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a Department of Veterinary Physiology and Pharmacology,
b Department of Large Animal Medicine and Surgery, Texas A&M University, College Station, Texas 77843
c Ultimate Genetics, Franklin, Texas 77856
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Day 30 pregnancy rates were similar for blastocysts derived from adult (6 of 26; 23%) or fetal (5 of 32; 16%) cells. Day 90 pregnancy rates were 3 of 26 for adult and 0 of 32 for the fetal cell lines. One viable bull calf derived from a 21-yr-old serum-starved adult skin fibroblast was born in August 1999. In summary, somatic NT embryo development rates were similar whether adult or fetal cells, from the same genotype, were used as donor cells. Serum starvation of these adult donor cells did not improve development rates of NT embryos to blastocyst, but when fetal cells were serum-starved, there was a significant increase in development to blastocyst.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Wakayama et al. [3] showed that cells from cloned mice can be used to generate more clones with apparent equal efficiency and therefore suggested that there were no changes in the cloned offspring that influenced subsequent cloning efficiency. The use of recloned fetuses was suggested by Cibelli et al. [2] to benefit transgenic animal production by increasing the number of population doublings available for gene targeting events such as homologous recombination. As bovine fetal fibroblasts usually have a finite life in culture, with senescence occurring after approximately 32 population doublings [2], a greater number of cell divisions can be achieved by rejuvenation of the fetal cell line by nuclear transfer (NT).
We have compared the development rates of cells from adult and from recloned fetal cells because the use of cells derived from an adult with a desirable phenotype generally precludes transgenic work due to the short life span of the adult cells until quiescence. A genetically identical fetus can be produced by NT using these adult cells, then surgically removed to create a rejuvenated cell line that may allow gene targeting experiments to continue [2]. However, the success rates of rejuvenated cells as donor cells for NT have not been reported for livestock species. In the study reported here, we compared the development of adult and fetal cells of the same genotype, and we also took this opportunity to observe the full-term development of a calf cloned from an uncommonly old (21-yr) adult bull.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adult The adult cell line was derived from a surgical excisional biopsy performed on a 21-yr-old Brahman bull (Fig. 1) in June 1998. Thin sections of the s.c. tissue were diced into 1- to 3-mm pieces using a razor blade, and explants were transferred into 25-mm2 flasks containing Dulbecco's modified Eagle's medium (DMEM-F12; Gibco Laboratories Inc., Grand Island, NY) + 10% v:v fetal bovine serum (Summit, Fort Collins, CO) + 1% v:v penicillin/streptomycin (Gibco; 10 000 U/ml penicillin G, 10 000 µg/ml streptomycin) and then cultured at 37°C in air containing 5% CO2. When confluence was achieved at 14 days, cells were trypsinized for 5 min, and total cell count was determined using a Coulter counter. The recovered cells were centrifuged, and the pellet was resuspended at a concentration of 1 million cells per ml. Aliquots were either frozen in DMEM-F12 containing 10% dimethyl sulfoxide (DMSO) before storage at -80°C, or 250 000 cells were transferred into a new 25-mm2 flask. As confluence was approached, the cells were passaged by trypsinization and again counted. The mean population doubling time for the first three passages (24 days in culture) was 44 h.
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Regenerated fetus A Day 40 fetus cloned from the adult cell line was surgically removed from the recipient cow's uterus. The head and viscera were removed, and the remainder of the fetal tissue was sliced into 2- to 5-mm pieces. These explants were then cultured as above. The mean population doubling time for the first eight passages (44 days in culture) was 27.4 h.
NT
Oocytes Recipient oocytes were slaughterhouse-derived from predominantly Brahman-cross cattle and matured for 17 h in Medium 199 (M199; Gibco) supplemented with 10% v:v fetal calf serum (FCS; Gibco), FSH (0.1 U/ml; Sioux Biochem, Sioux City, IA), LH (0.1 U/ml; Sioux Biochem), estradiol (1 µg/ml; Sigma, St. Louis, MO), pyruvate (28 µg/ml; Sigma), EGF (0.05 µg/ml; Sigma), and 1% penicillin streptomycin. The cumulus-oocyte complexes were vortexed at 17 h postmaturation (hpm) for 1–2 min, and then the oocytes were washed, placed in 0.05% w:v Pronase E (Sigma) for 3 min, and held in M199 + 10% FCS.
Enucleation Oocytes were enucleated at 19 hpm. Before enucleation, oocytes were placed for 15 min in Hepes-buffered synthetic oviductal fluid (H-SOF) [12] with 4 mg/ml BSA (Sigma) that contained 7.5 µg/ml cytochalasin B (Sigma) and 5 µg/ml Hoechst 33342 (Sigma). At this time, oocytes were selected for presence of a polar body and homogeneous cytoplasm. Suitable oocytes were enucleated in H-SOF with 7.5 µg/ml cytochalasin B using a beveled 25-µm outside-diameter glass pipette. Only oocytes in which the removal of both the polar body and metaphase nucleus was confirmed by observation under UV light were included in the experiment. Oocytes were then randomly allocated to be combined with either adult or fetal fibroblasts.
Donor cells Serum starvation of donor cells was achieved by culture in DMEM/F12 + 0.05% FCS for 1–5 days before NT. Fibroblasts were prepared by trypsinization of early-passage adult (passage [P] 3–4; Days 13–24 in culture) and fetal (P 3–4; Days 11–21 in culture) cells at 60–80% confluence.
Combining donor and recipient cells Fibroblasts of median diameter were combined with enucleated oocytes in 7.5 µg/ml cytochalasin B in H-SOF using a 30-µm outside-diameter glass pipette, then returned to M199 + 10% FCS. The oocyte-fibroblast couplets were manually aligned and fused in a 3.2-mm fusion chamber that contained Zimmerman cell fusion medium [13] using 2 x 20-µsec 1.6-kV/cm DC fusion pulses delivered by a BTX Electrocell Manipulator 200 (BTX, San Diego, CA). Oocyte activation was performed 3–5 h after fusion at 27 hpm, by a 4-min incubation in 5 µM ionomycin (Calbiochem, San Diego, CA) followed by 4 min in 3% BSA in Tyrode's lactate-Hepes and 4 min in H-SOF [14]. Fusion was assessed at this time by light microscopy before transfer into 100 µM butyrolactone-I (Biomol, Plymouth Meeting, PA) in SOF for 4 h followed by transfer to Charles Rosenkrans medium #1 with added amino acids (CR1aa) [15] + 10% FCS with buffalo rat liver coculture for 7 days.
Embryo development Embryos were classified as blastocysts according to their morphology on Day 7 or Day 8 following NT. Two or three blastocysts were nonsurgically transferred when synchronized recipients were available. Pregnancy status was assessed by transrectal ultrasonography (Aloka 500, 5-MHz transducer; Aloka Co., Tokyo, Japan) at 30 days post-NT and confirmed 10 days later, with pregnant recipients rechecked every 2 wk.
Microsatellite Analysis
The genomic DNA was compared between adult bull tissue (taken at the time of the bull's death in September, 1998), fibroblasts from the regenerated fetus, and blood from the newborn calf and surrogate Charolais dam using an ABI 310 automated DNA sequencer (Applied Biosystems, Inc., Foster City, CA) and the manufacturer's suggested protocols.
Statistical Analysis
For comparison of blastocyst development rates in adult and fetal donor cells (within plot factors), logistic regression using a generalized linear mixed model (Glimmix) in Statistical Analysis Systems was used to calculate the within-day fixed effect of donor cell type with adjustment for overdispersion by day [16]. To analyze the effect of serum starvation on blastocyst development rates (whole plot factors), logistic regression was used with correction for overdispersion by day according to the method of Williams [17]. The chi-square test was used for comparison of pregnancy rates.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The development of embryos reconstructed from either adult or fetal cells is summarized in Tables 1 and 2. Eight replicates were performed to compare developmental potential of adult and regenerated fetal cells cultured in 10% serum and five replicates under serum-starved conditions. Blastocyst production ranged from 9% to 47% of successfully fused oocytes for adult cell-derived NT embryos and from 0–60% for fetal cell-derived NT embryos. The total number of blastocysts produced per day ranged from 3 to 19, with no difference in morphology seen between blastocysts derived from adult or fetal cell NT (Fig. 2).
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There was no significant difference in development of fused oocyte-fibroblast couplets to blastocyst between fibroblasts derived from adult and from fetal cell cultures (Table 1). Similar NT embryo development rates were observed whether the donor cells were serum-fed (adult cell-derived blastocysts 28% vs. fetal cell-derived 12%; P > 0.1) or serum-starved (adult 28% vs. fetal 43%; P > 0.1) before NT. Oocyte-fibroblast fusion rates were higher for adult fibroblasts in the serum-starved (adult 60% vs. fetal 47%; P < 0.01) group and tended to be higher in the serum-fed group (adult 58% vs. fetal 38%; P = 0.06).
When the effect of donor cell treatment was analyzed within groups, serum starvation of fetal donor cells before NT was found to significantly improve NT blastocyst rates (serum-fed 12% vs. serum-starved 43%: P < 0.01). However, serum starvation of adult cells had no effect on NT blastocyst rates, which remained at 28% for both treatments.
Pregnancy Rates
When they were available, blastocysts were transferred into synchronized recipients, which resulted in an unequal distribution of embryos transferred from the serum-starved and serum-fed groups. Twenty-six embryos derived from adult cells (12 serum-fed + 14 serum-starved) were transferred into 11 recipients, and 32 embryos derived from regenerated fetal cells (30 serum-starved and 2 serum-fed) were transferred into 12 recipients (Table 3). Six fetuses produced from the adult line were detected (including presence of a heartbeat) by ultrasonography. Four were derived from serum-starved (0.05% FCS for 3–5 days before NT) and two from serum-fed fibroblasts. Five fetuses derived from the regenerated fetal cell line were detected (including presence of a heartbeat) by ultrasonography, and each was derived from a serum-starved cell.
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One fetus derived from the fetal cell line was surgically removed at Day 56 of pregnancy to confirm parentage by DNA analysis and to observe morphology. This fetus appeared to be normal, with a crown-rump length of 4 cm. The Day 90 pregnancy rates were 0 of 6 for fetal and 3 of 6 for adult cell-derived NT fetuses. Because 1 fetus from each of the adult and fetal cell derived groups was surgically removed in the second month of gestation, it is difficult to compare the Day 90 pregnancy rates.
Neonatal Observations and Treatments
One calf weighing 40 kg was born by Cesarian section at 290 days of gestation 30 h after administration of dexamethasone (20 mg i.m.) and prostaglandin (dinoprost, 25 mg i.m.) to the dam. The calf required intensive monitoring and therapy for a period of 5 days to treat lung dysmaturity and pulmonary hypertension, which were diagnosed by radiography and echocardiography. The cardiopulmonary abnormalities seen in this calf resembled those previously reported in cloned calves and fetuses derived from fetal cells [2, 18]. The calf was fed by oro-gastric tube until it began nursing its dam at 5 days of age. Type 1 insulin-dependent diabetes was diagnosed at 7 days of age after observation of polydipsia and polyuria. Further tests revealed elevated fasting serum glucose concentrations (> 200 mg/dl; normal 50–80 mg/dl), glycosuria, and low serum insulin levels (10 uU/ml; reference sample 65 uU/ml). Insulin administration (5 IU regular insulin twice every 12 h) was commenced, which consistently maintained fasting blood glucose levels between 100–120 mg/dl, and stopped the glycosuria. A generalized yeast skin infection, which is commonly associated with diabetes, was diagnosed and treated with an antimycotic shampoo. A mild, chronic iron deficiency anemia (serum iron 52 µg/dl; normal 57–162 µg) was treated with oral mineral and multivitamin supplements. White cell parameters remained within the normal range, although further analysis of lymphocyte subpopulations revealed that the calf's leukocytes expressed very low, almost undetectable levels of CD45 antigen. Four age-matched control calves showed normal expression of CD45, which has been shown to be critical for T cell activation [19]. The calf's demeanor and strength improved rapidly, and by 50 days of age it had gained 45 kg. Serum glucose levels were closely monitored, and insulin administration was discontinued at 2 mo of age, when normal endogenous insulin levels and normal blood glucose concentrations were maintained.
Microsatellite Analysis
The genomic DNA of the cloned calf and regenerated fetus matched that of tissue obtained from the adult bull at the time of his death and was distinct from that derived from the surrogate cow that carried the cloned calf (Table 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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One practical reason for recovering a regenerated fetus was to ensure an adequate supply of cells from the original adult bull genotype, as we were unsure how many cell divisions the 21-yr-old adult cells would achieve before senescence. It was also fortuitous that we did recover a fetus, as the frozen aliquots of adult cells were destroyed during a freezer malfunction. The bull had died from unknown causes before the freezer malfunction, and the tissue recovered post-mortem was unsuitable for cell culture, although DNA recovery for microsatellite analysis was successful.
The different pregnancy rates achieved in this study between fetal and adult cells may have been caused by factors such as altered karyotype in the fetal cells, although this remained stable throughout the period of cell culture. The fetal cells were derived from a Day 40 NT fetus that may have possessed undetected cellular changes that compromised NT fetal development, as there is a high incidence of abnormalities in cloned fetuses [1, 2, 5, 18]. It is also possible that the lower pregnancy rate was due to the small number of embryos transferred. Currently, cloning efficiencies are low, with around one live-born animal per 100 reconstructed NT embryos [9], and the developmental potential of somatic cell clones appears to vary according to the donor cell chosen. Previous studies have shown blastocyst development of NT embryos derived from fetal fibroblasts to be 18–43% [1, 2, 22] and of those from adult cells to be 9–49% [5, 7, 23]. In these studies, Day 40 pregnancy rates ranged from 9% to 47% (fetal cells) and from 15% to 80% (adult cells). The high rates of fetal loss following early pregnancy diagnosis seen in the present study have been observed in cloned pregnancies in cattle, sheep, and mice [1–3, 5, 9]. This high abortion rate appears to be related to abnormalities of placentation [3, 24]. The exception to this was the study of Kato et al. [7], who used granulosa and oviductal cells as NT donor cells and recorded an 80% pregnancy rate with all of these pregnancies progressing to term, although 50% of the calves died soon after birth.
It is also possible that in vitro cell culture conditions influence pregnancy rates [25]. We used a serum-containing medium and coculture system previously shown to result in NT embryo pregnancy rates of 20–30% [2]. Others have used similar serum-based coculture systems [2, 7, 23], in vivo sheep oviduct culture [1, 26], or serum-free defined media [5].
When donor cell nuclei are transferred into metaphase II oocytes, improved development occurs with diploid cells [27], and it is therefore preferable for donor cells to be in the G1/G0 stage of the cell cycle. Donor cell types such as fetal fibroblasts [2, 28] or granulosa [5, 29] or cumulus cells [3] have been chosen to maximize the percentage of cells in G0/G1. Alternatively, the G0 state can be reliably induced in a variety of cell types by a period of serum starvation [1, 26]. Population doubling times from the present study (adult 44 h; fetal 27.4 h) suggest that more adult than fetal cells were in G0/G1. As cell populations approach senescence, fewer cells cycle, and those that do spend more time in the G1 phase [20].
We compared the development rates of adult and fetal cells under either serum-starved [26] or serum-fed [2] culture conditions to establish which treatment resulted in the best blastocyst development rates for each cell type. There appeared to be no effect of serum starvation of adult cells on development to blastocyst (serum-starved and serum-fed 28%), whereas it markedly improved that of fetal cells (serum-starved 43% vs. serum-fed 12%). The subsequent in vivo development of blastocysts derived from either the serum-starved or serum-fed donor cells showed no difference, although the sample size was too small to allow any meaningful interpretation of pregnancy rates from the two treatments. Vignon et al. [23] found no effect of serum starvation of either adult- or fetal-derived fibroblasts on in vitro NT embryo development.
The cloned calf produced in this experiment possessed significant metabolic and cardiopulmonary abnormalities similar to those observed in previous studies [2, 5, 18]. Diabetes mellitus has seldom been reported in the bovine species [30], and the resolution of diabetes in this calf is likely to have been due to hyperplasia of an initially subnormal population of insulin-producing pancreatic beta cells. In humans and mice, juvenile-onset type 1 diabetes is an autoimmune disease in which the pancreatic beta cells have been destroyed by T lymphocytes [31]. The health of the cloned calf will continue to be monitored closely. Severe immunodeficiency due to lymphoid hypoplasia has been reported in a cloned calf [8], and the deficiency in the leucocyte common antigen, CD45, may impair immune response, although at present the calf is over 8 mo of age and is clinically normal.
Although much work is yet to be done comparing development from cells derived from different animals, our results and others [1, 5] suggest that it is possible to clone animals of advanced age, which bodes well for the preservation of superior livestock. Additionally, cells from individual animals with unique attributes such as disease resistance can be used to produce more animals of the same genotype for further research or for use in breeding programs. Regenerated fetuses will be of great use for enabling gene targeting using homologous recombination. This process will require many cell divisions to allow for selection of transgenic cells, and thus regeneration of the cell line by successive rounds of NT may assist in increasing the number of cell divisions [2].
In summary, our results indicate that cells from a very old male animal can be reprogrammed using NT to produce viable offspring and that cloned fetuses can be used to regenerate cell lines for use in successive rounds of cloning. The presence of significant cardiopulmonary pathology at birth, together with the juvenile-onset diabetes warrants further investigation. This study has important implications for the study of cell aging, and further investigations will pursue the activity of telomerase and telomere length both in the regenerated fetal cells and in the calf derived from the original adult cell line.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This research was supported by grants from the Department of Large Animal Medicine and Surgery to J.R.H. and the Texas Coordinating Board of Higher Education, Advanced Technology Program to M.E.W. 
2 Correspondence: Jonathan Hill, Box 34 Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853-6401. FAX: 607 253 3531; jrh35{at}cornell.edu
Accepted: December 6, 1999.
Received: September 22, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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