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BOR - Papers in Press, published online ahead of print February 19, 2003.
Biol Reprod 2003, 10.1095/biolreprod.102.013250
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BIOLOGY OF REPRODUCTION 69, 15–21 (2003)
DOI: 10.1095/biolreprod.102.013250
© 2003 by the Society for the Study of Reproduction, Inc.

Reproductive Technology

Telomerase-Immortalized Sheep Fibroblasts Can Be Reprogrammed by Nuclear Transfer to Undergo Early Development1

Wei Cui, Diana Wylie, Samena Aslam, Andres Dinnyes, Tim King, Ian Wilmut, and A. John Clark2

Department of Gene Expression and Development, Roslin Institute, Roslin, Midlothian EH25 9PS, United Kingdom


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Telomere shortening and lack of telomerase activity have been implicated in cellular senescence in human fibroblasts. Expression of the human telomerase catalytic reverse transcriptase subunit (hTERT) in these cells reconstitutes telomerase activity and immortalizes the cells without tumor transformation. In this report, we show that sheep fibroblasts are similar to human cells. They do not have detectable telomerase activity and undergo only a finite numbers of cell divisions before replicative senescence. Telomere lengths in sheep fibroblasts are similar to those reported for human cells and shorten at a rate of 50–200 base pairs (bp) each cell division. Expression of the human telomerase catalytic subunit restored the telomerase activity in the sheep cells and extended their proliferative life span. None of the telomerase positive sheep fibroblasts exhibited a transformed phenotype after 200 days of continuous culture, and the higher hTERT expressing cells maintained their telomere lengths and normal cell characteristics for more than 500 days in culture. In cloning experiments using one of these cell lines as a nuclear donor, the reconstructed karyoplasts were reprogrammed and developed to the blastocyst stage at a similar frequency to that observed with the parental, telomerase negative cell line. After embryo transfer the blastocysts exhibited a relatively high frequency of implantation, early fetal development, and organogenesis. No fetuses survived beyond 40 days of development, however, showing that although these cells could be substantially reprogrammed, they were not fully competent for nuclear transfer.

aging, nuclear transfer, embryo


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Normal human fibroblasts have a limited proliferative life span in culture and undergo only a finite number of divisions before they enter an irreversible nondividing state, termed replicative senescence [1, 2]. Evidence has accumulated that suggests that progressive loss of the telomeric repeats at the ends of chromosomes plays an important role in this mitotic clock mechanism [3–5].

Telomeres are nucleoprotein structures composed of tandem hexameric repeats that are conserved in all vertebrates as (TTAGGG)n. They are essential elements that protect chromosomal ends from nuclease degradation, interchromosomal fusion, and improper recombination, thereby contributing to genome stability [6, 7]. Because of the inability of DNA polymerase to completely replicate the chromosome ends [8], telomeric repeats are progressively lost each time somatic cells divide. The telomere hypothesis of cellular senescence proposes that when the telomeres shorten to a critical limit, they will lose their function, resulting in chromosomal fusion. This activates p53 and its associated signaling pathways, leading to cell arrest or apoptosis resulting in cell senescence or crisis.

Telomeres are elongated by a eukaryotic multiunit ribonucleoprotein complex, telomerase, which comprises two essential core units: a telomerase RNA unit (TR), which provides a template for the synthesis of hexameric repeats, and a telomerase reverse transcriptase (TERT) component, providing catalytic activity. Telomerase is inactive in normal human somatic cells but is active in the germ line, certain stem cells, and many tumor cells and stabilizes the telomeres in these cells by adding telomeric repeats to them [9, 10]. The key factor limiting telomerase activity in human cells has been demonstrated to be human TERT (hTERT) because the human TR (hTR) is present in all cell types, whereas hTERT is confined to cells that express telomerase activity [11, 12]. Direct evidence for this comes from experiments demonstrating that forced hTERT expression in primary human cells reconstitutes telomerase activity, stabilizes the telomeres, and extends their proliferative life span [13–15]. As predicted by the telomere hypothesis, hTERT-expressing cells do not exhibit changes associated with tumor transformation [16, 17] since telomerase activity is associated with cellular immortality, not growth deregulation.

Humans and mice show considerable differences with respect to the biology of their telomeres. The results from several laboratories indicate that there are fundamental differences in the regulation of telomerase and telomere length between human and mice [18–22].

To further our understanding of telomere biology in mammals, we have studied the relationship between telomere length, telomerase activity, and cellular life span in another species, the sheep. In particular, we have investigated whether expression of hTERT in sheep fetal fibroblasts extends proliferative life span and the extent to which the developmental capacity of these cells can be reprogrammed by nuclear transfer (NT). This is because replicative senescence in sheep somatic cells has a major impact on their genetic manipulation prior to NT, particularly if multiple genetic changes are required [23]. Our results reveal that sheep fibroblasts are very similar to human fibroblasts with respect to the biology of their telomeres. They have no detectable telomerase activity and undergo only a finite number of divisions in culture before they enter replicative senescence with a life span similar to that reported for human cells [3]. Expression of hTERT in these cells restores telomerase activity and extends proliferative life span, showing that the hTERT is compatible with the sheep TR (sTR) as well as the other sheep telomerase components required to assemble an active complex. Sufficient expression of hTERT maintained telomere length and preserved genome stability. Nuclei that were transferred into sheep oocytes from one of these cell lines retained a substantial degree of developmental plasticity and are reprogrammed by the oocytes to direct embryonic and early fetal development, although development to term was not observed. Cells derived from the NT fetuses exhibited telomerase activity and continued to maintain their telomere lengths, suggesting that constitutive expression of telomerase is compatible with reprogramming and early development.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture, Plasmids, and Transfection

Sheep fibroblasts were isolated from eviscerated fetuses [24] in which BW6F2 cells were recovered from a Day 35 Black Welsh fetus and OE644, IE011, and IE018 were recovered from NT-derived fetuses at the time of abortion. Cells were cultured in Glasgow Minimum Essential Medium (G-MEM, Sigma, St. Louis, MO) supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 1x nonessential amino acids, and 10% fetal calf serum (FCS, Globe Farm, Surrey, UK) in a humidified incubator at 37°C and 5% CO2.

Plasmid GRN145 was kindly provided by Geron Corporation (Menlo Park, CA) and contains hTERT cDNA driven by myeloproliferative sarcoma virus (MPSV) promoter and puromycin-resistant gene PAC driven by the SV40 promoter [13]. Control vector was constructed by EcoRI digestion of plasmid GRN145 followed by religation to remove the hTERT cDNA. Linearized plasmid was electroporated (250 µF/400 V) to BW6F2 cells at passage 6. Cells were then plated into 96-well plates at 5000 cells per well. Puromycin (750 ng - 1 µg/ml) was added into medium 48 h after transfection to select puromycin-resistant colonies.

Telomere Length Assay

Genomic DNA from cultured cells was isolated by phenol/chloroform extraction using tubes containing phase lock gel (Eppendorf, Hamburg, Germany). Telomere length was determined by telomere restriction fragment (TRF) Southern blot analysis [3], and mean TRF was calculated as described previously [25].

Telomerase Activity Assay

Telomerase activity of cell extracts was analyzed by telomeric repeat amplification protocol (TRAP) assay as described previously [10] with either 32P-labeled primer or staining the gel with SYBR green II (Molecular Probes, Inc., Eugene, OR) [26].

Senescence-Associated ß-Galactosidase Staining

The senescence-associated ß-galactosidase (SA-ß-gal) staining was performed as previously published [27]. Briefly, cells were washed in PBS and fixed in 2% formaldehyde/0.2% glutaraldehyde for 2–4 min at room temperature. Cells were washed again in PBS followed by incubation in SA-ß-gal staining solution overnight at 37°C.

Flow Cytometry

Cells were fixed in cold 70% ethanol after harvesting by trypsinization and washing in PBS. The cells were analyzed on a FACScan (Becton Dickinson, Franklin Lakes, NJ) after staining with propidium iodide (20 µg/ml). Ten thousands cells were acquired for each sample and analyzed by using CELLQUEST software (Becton Dickinson).

Nuclear Transfer

Nuclear transfer was done according to previous protocols [23, 24]. Embryos that developed to morulae or blastocysts in culture were transferred to the uteri of estrus-synchronized recipients (one to two embryos per recipient) and allowed to develop. Pregnancies were monitored using subcutaneous ultrasound scanning.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Sheep Fetal Fibroblasts Have a Limited Replicative Life Span

BW6F2, a sheep primary fetal fibroblast line, was continuously grown in culture, and the growth curve was recorded from passage 6. These fibroblasts divided at a rate of 17–24 h per population doubling (PD) until they reached approximately 100 doublings. After that, the rate of division slowed down, and the cells finally ceased dividing at PDs 113 (Fig. 1A). The morphology of the cells at later PDs was distinctly different to earlier passage cells (Fig. 1B); they became flattened and enlarged, with the typical appearance of senescent fibroblasts (Fig. 1C). These later passage cells were shown to be almost 100% positive using senescence-associated ß-gal staining (Fig. 1E), whereas no staining cells were seen at the earlier PDs (Fig. 1D). Telomere lengths in BW6F2 were measured by Southern blot analysis at early, middle, and late passages. This revealed that telomeres shortened with cell division and that the mean TRF shortened from almost 21 kb in early passage cells to about 10 kb in senescing cells (Fig. 1F), losing 50–200 bp telomeric sequence each cell division, which is in the same range reported for human fibroblasts [28]. Telomerase activity in the sheep fibroblasts was determined by TRAP assay and was negative (Fig. 2).



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  		FIG. 1. Sheep fibroblasts have a limited proliferative life span in culture. A) Growth curve of BW6F2 sheep fetal fibroblasts in G-MEM containing 10% fetal calf serum at 37°C, 5% CO2. The fibroblasts were resuscitated from liquid nitrogen and grown for 2 days, and then 3 x 105 cells were plated into a new flask (passage 6) to start the growth curve. The cells were maintained at log phase growth throughout the growth curve. B and C) Morphology of sheep fibroblasts at early passage (B) and at senescence (C). D and E) SA-ß-gal staining. Cells were stained for SA-ß-gal and are clearly positive when senescent (E) but negative in early passage cells (D). F) Sheep telomeres shorten with progressive cell division. DNA was extracted at various PD, and telomere length was measured by Southern blot analysis with (TAAGGG)3 probe after RsaI and HinfI digestion. White bars indicate mean TRF (telomere restriction fragment) length. (B–E, original magnification x 100)



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  		FIG. 2. Telomerase activity in hTERT-transfected clones. Reconstituted telomerase activity was measured by TRAP (telomeric repeat amplification protocol) assay with 32P-labeled primer. 1-1, 2-1, 2-5, 2-7, 2-8, 2-12, and 2-13 represent individual hTERT-transfected clones; BW6 represents parental cells BW6F2, and 293 is a transformed human kidney epithelial cell line used as a positive control. IC, internal control

Expression of hTERT Extends Proliferative Life Span

Forced expression of hTERT in a variety of normal human cells, including retinal epithelial cells, endothelial cells, and foreskin fibroblasts, results in reconstitution of telomerase activity and extension of replicative life span [13, 15]. To explore whether hTERT is compatible with sheep telomerase template RNA and other factors of the telomerase complex, we expressed hTERT in BW6F2 fibroblasts by stable transfection at passage 6. Seventeen clones were isolated after puromycin selection, and integration of hTERT cDNA was confirmed by PCR and Southern blot hybridization (data not shown). Eight of these had detectable telomerase activity as judged by TRAP assay (Fig. 2). One was found to be missing the Y chromosome at an early passage and so was excluded from further experiments. Additionally, five puromycin-resistant clones were obtained from BW6F2 cells transfected with control vector containing no hTERT cDNA. The proliferative life spans of these clones were examined and the results were shown in Figure 3A. The hTERT-expressing clones (Fig. 3A, open symbols) grew vigorously and maintained a similar growth rate to the early passage parental BW6F2 cells even after more than 180 PDs. Morphologically, they were indistinguishable from early passage fibroblasts, and they exhibited no positive staining with SA-ß-gal (Fig. 3, C and E). By contrast, the clones transfected with control vector and the clones that failed to express hTERT all had much slower growth rates (Fig. 3A, solid triangles), and all of them stopped dividing by 60 PDs. These cells revealed typical perinuclear staining with SA-ß-gal, a biomarker for senescence (Fig. 3, B and D). Telomere lengths were measured during an extended period of cell culture. Three of the cell lines maintained full-length telomeres, whereas the others exhibited progressive shortening and finally stabilized at characteristic lengths that were a function of hTERT expression levels [29]. These results show that the human telomerase catalytic unit is able to interact with sheep telomerase RNA template as well as with other components of the telomere complex to reconstitute telomerase activity, regulate telomere length, and extend proliferative life span.



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  		FIG. 3. Expression of hTERT in sheep fibroblasts extends proliferative life span. A) Growth curves of cells transfected with either vector containing hTERT or control vector. Each line represents a single clone. Clones expressing hTERT (open symbols) have maintained their growth rate and showed at least a 3-fold extension of proliferative life span. By contrast, clones having control vector only or not expressing the hTERT construct (solid triangles) showed a slower growth rate and stopped dividing at an earlier PDs. B–E) SA-ß-gal staining. Cells with hTERT expression did not show positive staining with SA-ß-gal even at high PDs (C and E), whereas cells transfected with the control vector were positive at early PDs (B and D) (B–E, original magnification x100)

hTERT-Expressing Cells Do Not Show a Transformed Phenotype after Extension of Their Natural Life Span

Loss of contact inhibition and acquisition of serum-independent growth are some of the characteristics of tumor progression in culture [30, 31]. It has been previously reported that hTERT-expressing human primary cells do not exhibit any changes associated with tumor transformation [16, 17]. To assess whether the hTERT-expressing sheep fibroblasts still maintain the normal cell characteristics, we examined their growth response both to low serum concentration and to contact inhibition. Cells that had been grown in culture continuously for about 230 days and had undergone over 150 PDs, at least 40 PDs more than the natural life span, were transferred to culture medium containing 0.1% serum for 7 days and then switched to 10% serum. All seven clones showed growth inhibition after 7 days in 0.1% serum (Table 1) and reentered the cell cycle synchronously after adding back serum to the media (Fig. 4). The cells also showed contact inhibition, and after being maintained at confluence for 3–4 days, the proportion of cells in S phase dropped 2–3-fold (Table 1). These data show that hTERT-expressing sheep fibroblasts maintain normal cell characteristics and do not exhibit a transformed phenotype after extension of their natural life span. Their behavior thus corresponds closely to a number of hTERT-immortalized human primary cells [16, 17]. After further continuous culture (>250 days), however, some of these cell lines exhibited karyotypic abnormality, and this was correlated with steady-state levels of telomere length and hTERT mRNA [29].


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  		TABLE 1. hTERT-expressing sheep fibroblasts respond normally to cell density and serum starvation



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  		FIG. 4. hTERT-expressing cells maintain a normal response to serum starvation. Cells were grown at subconfluence in G-MEME containing either 10% serum (SC) or 0.1% serum (SS) for 7 days, and then parallel flasks were changed to 10% serum and grown for another 24, 48, and 72 h (SR 24, SR48, and SR72, respectively). The cells were finally stained with propidium iodide and analyzed by FACScan. The results showed that cells stopped proliferation in 0.1% serum for 7 days (SS) but reentered the cell cycle after transferring into medium containing 10% serum (SR24, SR48, and SR72)

Nuclei from hTERT-Expressing Fibroblasts Support Early Development after Being Reprogrammed

Nuclear transfer provides a new method for producing transgenic animals and is also a powerful tool to evaluate the developmental potential of cell nuclei. In livestock, successful reports have used primary fetal fibroblasts as nuclear donors [24, 32, 33], and we wished to evaluate the extent to which hTERT-expressing fibroblasts would be competent for NT. To explore the developmental potential of the hTERT-immortalized sheep fibroblasts, cells from line 2-5 at passage 25 (about 87 PDs after transfection) were used as nuclear donors in nuclear transfer experiments. Line 2-5 was one of the three lines that have higher hTERT expression and maintained full-length telomeres and normal karyotype throughout an extended period (>200 PDs) of cell culture [29]. The results are summarized in Table 2. Nuclei derived from these cells supported early embryonic and fetal development. There was no significant difference in efficiency of preimplantation development between embryos derived by NT from hTERT-expressing cells (4.4% blastocysts) and parental, early passage BW6F2 cells (5.4% blastocysts). These results contrast with a report in cattle describing the use of a spontaneously immortalized mammary gland epithelial cell line as nuclear donor, which failed to support development to blastocyst of the reconstructed embryos [34]. However, no fetuses derived by NT from 2-5 survived beyond 40 days of pregnancy. This contrasted to the survival of fetuses derived by NT from early passage BW6F2 or from YH6, a late passage gene-targeted derivative [23], and in both these cases live-born lambs were obtained (Table 2). The failure to sustain pregnancies beyond day 40 with embryos derived from 2-5 cells contrasts sharply to the overall success rate of NT from both early and late passage cells in our laboratory. Thus, with transferring 517 blastocysts to a total of 241 recipients, the average frequency of establishing pregnancy at day 45 was 24.5%, significantly different from the results obtained with 2-5 (P < 0.001, {chi}2 test). Furthermore, although large differences in success between different cell lines and experiments were observed, full-term pregnancies were always established.


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  		TABLE 2. Development of nuclear transferred embryo from sheep fibroblasts

Five fetuses derived by NT from hTERT-expressing cells were aborted between days 44 and 51 when they were judged, by ultrasound scanning, to have died (Table 3). Postmortem examination showed that they all developed beyond gestation Day 20, and eyes, ears, limbs, and tails were clearly observed in some of the older ones. Primary cell lines were established from three fetuses. Interestingly, these cells all showed positive telomerase activity by TRAP assay at passage 1 (Fig. 5) as well as after passage 7 (data not shown). One of them (IE011) showed full-length telomeres even after 25 passages (about 90 PDs) in culture, and the other two cell lines showed a substantially reduced rate of telomere shortening, suggesting that hTERT continued to function during nuclear reprogramming by NT. These data showed that nuclei from telomerase-immortalized sheep fibroblasts retain a substantial degree of developmental plasticity and can be reprogrammed by the oocyte to direct embryonic and early fetal development.


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  		TABLE 3. Development of NT-derived hTERT embryos in vivo



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  		FIG. 5. Telomerase activity in cells derived from cloned fetuses generated by using hTERT-expressing cells as nuclei donors. The fetuses were aborted at Days 44–45 after NT. Fibroblasts cell lines were derived from the carcasses and grown in culture. Telomerase activity was examined by TRAP assay at passage 1 for each cell line and was all positive, though it may at a lower level in comparison with the GRN2-5 cell line before NT. IE011, IE018, and OE644 represent three cell lines derived from NT fetuses, and GRN2-5 is the hTERT expressing cell line used as donor cells for NT. IC, internal control


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this report we demonstrate that sheep fibroblasts have similar characteristics to human fibroblasts in terms of the length and rate of shortening of their telomeres but, in particular, that the forced expression of hTERT can reconstitute the telomerase activity and extend the proliferative life span of these cells.

It has been reported previously that expression of hTERT in bovine adrenocortical cells resulted in cell immortalization without affecting their efficiency of transplantation [35] and that hTERT expression in telomerase-negative rabbit tumor cells reconstituted telomerase activity [36]. These results suggested that hTERT can function in other mammalian cells, although neither of these studies showed to what extent hTERT expression could extend the proliferative life span of normal somatic cells in these species. Here we extend studies of the telomere, telomerase, and their relationship to proliferative capacity to another mammal, the sheep. We show unequivocally that expression of hTERT reconstitutes the telomerase activity in the sheep fibroblasts and as a consequence of this extends the proliferative life span. It has been demonstrated in human cells that reconstitution of telomerase activity by TRAP assay requires at least two core components—telomerase RNA (TR) and catalytic subunit (TERT) [37, 38]—while having functional telomerase to add telomeric repeats in vivo may even require more telomerase-related factors to form an active complex [39]. Our results indicate that hTERT is compatible with sheep telomerase RNA and any other telomerase-related factors required for functional telomerase activity in these cells. These results are consistent with the previous results and in support of the finding that telomerase RNA displays a strikingly similar secondary structure in 32 vertebrate phyla [39, 40]. They also suggest that regulation of telomerase and telomere length in sheep and human cells share a similar mechanism.

Human TERT-expressing sheep fibroblasts maintained the same response to serum starvation and contact inhibition as normal primary somatic cells after at least 40 generations beyond their natural life span, indicating that they are not transformed at the time. We attempted to monitor changes in cell cycle-associated proteins such as pRB, p53, p21, and p16 using the relevant human antibodies, but these did not detect their sheep counterparts and so could not be used for this purpose. However, we routinely karyotyped these cells, which were diploid until at least 220 PDs. This is more than three times the life span of the longest surviving nonexpressing clone. Thereafter, some of the hTERT lines did exhibit karyotypic abnormalities indicative of a possible transformation event, although three of the lines have now proliferated for more than 500 PDs without any apparent abnormalities [29].

Human cells whose proliferative life span has been extended by the forced expression of hTERT show the appropriate regulation of cell cycle genes as well as transcription profiles consistent with a lack of transformation [17, 39]. In sheep, NT provides a powerful alternative to assess the "normality" of such cells. To date, all successful NT in livestock has been carried out using cultured somatic cells. Successful cloning requires that the cell cycle of the donor nucleus be compatible with host cytoplast [41]. For early preimplantation development, the cycle of the donor nucleus must be responsive to the factors controlling cell division in the early embryo. Transformed cells have lost this capacity to respond to the normal cell cycle controls. This is evidenced by the fact that nuclei from a spontaneously immortalized bovine mammary epithelial cell line failed to support early cleavage and development to the blastocyst [34]. By contrast, nuclei from hTERT-immortalized sheep cells supported early cleavage and development to blastocyst at a reasonably high efficiency, showing that they were as responsive to the cell cycle controls of the early embryo as nuclei from normal primary somatic cells. The recovered NT fetuses from hTERT-expressing cells showed significant organ development (Table 3), showing that these cells are capable of postimplantation development. However, none of these fetuses developed beyond Day 40. This could be due to a number of possibilities, including the integration of the hTERT sequences disrupting a gene important for later fetal development. The constitutive expression of telomerase expression may not be compatible with later fetal development, particularly if this were to prevent normal processes requiring programmed cell death. Alternatively, the accumulated mutational load in these cells after the extended period of cell culture may block later development. Although it is possible to get full development from telomerase negative near-senescent cell lines, such as YH6, cell line 2-5 had been growing in culture for more than twice the number of PDs prior to NT.

NT provides a new method for making transgenic animals, in which the modified DNA is transfected and integrated into the genome of cultured cells before NT. This is important because in the absence of ES cells in livestock, it provides the only feasible route for carrying out gene targeting in these species. This has been achieved recently in sheep [23, 42]. A major limitation to gene targeting somatic cells, however, is the overall proliferative life span of the cells. Selection and expansion of targeted clones requires long-term culture, and in some cases this may approach or even exceed the natural life span of the cell [43]. Thus, we have found in sheep that a very high proportion of targeted cell clones senesce prior to NT, and this dramatically reduces the efficiency of effective gene targeting [23]. Methods to extend life span of donor cells, therefore, have tremendous implications for the genetic engineering of these species. Although full-term development was not achieved with NT from hTERT cells, this may simply reflect that, so far, we have sampled only one clonal line. If the failure to achieve full-term development was due to the integration of hTERT disrupting a gene important for late development or accumulation of deleterious mutations in this one line, then other lines should prove more successful and, in addition, it should be possible to improve culture conditions to reduce the mutational load in vitro. If, however, the constitutive expression of telomerase was the cause of developmental failure, then producing cells in which hTERT was conditionally regulate might provide the solution.


   ACKNOWLEDGMENTS
 
We acknowledge technical assistance from the embryology and large animal surgery teams at Roslin and would like to thank Calvin Harley and Jane Lebkowski for critical reading of the manuscript.


   FOOTNOTES
 
1 This work was supported by the BBSRC and the Geron Corporation. Back

2 Correspondence. FAX: 44 0 131 440 0434; john.clark{at}bbsrc.ac.uk Back

Received: 12 November 2002.

First decision: 6 December 2002.

Accepted: 5 February 2003.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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