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Generation of Dwarf Goat (Capra hircus) Clones Following Nuclear Transfer with Transfected and Nontransfected Fetal Fibroblasts and In Vitro-Matured Oocytes¹

^a Nexia Biotechnologies Inc., ^b Ste Anne de Bellevue, Quebec, Canada H9X 3R2 Department of Animal Science, McGill University, Ste Anne de Bellevue, Quebec, Canada H9X 3V9

ABSTRACT

The developmental potential of caprine fetal fibroblast nuclei after in vitro transfection and nuclear transfer (NT) into enucleated, in vitro-matured oocytes was evaluated. Fetal fibroblasts were isolated from Day 27 to Day 30 fetuses from a dwarf breed of goat (BELE: breed early lactate early). Cells were transfected with constructs containing the enhanced green fluorescent protein (eGFP) and neomycin resistance genes and were selected with G418. Three eGFP lines and one nontransfected line were used as donor cells in NT. Donor cells were cultured in Dulbecco minimum Eagle medium plus 0.5% fetal calf serum for 4–8 days prior to use in NT. Immature oocytes were recovered by laparoscopic ovum pick-up and matured for 24 h prior to enucleation and NT. Reconstructed embryos were transferred as cleaved embryos into synchronized recipients. A total of 27 embryos derived from transgenic cells and 70 embryos derived from nontransgenic cells were transferred into 13 recipients. Five recipients (38%) were confirmed pregnant at Day 35 by ultrasound. Of these, four recipients delivered five male kids (7.1% of embryos transferred) derived from the nontransfected line. One recipient delivered a female kid derived from an eGFP line (7.7% of embryos transferred for that cell line). Presence of the eGFP transgene was confirmed by polymerase chain reaction, Southern blotting, and fluorescent in situ hybridization analyses. Nuclear transfer derivation from the donor cells was confirmed by single-strand confirmation polymorphism analysis. These results demonstrate that both in vitro-transfected and nontransfected caprine fetal fibroblasts can direct full-term development following NT.

assisted reproductive technology, developmental biology, embryo, gamete biology, gene regulation, ovum

INTRODUCTION

Live offspring have been obtained following nuclear transfer (NT) of embryonic, fetal, and adult cells in cattle, sheep, goats, pigs, and mice [1–11]. This cloning process involves transfer of a donor cell nucleus into an enucleated oocyte. The source and treatment of the donor cell and recipient oocyte are key factors in the successful outcome of this process. In vivo-matured oocytes were used initially as the recipients for blastomeres for embryo cloning in cattle [1, 12]. However, the high cost of in vivo-sourced bovine oocytes propelled the switch to in vitro-matured oocytes [13, 14]. Cloning in cattle now involves a totally in vitro approach: Recipient oocytes are derived from in vitro-matured oocytes obtained from slaughterhouse ovaries and resulting NT embryos are cultured to the blastocyst stage in vitro prior to transfer [7, 15]. In other species, including sheep, goat, and mice, in vivo-matured oocytes are used predominately [8, 9, 16]. Although the use of in vitro-matured ovine oocytes in NT was reported in one study, a lower production of lambs from NT embryos derived from in vitro-matured oocytes was achieved than from in vivo-matured oocytes (2.3% vs. 6.5% of embryos transferred;[17]). Despite these results, the use of in vitro-matured oocytes in small ruminants could provide similar advantages as those seen in the bovine system. While the quality and quantity of oocytes obtained from slaughterhouse-derived ovaries of small ruminants can be affected by season, immature oocytes can be readily obtained year round from hormone-primed animals by either laparoscopy or laparotomy [18]. As we show in this study, the laparoscopic approach provides a minimally invasive, efficient means of obtaining immature oocytes for subsequent use in NT.

Freshly isolated cells [8], short-term culture of fetal and adult cells [5, 15, 16], and established embryonic stem cell lines [10] have all been successfully used in NT. In a few cases, transgenic cells have been used as donor cells in order to produce transgenic offspring. This includes propagation of transgenic animals (goat [9]) and the in vitro transfection and selection of transgenic donor cells (sheep [19] and cattle [7]). Furthermore, there has been a recent report of transgenic lambs resulting from the application of homologous recombination techniques to the donor cells [20]. In transgenic animal production programs, the ability to transfect and select cells prior to animal production results in transgenic animals of the desired gender and overcomes the problem of founder animals being mosaic. Therefore, in contrast to production of transgenic animals by pronuclear injection that results in less than 10% of offspring carrying the transgene, production of a transgenic animal can be ensured by confirming integration site(s) prior to use in NT.

In this study, we demonstrate that NT embryos derived from goat fetal cells transfected in vitro can support development to term following NT using in vitro-matured oocytes. Furthermore, we demonstrate that the BELE (breed early lactate early) system of transferring dwarf goat embryos into standard-sized goats (e.g., Alpine, Saanen, and Nubian) is suitable for the production of NT-derived, transgenic offspring. In earlier studies, the advantages of this system were demonstrated by the production of transgenic dwarf offspring following transfer of pronuclear-injected, dwarf zygotes into standard-sized dairy goats [21]. These dwarf goats provide advantages in their small size (reduced housing and feed) and early sexual maturity (reduced time lines) [22]. Furthermore, the ability to utilize both standard dairy breeds and dwarf breeds as recipients simplifies recipient herd management.

MATERIALS AND METHODS

Isolation of Donor Cell Lines

Fetal cells were isolated from Day 27 to Day 30 fetuses recovered surgically from a dwarf breed of goat (BELE). After removal of the head and internal organs, the remaining tissues were mechanically dissociated. Explants were cultured in Dulbecco modified Eagle medium (DMEM; Gibco, Canadian Life Technologies, Burlington, ON, Canada) supplemented with 20% fetal bovine serum (FBS) and 20 µg/ml gentamycin at 38°C in 5% CO₂. While the explant cultures contained a mixed population of cells, fetal fibroblasts were predominant. When the cells from the explants reached 70% confluency, they were removed with 0.05% trypsin-EDTA treatment, counted, and frozen into aliquots in 10% DMSO + 90% FBS, or plated in chamber slides (Labtek, Canadian Life Technologies, Burlington, ON, Canada) for cytogenetic analysis. Frozen aliquots of cells with a normal chromosome number were thawed and either prepared for use in NT or were transfected with the DNA construct to generate stable lines.

Generation of Transfected Cells

Stable cell lines were generated through lipid-mediated gene transfer. The CEeGFP plasmid (kindly provided by Dr. T. Takada, National Childrens' Medical Research Center, Tokyo, Japan) contained the enhanced, humanized version of the green fluorescent protein reporter gene driven by the human elongation factor-1 promoter and cytomegalovirus enhancer, and the neomycin selection marker under the control of the simian virus-40 promoter [23, 24]. This plasmid was delivered into the cells using lipofectamine (Gibco) according to the manufacturer's instructions. A number of stable clones were generated by selection under G418 for 20 days and assessed for expression of the reporter gene by visualization of the fluorescent signal under blue light (Zeiss Filter Set 09; Carl Zeiss Canada Ltd., North York, ON, Canada). Stable lines were either used as donor cells in NT or were frozen into small aliquots for later use. Four lines were subsequently used in NT: a nontransfected male line (FF4), a transfected male line (FF4-eGFP), and two transfected female lines (FF1-eGFP and FF3-eGFP; Fig. 1A).

View larger version (72K): [in this window] [in a new window]   FIG. 1. A) The in vitro-transfected fetal fibroblast line FF3-eGFP demonstrating GFP expression under blue light. B) Expression of GFP in donor FF3-eGFP cells at time of NT. C) Expression of GFP after 5-azacytidine treatment of cells obtained from the transgenic female. D) Fluorescence in situ hybridization analysis using a biotin-labeled probe containing the CEeGFP plasmid demonstrating integration of the transgene in cells obtained from the transgenic FF3-eGFP female clone. Original magnification x25 (A), x200 (B and C), x1000 (D)

Donor Cell Preparation

Fetal fibroblast cells (0.5 to 1 x 10⁵) were plated into 24- or 96-well plates and cultured in DMEM + 10% FBS until they reached 100% confluency. The medium was then replaced with low serum media (DMEM + 0.5% FBS + 20 µg/ml gentamycin), and the cells were incubated at 38°C, 5% CO₂ for 4–8 days until day of NT [25]. Just prior to cell transfer, the donor cells were collected by trypsinization using 0.05% trypsin-EDTA, washed twice, and resuspended in EmCare containing 1% BSA.

Oocyte Maturation and Preparation

Ten to fifteen cumulus-oocyte complexes (COCs) were cultured per 50-µl drops of maturation medium covered with an overlay of mineral oil (Sigma, St. Louis, MO) and incubated at 38.5–39°C in 5% CO₂. The maturation medium consisted of M199 supplemented with bovine LH (0.02 U; Sioux Biochemicals, Sioux Center, IA), bovine FSH (0.02 U; Sioux Biochemicals), estradiol-17ß (1 µg/ml; Sigma), 0.2 mM sodium pyruvate, kanamycin (50 µg/ml), and 10% heat-inactivated goat serum (Sigma), or FBS (Immunocorp, PQ, Canada). The FBS was used as the serum source in five sessions, whereas, goat serum was used in seven sessions. After 23–24 h of maturation, the cumulus cells were removed from the matured oocytes by vortexing the COCs for 1–2 min in EmCare (Immuno-Chemical Products, Auckland, NZ) containing 1 mg/ml hyaluronidase (Sigma). The denuded oocytes were washed in handling medium (EmCare supplemented with 1% FBS) and were returned to maturation medium. The enucleation process was initiated within 1 h of oocyte denuding. Prior to enucleation, the oocytes were incubated for 20–30 min in 5 µg Hoeschst 33342 (Sigma) per ml handling medium at 30–36°C in air atmosphere.

Enucleation

Oocytes stained with Hoechst were enucleated following brief exposure of the cytoplasm to UV light (Zeiss Filter Set 01) to determine the location of the chromosomes. Oocytes were manipulated at room temperature (24–26°C) in handling medium (EmCare supplemented with 1% FBS) without the addition of cytochalasin B. Stage of nuclear maturation was observed and recorded during the enucleation process. The removed cytoplasm was checked for the presence of chromosomes and polar body by exposure to UV light.

Donor Cell Transfer and Fusion

The enucleated oocytes and dispersed donor cells were manipulated in handling medium. Small (<20 µm) donor cells with smooth plasma membranes were picked up with a manipulation pipette and slipped into the perivitelline space of the enucleated oocytes. Cell-cytoplast couplets were fused immediately after cell transfer. Groups of four to six couplets were manually aligned between the electrodes of a 500-µm gap fusion chamber (BTX, San Diego, CA) overlaid with sorbitol fusion medium (0.25 M sorbitol, 100 µM calcium acetate, 0.5 mM magnesium acetate, 0.1% BSA). A brief fusion pulse (15 µsec) at 2.39 kV/cm was administered by a BTX Electrocell Manipulator 200. After the couplets had been exposed to the fusion pulse they were placed into 25-µl drops of culture medium overlaid with mineral oil (Sigma). Culture medium consisted of either a low phosphate (0.35 mM) modification of SOFaa [26] or G1.2 (Colorado Center for Reproductive Medicine, Englewood, CO [27]) supplemented with 8 mg/ml BSA. Fused couplets were incubated at 38.5–39°C in 5% CO₂, 7% O₂, 88% N₂. After 1 h, couplets were observed on a stereomicroscope for fusion. Unfused couplets were administered a second fusion pulse as described above.

Activation and Culture

Two to three hours after application of the fusion pulse, the couplets were activated using the calcium ionomycin and 6-dimethylaminopurine (6-DMAP; Sigma) method of Susko-Parrish et al. [28]. Activated couplets were cultured for 2.5–4 h in DMAP, then washed in handling medium, and placed into 25-µl culture drops consisting of low phosphate SOFaa or G1.2 under an oil overlay. While group size ranged from 4 to 27 embryos per drop, the average number was 10. Cleavage development (2- to 4-cell stages) was observed at 36 h. Embryos were transferred on Day 2 (Day 0 = day of fusion) into synchronized recipients on Day 1 of their cycle (D0 = estrus). Visual observations of the reconstructed embryos for GFP expression were not made prior to embryo transfer to reduce further manipulations of the embryos.

Donor and Recipient Animals

Oocyte recoveries and embryo transfers were performed during the months of October, November, and December 1998. The donor herd consisted of 32 does (Alpine, Saanen, Nigerian Dwarf, and Pygmy). Twenty of the donors underwent two or three laparoscopic ovum pick-up (LOPU) sessions. Intravaginal sponges containing 60 mg of medroxyprogesterone acetate (Veramix; Upjohn Co., Orangeville, ON, Canada) were inserted into the vagina of donor goats and left in place for 7–10 days with an injection of 125 µg cloprostenol (Estrumate; Schering Canada Inc., Pointe-Claire, PQ, Canada) given 36–48 h before sponge removal. Priming of the ovaries was achieved by the use of gonadotropin preparations including FSH and eCG. The following hormonal regimes were used: a total dose equivalent to 120 mg NIH-FSH-P1 of Ovagen (Immuno-Chemical Products, Auckland, New Zealand) given twice daily in decreasing doses (35, 35, 25, and 25 mg) starting 48 h before sponge removal, or a total dose equivalent to 70 mg NIH-FSH-P1 (Follitropin-V; Vetrepharm, London, ON, Canada) given together with 400 IU of eCG (Equinex; Ayerst Laboratories, Canada) 36–48 h before oocyte recovery. Both regimes resulted in similar numbers of follicles and COCs recovered (data not shown).

Recipients (Alpine, Saanen, Nubian, LaMancha, and Toggenburg) were synchronized using intravaginal sponges containing 60 mg of medroxyprogesterone acetate. An injection of 125 µg cloprostenol was given 36–48 h before sponge removal. An injection of 400 IU of eCG was given at sponge removal on the same day as LOPU.

Laparoscopic Oocyte Pick-Up and Embryo Transfer

Donor goats were fasted 24 h prior to laparoscopy. Anesthesia was induced with i.v. administration of diazepam (0.35 mg/kg body weight) and ketamine (5 mg/kg body weight) and was maintained with isofluorane via endotrachial intubation. Cumulus-oocyte complexes were recovered by aspiration of follicular contents under laparoscopic observation [18].

Recipient goats were fasted and anesthetized in the same manner as the donors. A laparoscopic exploration was performed to confirm if the recipient had one or more recent ovulations or corpora lutea (CL) present on the ovaries. Four to 11 cleavage-staged embryos (2-cell to 8-cell stages) were transferred into the oviduct ipsilateral to ovulation(s) by means of a TomCat catheter threaded into the oviduct by way of the fimbria. Donors and recipients were monitored following surgical procedures. Antibiotics and analgesics were administered according to approved procedures.

Determination of Fetal Survival and Kidding

All recipient goats were examined by ultrasonography on Day 35 and 60 of gestation to record fetal development. Parturition was induced with three injections of 8 mg dexamethasone (Azium) given at 12-h intervals with 125 µg of clorprostenol (Estrumate) given with the third injection. The first injection was administered on Day 147 or 148 of gestation. Cotyledon number and birth weights were recorded.

Cytogenetic Analysis (Chromosome Counts, Sexing, and Fluorescence In Situ Hybridization)

Normal chromosome number (2n = 60) was determined prior to use of cells in NT. Slides for cytogenetic analysis were prepared by standard techniques [29, 30]. Briefly, fetal fibroblasts were cultured with colcemid in order to increase the number of cells in mitotic metaphase. Spreads were stained with Giemsa and assessed for chromosome number and sex.

Chromosomal integration of the transgene into transfected stable lines was demonstrated by fluorescent in situ hybridization (FISH) techniques. The transgene hybridization signal was visualized using a biotin-labeled probe containing the entire CEeGFP construct. The chromosome spreads were stained with propidium iodide [29, 31, 32].

Genotyping and Polymerase Chain Reaction Analysis of Cloned Animals

Genomic DNA was isolated (QIAmp DNA Blood Mini Kit; Qiagen Inc., Mississauga, ON, Canada) from blood samples collected from the cloned animals, the surrogate dams, the biological dams and sire, and the donor cells. Each nontransgenic animal was analyzed by polymerase chain reaction (PCR)-single strand confirmation polymorphism (SSCP) to confirm that the clones were derived from the NT procedure. Briefly, a 286-base pair (bp) fragment of the goat major histocompatibility complex (MHC) class II DRB gene [33] was amplified using two primers (ACB0445, 5'-TATCCCGTCTCTGCAGCACATTTC-3'; ACB0446, 5'-ATCGCCGCTGCACACTGAAACTC-3'). The identity of the PCR product was confirmed by DNA sequencing analysis (Mobix, Hamilton, Canada). The PCR product was analyzed by SSCP [34, 35] in order to identify the relationship between animals.

Genomic DNA from the goat derived from a transgenic cell line was analyzed by PCR for the presence of the transgene using two different primer sets. The first set (ACB411, 5'-AGACTGAAGTTAGGCCAGCTTGG-3' and ACB412, 5'-GTCTTGTAGTTGCCCGTCGTCCTT-3') amplified a 490-bp fragment that spanned the elongation factor-1 promoter and the GFP reporter gene. A second set of primers amplified a 360-bp portion of the endogenous ß-casein that provided a positive control to ensure that the extracted DNA was amplifiable by PCR.

Induction of GFP Expression

A skin biopsy was obtained from the lower edge of the ear using a 6-mm biopsy punch. The cells were dispersed by cutting the sample into small fragments and incubating them in digestion medium (Gibco) at 37°C in a 5% CO₂ incubator. After 16 h, the tissue digest was pipetted up and down, and the dissociated cells were centrifuged, washed, resuspended in DMEM supplemented with 10% FBS and 0.02 mg/ml gentamycin (Gibco), and plated in a culture vessel. Once the cells reached 70–80% confluency, they were trypsinized, counted, centrifuged, resuspended in cryomedia (90% dimethylsulfoxide, 10% FBS) at a concentration of 1 x 10⁶ cells per ml, and frozen in aliquots. An aliquot was thawed and the cells incubated at 37°C in 5% CO₂. Upon reaching 70–80% confluency, the cells were trypsinized, counted, and plated for drug treatment in 12-well plates at a concentration of 1 x 10⁴ cells per well. These cells were treated with either 5-azacytidine, a cytidine analog that inhibits methylation when incorporated into DNA [36], or sodium butyrate that nonspecifically inhibits histone deacetylase enzyme [36, 37]. Twenty-four hours after plating, 5-azacytidine (Sigma) was added to the cells at either 0, 3, 6, or 10 µM concentrations. After an additional 24 h, the drug was removed from the cells, and they were observed daily under blue light for the presence of GFP fluorescence. Similarly, the cells were plated in 12-well plates at a concentration of 1 x 10⁴ cells per well, and 24 h later they were treated with either 0, 50, or 100 mM sodium butyrate (Sigma). The drug was removed 24 h later, and the cells were observed daily under blue light for the presence of GFP fluorescence.

Animal Ethics

This project was approved by the McGill Animal Ethics and Animal Care Committees.

Statistical Analysis

The proportional data for oocyte nuclear maturation, cell fusion, in vitro development, pregnancy rates, and NT efficiencies (offspring produced per embryo transferred) were analyzed by contingency tables using InStat (GraphPad, San Diego, CA). Birth weight and cotyledon number were analyzed using the Student's t-test (GraphPad).

RESULTS

In 11 sessions, 781 oocytes were recovered from 57 donor aspirations (13.7 per donor). Following maturation, 734 in vitro-matured oocytes were denuded of cumulus cells and assessed for nuclear maturation. Significantly fewer oocytes matured to metaphase II in maturation medium supplemented with FBS, than for oocytes matured with goat serum, 69% (235/338) versus 86% (341/396), respectively (P < 0.01). A total of 366 karyoplast-cytoplast couplets were produced using donor cells from one nontransfected and three eGFP-transfected fetal fibroblast cell lines (Table 1). At the time of cell transfer the donor cells from all three lines were mosaic for expression of the transgene as determined by visual observation (Fig. 1B).

Due to the small size of the donor cell, fusion was difficult to assess; however, 54% of the karyoplast-cytoplast couplets appeared to have fused. As culture group size was known to affect developmental rates [26], fused and nonfused couplets were not separated at assessment and were cultured together in order to prevent the generation of small subsets of embryos. Forty-three percent of the couplets underwent initial cleavage (2-cell to 8-cell stages). No differences were observed in fusion or initial cleavage rates when NT embryos were cultured in SOF or G1.2 (P > 0.05; data not shown). A total of 97 reconstructed embryos were transferred into 13 recipients (Table 1) with each recipient receiving on average 7.5 ± 0.6 embryos. Number of embryos transferred per recipient did not significantly affect the pregnancy rate.

Five of the recipients (38%) receiving cleavage-staged embryos were confirmed pregnant by ultrasound. These pregnancies resulted from transfers of reconstructed embryos from two of the four cell lines. In both cases the pregnancy rate was 50%, four of eight recipients receiving embryos derived from the nontransfected FF4 male line and one of two recipients receiving embryos derived from the FF3-eGFP positive female line (Table 1). Transfers involving the other two cell lines did not result in pregnancies. The FF4 pregnancies resulted from transfer of cleavage stage NT embryos derived from donors cells at passage 2 that had been maintained in low serum for 4 days. The FF3-eGFP pregnancy resulted from transfer of cleavage-stage NT embryos derived from donor cells at passage 5 that had been maintained in low serum for 6 days. The source of the oocyte, Dwarf/Pygmy or Saanen/Alpine, affected neither the initial pregnancy rate (37.5% vs. 25%, respectively) nor the NT efficiency (4.6% vs. 4.5%, respectively, P > 0.05).

One kid was delivered by cesarean section on Day 150; the remaining kids were born on Days 149 and 150 by vaginal delivery approximately 36–48 h following initiation of induction (Table 2). Four of the recipients delivered five male kids derived from the nontransfected FF4 line. Average birth weight (±SEM) was 2.36 ± 0.27 kg (range 1.5–3.1 kg). Four placentas were recovered and had an average of 57 cotyledons (range 32–83). One kid subsequently died within 24 h of birth, one died at 1 mo of age, and one died at 3 mo of age, all three succumbed to bacterial infections affecting the lungs. The remaining two males are healthy (Fig. 2B). One recipient delivered a healthy female kid (2.1 kg birth weight) derived from the eGFP-positive line (Fig. 2A). The placenta was recovered and had 48 cotyledons. The birth weights and cotyledonary numbers were not significantly different from those of dwarf goat kids produced by natural breeding at Nexia Biotechnologies Caprine Production Facility that averaged 2.35 ± 0.15 kg at birth (n = 11) with recovered placentas having an average cotyledon number of 60 (n = 5; N. Kafidi, personal communication).

View larger version (110K): [in this window] [in a new window]   FIG. 2. A) Female kid produced by NT using in vitro-transfected FF3-eGFP fetal fibroblasts. B) Male FF4 kids produced by NT using fetal fibroblasts. C) A PCR-SSCP analysis of the second exon of the caprine MHC class II DRB gene: lanes 1–4, male FF4 kids; lane 5, FF3-CEeGFP kid; lane 6, FF3 cell line; lane 7, FF4 cell line; lane 8, biological dam of FF3 and FF4 fetuses; lanes 9, 10, and 11, surrogate dams

Chromosome Number, SSCP, FISH, PCR, and GFP Expression Analyses

All four cell lines used in NT had a normal chromosome count consisting of 60 chromosomes (60XX or 60XY). The PCR-SSCP analysis for the polymorphic MHC class II DRB gene confirmed that all the cloned kids were derived from their donor cell lines, whereas the surrogate dams carried different alleles. The cloned goats differ from the surrogate dams at the analyzed locus but have the same allelic information as the donor lines and biological dam (Fig. 2C).

Presence of the CEeGFP transgene in the genomic DNA of the transgenic female was confirmed by PCR, Southern blotting (data not shown), and FISH (Fig. 1D). The Southern blotting and FISH analyses both indicated that there were three integration sites. Based on banding patterns, the transgene integration sites were located on 10q34, 23q12, and 25q15. However, neither epithelial cells taken from a mouth swab nor skin fibroblasts expressed the GFP protein as determined by observation under blue light. Western blot analysis of cytoplasmic extracts of cultured fibroblasts from skin biopsies also indicated that the protein was not expressed (data not shown). Expression of GFP was induced in a subpopulation of the skin fibroblast cells (<1%) after a 24-h drug treatment with either 3, 6, or 10 µM 5-azacytidine (Fig. 1C). This expression persisted for over a week and then declined slowly if drug treatment was not repeated. Cells that were treated repeatedly retained GFP expression in a subpopulation of cells. Expression of GFP was not observed in any of the cells that were treated with sodium butyrate or in the controls (data not shown).

DISCUSSION

In this study, in vitro-matured caprine oocytes supported full term development when used as recipient cytoplasts in NT. Both in vitro-transfected and nontransfected fetal fibroblasts were used successfully as donor cells. Polymerase chain reaction and FISH analyses confirmed the presence of the transgene in the FF3-eGFP-derived female; however, expression was not detected either visually or by Western analysis in skin fibroblastic cells. One possible explanation for lack of expression could be that the transgene had been silenced by epigenetic regulatory mechansims (e.g., methylation, histone deacetylation) [36]. Treatment of cells with 5-azacytidine, a cytidine analogue that inhibits methylation when incorporated into DNA, resulted in expression of the transgene in a cell subpopulation. Although position-dependent expression cannot be ruled out, this expression indicates the presence of a functional transgene that may have been silenced by epigenetic mechanisms. Furthermore, at the time of NT the donor cells demonstrated mosaic expression of the transgene as determined by visual observation (Fig. 1B). The mosaic expression observed at the time of NT may indicate that the process of transgene methylation had commenced during in vitro culture. At this time it is not certain if the epigenetic mechanisms affected the promoter or the GFP transgene or both. Incorporation of insulator elements, locus control regions, matrix-attachment regions into the construct design, or the use of artificial chromosomes or gene targeting could alleviate problems of nonexpression due to transcriptional regulation [36, 38]. While the GFP protein was not expressed in the animal itself, these results demonstrated the production of a transgenic goat through in vitro transfection and NT confirming similar work in cattle and sheep [7, 19].

Fetal fibroblasts were chosen as the source of donor cells owing to their potential for longer term culture required in the transfection and selection processes. Optimization of culture, transfection conditions, and selection treatments may permit utilization of other sources, adult or fetal, as the targeted donor cell. Recent reports indicate that some adult cell lines can be maintained sufficiently long enough for homologous recombination events to take place [20, 39, 40]. In this study, serum starvation was utilized in order to synchronize the donor cells in G₀/G₁ arrest [25]. While there are conflicting reports on the necessity of serum starvation, G₀/G₁-arrested cells may be more receptive to reprogramming than other stages of the cell cycle [7, 25, 41, 42]. The number of recipients per cell line was too small in this study to determine any real differences in NT efficiencies between the cell lines; however, the longer period of culture necessitated for in vitro transfection and/or the in vitro transfection treatments themselves may have affected the viability of the transfected lines. The ability to correlate success (live offspring) with type, stage, or donor cell treatment has been limited by the low efficiencies currently achieved in NT [43]. However, with the current level of interest and the application of NT techniques to divergent species, some of the intriguing questions regarding cell cycle and nuclear reprogramming should be resolved.

A 50% pregnancy rate was achieved following embryo transfer of NT embryos derived from both the transgenic FF3-eGFP line and the FF4 nontransgenic line. This is similar to the pregnancy rates reported for standard-sized recipients receiving DNA-injected dwarf goat zygotes [21]. Unlike reports in sheep and cattle [17, 19, 42], we did not observe any prenatal loss. This is similar to the report by Baguisi and coworkers [9] for somatic cell NT in goats, in which no losses were observed after confirmation of fetal heartbeats at Day 40. However, at earlier observations (Day 30) a high proportion of recipient does (55–78%) exhibited vesicle formation that were subsequently resorbed. In their work, NT-derived kids were produced using in vivo-matured oocytes and transgenic fetal fibroblasts obtained from a transgenic donor animal. In both their report and in our study, NT-derived embryos were transferred at early cleavage stages. In sheep and cattle, an increased incidence of large offspring following transfer of embryos cultured in the presence of serum has been observed [44, 45], demonstrating the possible effects of in vitro culture on fetal development. It may be that avoidance of longer term culture may have alleviated some of the detrimental effects of in vitro culture. Contrarily, this lack of prenatal loss may be a species-related phenomenon reflected in the cytoplast's ability to reprogram the donor nucleus. Compatibility between nucleus and cytoplasm may be limited to closely related breeds such as those used in this study and those involving Enderly Island [6], Zebu [46], and Holstein breeds, while more diverse relationships may result in early embryonic loss [47, 48]. In this study, both dairy and dwarf goat oocytes were used as recipient cytoplasts with similar efficiencies. In mice, gene expression and adult body weight were affected by epigenetic events resulting from pronuclear exchange between two different strains [49]. Furthermore, strain has been shown to have significant effects on efficiencies following NT [50]. As compatibility between the recipient ooplasm and the donor nucleus must be reflected in some part in the overall effectiveness of nuclear reprogramming, nucleocytoplasmic interactions may be responsible for some of the detrimental effects observed following NT.

All pregnancies were full term with recipients showing mammary gland development prior to initiation of induction. The first cloned kid was delivered by c-section due to concern for the well-being of the kid and not to any difficulties associated with the dam itself. Birth weights and number of cotyledons on the recovered placentas were within normal range for dwarf goats (Table 2). While the shape and distribution of the cotyledons appeared slightly irregular, further studies are needed to confirm these observations. Despite the apparent normalcy of the pregnancies and births, a high postnatal loss (50%) was observed. While three of the six cloned kids were lost, only one of these occurred within a day of birth. The other two occurred at 1 and 3 mo of age. These postnatal losses are similar to those reported in sheep [16, 19] and cattle [51, 52]. Renard and coworkers [52] reported the sudden loss of a cloned calf after 6 wk of apparent normal development. While these losses may be overcome by application of intensive perinatal monitoring and kid management [11], continued long-term monitoring of resulting clones is needed.

Nuclear transfer may be successful in some cases due to the convergence of several factors including oocyte viability, nucleocytoplasmic compatibility, and cell cycle synchronization. In other cases, reprogramming may be incomplete, resulting in embryonic loss, abortion, or abnormal development. It is remarkable that efficiencies of animal production based on number of NT embryos produced and transferred are similar in many reports. Average efficiencies in cattle, sheep, and mouse have ranged between 1% and 10% of transferred NT embryos [5, 8, 10, 15, 43]. While a few reports present higher efficiencies, these generally represent small sets of data [51]. Whether the low efficiencies observed for NT-derived embryos is due to incomplete reprogramming and/or to procedural elements remains to be resolved. Nevertheless, NT is a viable technique for the production of transgenic animals. Further studies are needed in order to gain a better understanding of the intricacies involved in the processes of NT, reprogramming, and epigenetic regulatory mechanisms.

ACKNOWLEDGMENTS

We gratefully acknowledge Dr. T. Tanaka for the gift of the GFP vector. We also gratefully acknowledge the technical and animal health staff on the Macdonald farm campus and from Nexia Biotechnologies, Inc. for their assistance. We are especially grateful to Nathalie Chrétien, Denyse Laurin, Mélanie Gauthier, and Dr. N. Kafidi for their assistance.

FOOTNOTES

First decision: 15 September 2000.

¹ Preliminary reports of this work were presented at the 32nd Annual Meeting of the Society for the Study of Reproduction, July 1999, Pullman, WA, and the Transgenic Animal Research Conference, August 1999, Tahoe City, CA.

² Correspondence: Carol L. Keefer, Nexia Biotechnologies Inc., 21025 Trans Canada Highway, Ste-Anne-de-Bellevue, PQ, Canada H9X 3R2. FAX: 514 457 6151; ckeefer{at}nexiabiotech.com

Accepted: October 11, 2000.

Received: August 16, 2000.

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