morulae/blastocysts. Both cleavage and morula/blastocyst development rates were significantly lower than those with the embryos reconstructed with ES cells of high confluence. Only 1 of 43 (2.3%) blastocysts developed to term, and that pup died 1 h after delivery.
Cloned Mice from R1 ES Cells
The two factors influencing development of cloned embryos from R1 ES cells were passage number and confluence of the culture. As shown in Table 3, with cell passage 19, 66.8% of cloned embryos cleaved, and 47.2% of embryos developed to morulae/blastocysts. Nine of 178 (5.1%) transferred embryos developed to term, and 3 of these were alive at birth. One that survived to adulthood was agouti, as expected of the ES cell line R1, which is an XY line derived from 129/Sv x 129/Sv-CP (Fig. 4B). This pup has been proved to be fertile after mating with female mice. In striking contrast, with cell passages 22–25, only 47.5% of activated oocytes cleaved, and 27% of embryos developed to blastocysts. Only 1 of 537 (0.2%) transferred embryos developed to term.
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Similar to HM-1 ES cells, the cell confluence at culture dramatically affected the development of cloned embryos reconstructed with R1 ES cells. As shown in Table 4, when using cell passage 19, 52.7% of activated embryos developed to morulae/blastocysts using 80–90% cell confluence, and 9 of 146 (6.2%) transferred embryos developed to term. In contrast, using 60–70% cell confluence, only 29.7% of activated embryos developed to the blastocyst stage, and none developed to term after transfer to surrogate mothers.
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Effects of Cell Confluence on Cell Cycle Stage
The effects of cell confluence on the cell cycle of ES cells used for nuclear transfer were determined by flow-cytometric cell cycle analysis. Under each experimental condition, three replicates were carried out, and more than 10 000 events were counted for each sample. As shown in Table 5 and Figure 6, no difference was observed in the percentage of G1 cells between high confluence and subconfluence in both HM-1 and R1 ES cells analyzed by two-way ANOVA.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Compared to the previous study [10], in which all cloned mice from inbred ES cells died within a few minutes after birth, 5 of 18 (27.8%) cloned mice from the HM-1 cell line survived to become adults in our study. The data from mice cloning with somatic cells showed that both inbred mice- and hybrid mice-derived cells can be used to make clones and that the cloned mice could survive to adulthood [16]. The very interesting point is not only that cumulus cells from inbred 129/Sv mice can be used to make cloned mice but that these clones can survive. No cloned mice produced from the 129/Sv strain-derived ES cell lines survived [9, 10]. The only cloned mouse to survive from an inbred ES cell line is "Hooper." The ES cell line known as E14 is derived from the 129/Ola [8]. The inbred ES cell line HM-1, employed in the present study, is also derived from the 129/Ola. It is therefore necessary to study whether the 129/Ola-derived ES cell lines are more effective nuclear donors or whether HPRT deficiency in HM-1 is important for the survival of cloned mice.
Cell confluence has been shown to dramatically affect cloning efficiency using ES cells for nuclear transfer in the present study, in contrast to results from a previous study on bovine nuclear transfer from fibroblast cells, in which cell confluence showed no effect on development of reconstructed embryos [23]. The mechanism is undetermined. With both ES cell lines used in the present study, a greater proportion of embryos developed to blastocyst and term when donor cells were at high confluence. Initially, we considered that the cell cycle stage might be affected by the degree of confluence, but analysis of cell cycle stage by flow cytometry found no difference between high and low confluence in either of the cell lines used. This may indicate that the probability of selecting G1 cells is equivalent and that cell cycle stage is not the only factor responsible for successful cloning in the present study. The only difference between high and low confluence that was observed was in cell size. More small-sized cells were observed with high confluence compared with low confluence, and we suggest that this is associated with a difference in cloning efficiency.
The exact mechanism behind this phenomenon and differences in gene expression may be important. In somatic cell culture, the gene expression of both insulin-like growth factor (IGF)-I and IGF-II is significantly affected by cell density during in vitro culture [24, 25]. The IGF-I mRNA transcripts increased more than 200-fold when C6 glioma cells grew to postconfluence, and IGF-II mRNA increased approximately 20-fold when BRL-3A cells were cultured at higher cell density. The results from a recent study indicate that both serum starvation and high confluence cause overexpression of both IGF-II and H19 genes in ES cells [26]. It has been demonstrated that both IGF-I and IGF-II are necessary for normal embryonic growth [27, 28]. Transcriptional regulation of both maternally and paternally imprinted genes is suggested to be important for development of nuclear transfer embryos. It might be that expression of these specific genes is more appropriate after nuclear transfer from confluent cells or that other differences exist in chromatin structure and gene expression that are important for development.
By using high-confluence ES cells for nuclear transfer, the morula/blastocyst rate was shown to be higher than in the results of previous reports using the same method [8–10]. In those studies, the morula/blastocyst rate of cloned embryos was approximately 20–30%, whereas more than 45% reached these stages with both ES cell lines in the present study. Our results are comparable to the studies using ES cells arrested at metaphase for nuclear transfer [29–31].
In the present study, we also found that the number of cell passages dramatically affected cloning efficiency, which confirms a previous report [29], although preimplantation development was not affected using ES cells at higher passage number for nuclear transfer in that study. One possible reason for the low development is progressive loss of karyotype stability when ES cells are cultured to higher passages [32]. In particular, the results from this study proved that very few R1 ES cells beyond passage 20 are clonable.
Developmental changes in the transferred nuclei were different from those described previously [3, 8]. One to three hours after nuclear transfer, the ES nucleus condensed into chromosomes as in the previous study [3, 8], but in these experiments, the chromosomes aligned on the metaphase equator instead of being disorganized. Furthermore, in the present study, only two pronuclei formed in all embryos, in contrast to the previous report of three or more pronuclei in 36% of embryos reconstructed with cumulus cells [3]. Preculture of the reconstructed oocytes for at least 1 h might be important for successful cloning, because a well-formed, "metaphase II"-like spindle can be formed.
In conclusion, our present data demonstrate not only that hybrid ES cells can be used to make cloned mice that survive but that the inbred ES cell line HM-1 also can be used to make clones (i.e., 5 of 18 of these cloned mice survived to become adults with normal reproductive capacity). If donor cells were highly confluent, a significantly greater proportion of cloned embryos developed to blastocysts or term. Finally, cloning success can be improved by limiting the passage number of R1 ES cells to fewer than 20.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence. FAX: 44 131 527 4493; ian.wilmut{at}bbsrc.ac.uk
Received: 26 March 2002.
First decision: 15 April 2002.
Accepted: 27 August 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-tubulin. Dev Biol 2001 239:281-294[CrossRef][Medline]This article has been cited by other articles:
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  A.M. Powell, N.C. Talbot, K.D. Wells, D.E. Kerr, V.G. Pursel, and R.J. Wall Cell Donor Influences Success of Producing Cattle by Somatic Cell Nuclear Transfer Biol Reprod, July 1, 2004; 71(1): 210 - 216. [Abstract] [Full Text] [PDF]
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 L. J. Mullins, I. Wilmut, and J. J. Mullins Nuclear transfer in rodents J. Physiol., January 1, 2004; 554(1): 4 - 12. [Abstract] [Full Text] [PDF]
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K. Inoue, N. Ogonuki, K. Mochida, Y. Yamamoto, K. Takano, T. Kohda, F. Ishino, and A. Ogura Effects of Donor Cell Type and Genotype on the Efficiency of Mouse Somatic Cell Cloning Biol Reprod, October 1, 2003; 69(4): 1394 - 1400. [Abstract] [Full Text] [PDF]
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