Transgenic Research 13: 215–224, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands.
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Viable transgenic goats derived from skin cells Esmail Behboodi, Erdogan Memili, David T. Melican, Margaret M. Destrempes, Susan A. Overton, Jennifer L. Williams, Peter A. Flanagan, Robin E. Butler, Hetty Liem, Li How Chen, Harry M. Meade, William G. Gavin & Yann Echelard∗ GTC-Biotherapeutics Inc., 5 Mountain Road, Framingham, MA, 01701, USA Received 1 September 2003; accepted 24 December 2003
Key words: cell cycle, cyclin D1 , embryo, goat, nuclear transfer, transgenic
Abstract The current study was undertaken to evaluate the possibility of expanding transgenic goat herds by means of somatic cell nuclear transfer (NT) using transgenic goat cells as nucleus donors. Skin cells from adult, transgenic goats were first synchronized at quiescent stage (G0 ) by serum starvation and then induced to exit G0 and proceed into G1 . Oocytes collected from superovulated donors were enucleated, karyoplast–cytoplast couplets were constructed, and then fused and activated simultaneously by a single electrical pulse. Fused couplets were either co-cultured with oviductal cells in TCM-199 medium (in vitro culture) or transferred to intermediate recipient goat oviducts (in vivo culture) until final transfer. The resulting morulae and blastocysts were transferred to the final recipients. Pregnancies were confirmed by ultrasonography 25–30 days after embryo transfer. In vitro cultured NT embryos developed to morulae and blastocyst stages but did not produce any pregnancies while 30% (6/20) of the in vivo derived morulae and blastocysts produced pregnancies. Two of these pregnancies were resorbed early in gestation. Of the four recipients that maintained pregnancies to term, two delivered dead fetuses 2–3 days after their due dates, and two recipients gave birth to healthy kids at term. Fluorescence in situ hybridization (FISH) analysis confirmed that both kids were transgenic and had integration sites consistent with those observed in the adult cell line. Introduction Recent advances in the large scale production of human recombinant proteins have had a significant impact on the pharmaceutical industry. The mammary gland of large animals is well suited for production of these proteins (Clark et al., 1998; Meade et al., 1998). Therapeutic proteins, such as alpha-1 antitrypsin, antithrombin, and several monoclonal antibodies (Wright et al., 1991; Edmunds et al., 1998; Pollock et al., 1999) have been produced in the milk of transgenic animals. Among the benefits of this technology are high production yields, low capital investment, and the elimination of reliance on products derived from human blood. ∗ Author for correspondence E-mail:
[email protected]
Until recently, the only reliable method available for producing transgenic farm animals has been pronuclear microinjection. The success rate of this technique has been low, with 0.5–3% of microinjected embryos giving rise to transgenic offspring (Hammer et al., 1985; Bondioli et al., 1991; Ebert et al., 1994; Gavin, 1996; Behboodi et al., 2001). The emerging use of transfected cultured somatic cells as karyoplast donors for nuclear transfer (NT) has several advantages over microinjection, and has facilitated the generation of transgenic animals (Campbell et al., 1996; Wilmut et al., 1997; Cibelli et al., 1998; Baguisi et al., 1999; Onishi et al., 2000; Polejaeva et al., 2000). NT using transfected somatic cells allows the prescreening of cells for desirable genotypic characteristics which can reduce the number of animals (donors and recipients) used during the production of transgenic animals. In cattle, sheep and goats, both fetal and adult
216 primary cells have been used as karyoplast donors in somatic cell NT (reviewed in (Campbell, 2002; Foote, 2002; Mollard et al., 2002; Tsunoda & Kato, 2002; Yanagimachi, 2002)). The use of adult cells, such as skin cells, allows the cloning of individual animals for the propagation of desirable qualities such as superior conformation, disease resistance, and milk production (Kato et al., 1998; Wells et al., 1999; Westhusin et al., 2001). Adult skin cells also have the added advantage of being easier to obtain than fetal or cumulus cells. The efficiency of NT using these adult cells appears to be comparable to that seen with other somatic cell nuclei donors reviewed in (Yanagimachi, 2002). There is a possible reduction in live birth frequency in use of skin cells compared to fetal cells due to increased late-pregnancy losses (Heyman et al., 2002). The current study describes a further advance in the technology available for the generation of transgenic animals. Skin cells of an existing transgenic goat were collected, cultured, and used as nuclear donors in NT experiments. This technique results in transgenic progeny possessing the same transgene copy number and integration site(s) as in the donor cells. It is thus possible, based on the protein production characteristics of the transgenic goat from which the donor cells came, to predict what those characteristics will be in the cloned progeny. Cloning of existing transgenic animals can result in a highly focused initial expansion of a transgenic herd capable of large scale production of therapeutic proteins. The efficiency of NT has been shown to be influenced by the cell cycle status of the donor cells (Campbell, 2002). Live offspring have been produced both from cells that were serum starved and likely to be quiescent (G0 ) (Wilmut et al., 1997), and from cycling cells likely to be in the G1 phase of the cell cycle (Cibelli et al., 1998; Kasinathan et al., 2001a). Experiments evaluating the effect of the cell cycle phase on the ability of somatic donor cells to promote development of NT embryos have yielded somewhat contradictory results (Zakhartchenko et al., 1999; Kasinathan et al., 2001a, b; Cho et al., 2002). However, it is likely that cells that are in either G0 or G1 are competent (Wells et al., 2003). Therefore, to optimize NT conditions it may be more important to devise protocols that allow efficient synchronization of the donor cell population without inducing apoptosis (Lee & Piedrahita, 2002). In that respect, adult cell populations derived from skin biopsies are challenging, since they are heterogeneous and composed of sub-groups of cells likely to have varying cell cycle
characteristics. However, culturing these cells for several passages can result in a more homogeneous cell population whose cycle can be determined. In this study, we describe the use of adult, transgenic goat cells for somatic cell NT. A cell line was established from the skin of a mosaic, transgenic doe. A synchronization protocol was tested which resulted in a cell population with a majority of adult donor cells in the G1 phase of the cell cycle. In addition, the developmental potentials of goat NT embryos cultured to morulae and blastocyst stages in vitro and in vivo were evaluated. These experiments resulted in the generation of live, transgenic offspring.
Materials and methods Goats The herds of pure and mixed-breed Alpine, Saanen, and Toggenburg dairy goats used for this study are certified Scrapie-free and are maintained under good agricultural practice (GAP) guidelines at the GTCBiotherapeutics, Inc. farm in Charlton, Massachusetts, USA. Preparation of donor cells Skin biopsies were obtained from an adult transgenic Saanen doe previously derived by the pronuclear comicroinjection of two transgenes targeting the expression of antibody heavy- and light-chain fusion proteins to the mammary gland (Meade et al., unpublished data). Samples were finely minced and tissues were plated in a 60-mm culture dish with TCM-199 medium (Gibco BRL, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS) (JRH Biosciences, Lenexa, KS, USA), 2 mM L-glutamine and 50 IU/ml (50 µg/ml) penicillin–streptomycin and cultured in a humidified 5% CO2 incubator at 38◦ C. Established sub-confluent skin cell cultures were transferred to a 4well culture dish, maintained in TCM-199 with 0.5% FBS for 4 days (serum starvation), and then cultured in TCM-199 with 10% FBS (serum stimulation) for 3–6 h before NT. Cell cycle analysis: cell culture, preparation of cells for fluorescence-activated cell sorting (FACS) and northern blot analysis The cells were cultured and expanded in 100-mm cell culture dishes (Corning, NY) to 80–90% confluency
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Figure 1. Determination of timing of G0 → G1 transition in goat fibroblast cells by northern blot (A), and quantification of the northern data with phosphoimager (B). Bars are mean values ± SD of two experiments, bars with different superscripts are significantly different (P < 0.05). Cells were cultured the same way as for the FACS analyses. Cyclin D1 mRNA expression was at a very low level in serum starved cells (0 h), increased significantly upon serum stimulation for 5 and reached high level at 10 h of serum stimulation. Level of cyclin D1 mRNA expression in control cells was higher than serum starved cells, but not from that of serum stimulated cells for 10 h.
and then serum starved by culturing in medium containing 0.5% FBS for 4 days. The serum starved cells were then stimulated to resume proliferation by culturing in media supplemented with 10% FBS and were processed for FACS analysis after 0, 5 and 10 h. Cells in a separate culture dish that had not been serum starved were maintained at sub-confluency (70–80% confluency) and processed for FACS as a control. Processing for FACS analyses was as follows: cells were trypsinized (0.25% trypsin and 5.3 mM EDTA, both from Sigma), washed with PBS (Sigma, St. Louis, MO), and stored in 40% ethanol at 4◦ C for 3 days, after which the cells were centrifuged, washed with PBS, and resuspended in 500 µl of RNAse A (Sigma) solution (50 µg/ml in PBS containing 0.1% Triton X-100) at 37◦C for 45 min. The cells were then centrifuged and resuspended in 500 µl of propidium iodide (Sigma) solution (30 µg/ml in 6.9 ×10−5 M sodium citrate, from Sigma) and stored at 4◦ C overnight. FACS analyses of all samples were performed the following day. Ten-thousand events per sample were analyzed and the distribution of cells in G0 /G1 , S and G2 /M phases was determined. Northern blot analyses were performed to detect the initiation of transcription of mRNA encoding cyclin D1 , a G1 phase-specific marker. Cells were cul-
tured in duplicate under the same conditions used for cells analyzed by FACS. Using TRIZOL reagent (Invitrogen Life Sciences, Carlsbad, CA), total cellular RNA was isolated from one duplicate plate for each time point. Cells in the corresponding plate were trypsinized and counted using hemocytometer. RNA concentration was normalized to the cell number for each sample. Equal volumes of each RNA sample (representing equal numbers of cells) were electrophoresed on 1% agarose gels containing 6.7% formaldehyde as a denaturant. The RNA was transferred to Duralon-UVTM membranes (Stratagene, Cedar Creek, TX) by capillary action in 10 × SSC (1.5 M NaCl, 0.15 M Na citrate, pH 7.0). After transfer, the membranes were rinsed in 2 × SSC and cross-linked in a UV Stratalinker 2400 (Stratagene). Prehybridization was done for a minimum of 1 h at 65◦ C in a buffer containing 0.125 M Na2 HPO4 , pH 7.2; 0.25 M NaCl; 7% sodium dodecyl sulfate (SDS); 1 mM [ethylenedinitrilo]-tetraacetic acid (EDTA); and 0.1 mg/ml E. coli tRNA (Sigma). The probe used for blot hybridization was a 32 P-labeled mouse cyclin D1 DNA fragment, labeled using a Prime-It II Random Primer Labeling Kit (Stratagene) according to the manufacturer’s instructions. Preliminary experiments had shown that the mouse cyclin D1 probe
218 would cross-hybridize to goat cyclin D1 mRNA. Hybridization was at 65◦C overnight. After hybridization, the membranes were washed twice in 2 × SSC, 0.1% SDS at room temperature and twice in 0.2 × SSC, 0.1% SDS at 65◦C. The membranes were analyzed on a Storm 860 phosphoimager (Molecular Dynamics, Sunnyvale, CA). Northern blot experiments were repeated three times and results were analyzed by student’s t-test. A representative picture of northern blot along with a graph showing analyzed levels of cyclin D1 mRNA is exhibited in Figure 1. Oocyte donors – estrus synchronization/ superovulation Donor synchronization and superovulation were achieved by implanting a Norgestomet implant (Synchromate-B, Rhone Merieux, Athens, GA) into the ear on day 0. A single injection of prostaglandin (Lutalyse , Pharmacia and Upjohn, Kalamazoo, MI) was administered on day 7. Starting on day 12, follicle-stimulating hormone (FSH, Folltropin , Vetrepharm, Ontario, Canada) or (Ovagen , Immuno Chemicals Limited, Auckland, New Zealand) was administered by injection twice daily over four consecutive days. The ear implant was removed on day 14. Twenty-four hours following implant removal, the donor animals were mated several times daily to vasectomized bucks over two consecutive days. A single injection of gonadotropin-releasing hormone (Cystorelin , Rhone Merieux, Athens, GA) was administered following the last FSH injection coinciding with the first day of estrus/mating. NT After collection, oocytes were washed in TCM199 containing 10% fetal calf serum (FCS) (Gibco), and incubated in TCM-199 containing 10% FCS, 5 µg/ml cytochalasin-B and 5 µg/ml Hoechst 33342 stain (Sigma) for 10 min. Oocytes were enucleated in a 30-µl droplet of micromanipulation medium (TCM-199/10% FCS supplemented with 5 µg/ml cytochalasin-B) overlaid with mineral oil on the stage of an inverted microscope (Nikon Diaphot) equipped with Hoffman modulation contrast objectives, Narishige micromanipulators, and epifluorescent illumination. Oocytes were held with a 50 µm diameter pipette. After a brief exposure to ultraviolet light to visualize the metaphase plate, both the polar body and the metaphase plate were removed with a 20–25 µm
diameter beveled suction pipette. Following the enucleation, the oocytes were placed in Hoechst 33342 and exposed to epifluorescent illumination to verify by the absence of chromatin then the oocytes reconstructed immediately. A single fibroblast (15–20 µm diameter) was injected into the perivitelline space of each enucleated oocyte in a 30 µl droplet of micromanipulation medium using a 25–35 µm diameter pipette. Karyoplast-cytoplast couplets were manually aligned between two stainless steel electrodes (500 mm in gap) in a micro slide fusion chamber filled with fusion buffer (0.3 M mannitol, 0.1 mM MgSO4 , 0.05 mM CaCl2 , 0.5 mM Hepes, and 4 mg/ml bovine serum albumin, BSA) and fused by a single DC pulse (1.3 kV/cm for 20 ms) delivered by a BTX Electro cell Manipulator 2001 (Genetronics, San Diego, CA). Couplets were evaluated for fusion after 30–60 min of incubation in TCM-199/10% FCS. Fused couplets were washed extensively in TCM-199/10% FCS and then incubated for 3 h in 6-dimethylaminopurine (2 mM, from Sigma) prepared in TCM-199/10% FCS medium prior to transfer to intermediate recipient females or embryo culture medium. Embryo culture in vitro and in vivo The fused embryos were either co-cultured on monolayers of primary goat oviduct epithelial cells in 50 µl droplets of TCM-199 with 10% FBS overlaid with mineral oil at 38◦ C in a humidified 5% CO2 incubator or transferred to the oviducts of an intermediate recipient goat. Intermediate recipient goats were euthanized 5 days after embryo transfer and the reproductive tracts were collected immediately in warm saline solution. Embryos were recovered by flushing the oviducts and uterus with 15–20 ml of PBS supplemented with 10% FCS. Embryo transfer Morulae/blastocyst stage embryos were surgically transferred to synchronized recipients on day 5–7 of their estrus cycle (day 0 = estrus). Immediately prior to transfer to recipients, embryos were placed in equilibrated Ham’s F-12 medium (Gibco BRL) supplemented with 10% FBS. For morulae/blastocyst transfers, a perforation ipsilateral to a corpus luteum of the uterine horn of a recipient doe was made using an 18-gauge needle. Utilizing a 3.5 Fr. Tom Cat Catheter attached to a 1-cc syringe, 1–3 embryos were transferred into the uterine horn of each recipient at a
219 Table 1. Development of NT-produced morulae/blastocysts in goats Culture conditions In vivo (n = 8) In vitro (n = 7)
No. of NT couplet 102 142
No. of morulae blastocysts (%) 20(20)a 22(15)b
No. of embryos transferred 20 15
No. of recipients 9 7
No. of pregnancies 30–60 days
60 days-term
Live birth
6 0
4 0
2 0
Values with different superscripts are significantly different (P < 0.05).
location that was 2–4 cm from the utero-tubal junction. Pregnancy was determined by ultrasonography starting on day 30 after the first day of standing estrus. For the pregnancies that continued beyond 152 days, parturition was induced with 5 mg of PGF2α (Pharmacia and Upjohn, Kalamazoo, MI), and it occurred 22–36 h thereafter. Genotyping of NT animals Shortly after birth, blood samples were obtained from the cloned kids (D108 & D109) and the donor (C690) of the nuclei used for the NT. These bloods were used to establish lymphocyte suspension cultures. In addition, the mosaic NT donor’s (C690) skin cells were cultured in situ on Lab Tek Chamber slides. In order to allow for replication banding, 5-bromo2 deoxyuridine, BrdU (Sigma) was added to the cultures. Post culture processing and fixation were performed following standard cytogenetic techniques. In situ fibroblast and lymphocyte slides were pretreated and hybridized, as previously described (14), using a digoxigenin labeled DNA probe that was specific for a portion of the transgene. The bound probe was amplified using anti-digoxigenin peroxidase and detected with fluorescein tyramide deposition. Nuclei and metaphase chromosomes were counterstained with 4 , 6-diamidino-2-phenylindole, DAPI (Sigma). Images were captured using a Zeiss Axioskop microscope, a Hamamatsu digital camera, and Image Pro-Plus software.
Results Cell cycle analysis According to the FACS analyses, serum starvation of cells for 4 days followed by serum stimulation resulted in 94% of the cells in G0 /G, 1.5% of the cells in S phase, and 4% of the cells in G2 /M phases of the cell cycle. The proportion of serum starved and stimulated cells in G0 /G1 and in other phases of the cell cycle did not differ significantly with the length of serum stimulation (P < 0.05). Eighty-nine percent of sub-confluent, non-starved cells were in G0 /G, 4% in S phase and 6.5% in G2 /M phases which were not significantly different than that of serum starved cells (P < 0.05). Since the FACS cannot distinguish cells in G1 from cells in G0 , we next determined the relative levels of cyclin D1 mRNA, which is expressed in G1 but not in G0 . Northern blot analyses showed that cyclin D1 mRNA was detectable at trace levels in serum starved cells, and increased significantly at 5 and keep increasing by 10 h after the start of serum stimulation (Figure 1). The level of cyclin D1 mRNA was also high in the sub-confluent, non-starved cells (control), and not significantly different from that of 10 h serum stimulated cells. The small amounts of cyclin D1 mRNA in serum starved cells could be from a small
Statistical analysis Results of cell cycle analysis by FACS and northern blot, embryo development outcome, and birth and weaning weights of cloned goats were analyzed by student’s t-test. A value of P < 0.05 was considered to be significantly different.
Figure 2. The two cloned goats.
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Figure 3. FISH images showing the transgene integrations in the donor cell line and two cloned goats. Interphase nuclei were captured using a 40 × objective, metaphase spreads with a 100X objective. Nuclei and chromosomes were stained with DAPI, transgenes were detected with fluorescein. Row A: Considerable variation of the signal intensity was observed in both the cell line and blood for the skin cell donor, indicating that integrations of various copy numbers were present. The nuclei in kid D108 showed one of the smaller transgene signals while nuclei from kid D119 showed a higher copy number integration. Row B: Partial metaphases showed the higher copy number integration site that was present in donor skin, donor blood, and in kid D119. Row C: Partial metaphases showed the lower copy number integration site that was present in donor skin, donor blood, and in kid D108.
number of early S phase cells that had not yet degraded the message, and/or from a small number of cells in G1 . The relatively high level of expression of cyclin D1 mRNA in the sub-confluent, non-starved cells indicates that a large proportion of the G0 /G1 cells (∼89%) are in fact in G1 . The significant increase in levels of cyclin D1 mRNA in serum starved cells that were serum stimulated for 10 h indicates that most cells have already made the transition from G0 to G1 during this time (Figure 1). Embryo development Culture conditions had a significant effect on the number of NT couplets that developed to morulae/ blastocyst stages: 20/120 (20%) of in vivo cultured embryos versus 22/142 (15%) of in vitro cultured embryos, shown in Table 1. A total of 16 recipient does received 35 embryos (an average of 2.1 in vitro and 2.2
in vivo developed embryos per recipient). The early pregnancy rate (the first 30 days of gestation) following embryo transfer was significantly different for in vivo and in vitro derived embryos. No pregnancies resulted from embryos cultured in vitro, whereas in vivo cultured embryos produced six pregnancies (Table 1). Fetal development In this study only in vivo cultured NT embryos generated pregnancies. The two NT offspring born were healthy, with birth weights within normal range (3.8 and 4.1 kg) for their breed, Saanen (Figure 2). Birth weights of the two NT goats produced in this study were 3.8 and 4.1 kg. Kids were weaned from milk at the age of approximately 8 weeks with the weaning weights 14.5 and 18.1 kg. The average weaning weights were 14.88 ± 1.98 kg for goats obtained by
221 natural breeding, and 12.92 ± 2.96 kg for transfected fetal cell derived NT. In this study, four recipients lost pregnancies, two of the losses occurring after 30 days of gestation and the other two on days 152 and 153. The pathology report on the two fetuses with viable tissues showed diffuse atelectasis in the lungs in addition to the presence of amniotic fluid and epithelial cells in the alveoli. The laboratory results on both dead fetuses showed no bacterial or viral causes of fetal death. Genotype analysis Interphase and metaphase FISH analyses showed that the donor skin cell line was mosaic and had multiple integrations with different copy numbers. On the initial analysis, 94% of the skin cells were positive for at least one transgene integration. FISH was also performed on lymphocytes from the donor. The blood was also mosaic, had multiple integration sites, and displayed FISH signals of different sizes indicating that different copy number integrations were present. FISH was also performed on blood samples from each of the two live born NT kids, and results indicated that the two kids were not genotypically identical and therefore were the results of genotypically different cells in the skin cell line having been used for NT. Kid D108 appeared to have a lower copy number integration on chromosome 16 while kid D119 seemed to have a higher copy number integration on chromosome 13 (Figure 3). Both of these sites were observed in different metaphase spreads and interphase nuclei in the original donor skin cell line. Both NT kids were also shown to be transgenic by PCR and Southern blot analyses. Discussion In somatic cell NT, the appropriate cell cycle stage of the donor cells is thought to be an important factor in the development of NT embryos. Successful NT embryo development to term has been achieved by using donor cells that are either in G0 (quiescent, serum starved cells), or in G1 (confluent cells or cells obtained by shake-off of sub-confluent cells) (Wilmut et al., 1997; Cibelli et al., 1998; Kasinathan et al., 2001a, b) However, the relative advantages of using donor cells that are quiescent or in G1 phase have not been studied in detail. The two main reasons for using G1 cells as donors are: the presence of fewer apoptotic cells in the G1 cell population, and the pos-
sibility of better cell cycle synchrony between the G1 nucleus and the oocyte cytoplasm (Kurosaka et al., 2002). When confluent cells are serum starved, some cell death occurs (Lee & Piedrahite, 2002; Yu et al., 2003). To minimize the presence of dead cells, the cells used in our study were washed 3–4 times with medium after serum starvation, followed by another series of washes after 10 h of serum stimulation. FACS analyses showed that transgenic fibroblast cells were highly synchronized at G0 /G1 by 4 days of serum starvation, by which time more than 94% of the cells were in G0 /G1 phases. Cdk4 and cdk6 activities are first detected during mid-G1 phase and increase as cells approach the G1 /S boundary (Meyerson & Harlow, 1994; Sherr et al., 1995). D-type cyclins (cyclins D1−3 ) are important elements of cell cycle regulation, especially during mid-to-late G1 phase (Sherr et al., 1995). When the quiescent cells exit G0 and enter G1 , D-type cyclins are synthesized and assembled with cyclin dependent kinases (cdk) 4 and 6 (Matsushime et al., 1991, 1992). Therefore, induction of cyclin D1 mRNA transcription precedes its assembly with the cdks during midto-late-G1 phase. In order to obtain a cell population highly enriched in G1 , fibroblasts were serum starved for 4 days and then serum stimulated for 10 h. The fact that these cells were highly synchronized at G1 was demonstrated by northern blot detection of significantly high levels of cyclin D1 mRNA (expressed by cells that are already in G1 ) in cells cultured in the presence of 10% FBS, compared to the trace amount of cyclin D1 message in starved cells (Figure 1). The results presented here are consistent with previous studies in which it took 3T3 fibroblast cells, that had been serum starved and then stimulated with serum, at least 12 h to exit G0 and enter into S phase (Pardee, 1989). We also compared the development rates of goat NT embryos cultured in vivo (in goat oviducts) to those co-cultured with oviductal cells in vitro. The results of these experiments clearly indicate that in vivo culture better supports caprine NT embryo development to morulae and blastocyst stages, and subsequently to live birth (Table 1). However, the poor NT embryo development could be due to the co-culture conditions (oviductal cells used for co-culture). Goat NT embryo in vivo culture results were reported recently by (Xiangang et al., 2002) in which early goat NT embryos were cultured in oviducts of intermediate recipients and then transferred to final recipients after collection. NT embryos co-cultured in vitro with
222 oviductal cells produced significantly fewer morulae and blastocysts, with no pregnancies after transfer to recipients. The failure of successful goat embryo development in vitro in the present experiment is consistent with the results previously reported on in vitro culture of goat IVF embryos in Ham’s F-10 medium, Human Tubal fluid, and B2 medium (Blakewood et al., 1990). In contrast, (Prichard et al., 1992) reported 4070% goat blastocyst development of in vivo fertilized embryos co-cultured with oviductal cells in TCM-199. Since production of the first cloned sheep, Dolly (Wilmut et al., 1996), it is widely acknowledged that somatic cell NT results in increased abortion, high birth weight, hydrops, and postnatal loss in several species including cow, sheep, mouse and goat (Wells et al., 1997; Vignon et al., 1999). In this study, nonviable early pregnancies were found in 2/6 (33%) of recipients. This rate is similar to earlier reports from our laboratory and others with respect to early goat NT pregnancy loss of animals generated using transfected fetal donor cells (Keefer et al., 2002; Xiangang et al., 2002). Two of the remaining four NT goats were lost at term; neither of these evidenced high birth weight. The first doe was not exerting any effort to deliver the kid despite being 3 days past her due date (153 days). A full term, dead female kid weighing 4.2 kg was removed by elective caesarian section. The second term NT pregnancy that resulted in a dead kid had a similar presentation. The recipient was 2 days overdue and was injected with prostaglandin (lutalyse), which did not induce labor. An elective caesarian section was performed and a dead kid (1.2 kg) was delivered. Wells et al. (1997), suggest that in some NT pregnancies a breakdown occurs in communication between the fetus and the dam, resulting in a lack of appropriate signaling in preparation for birth. Such a communication breakdown could be responsible for the failure of two of the recipient does in our study to commence labor, despite being past their due dates. The final two NT goats produced in this study were born alive, had normal birth and weaning weights, and have both conceived and delivered healthy kids (data not shown). In summary, the results presented here indicate that readily available skin cells from an adult goat can be reprogrammed and successfully generate cloned offspring after NT. However, the significant loss of cloned animals observed in early and late-pregnancy needs further investigation.
Figure 4. Birth and weaning weights of NT and naturally bred goats. Birth weights. (a) Natural bred (n = 11, mean 3.48 ± 0.61); (b) Transfected fetal NT (n = 11, mean 3.25 ± 0.66); (c) Skin cell NT (n = 2, 3.8, 4.1). Weaning weights. (A) Natural bred (n = 11 mean 14.88 ± 1.98); (B) Fetal NT (n = 11, Mean 12.92 ± 2.96); (C) Skin cell NT (n = 2, 14.5, 18.1).
Our results show that NT loss can occur at the prenatal stage in goats as it does in cattle and sheep, and is likely a consequence of the cloning process. The weights of the two live cloned goats were within normal range both at birth and after weaning as compared to goats obtained by natural breeding (Figure 4), in contrast to previously reported large offspring syndrome in cloned cows and sheep. Another area for future study lies in the development of an efficient method for in vitro goat embryo culture, which would greatly increase the success rate of NT programs and subsequently benefit both agriculture and biomedicine. Our results show the utility of NT using transgenic somatic cells as nucleus donors to rapidly expand a pool of transgenic animals, as well as to reproduce individual animals with superior genetics. However, in order to better use this technology for commercial production, research is needed to study the reprogramming of differentiated cells for improvement of maintenance of pregnancy and production of healthy cloned animals.
Acknowledgements The authors gratefully acknowledge the help of Michael Schofield, DVM and veterinary staff members. We thank Catherine Jameson for help in collecting data. Finally we gratefully acknowledge the support and assistance of Carol A. Ziomek, PhD throughout this project.
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