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Reproductive Technology |
Centre de recherche en reproduction animal,3
Département de sciences cliniques,4 Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, Quebec, Canada J2S 7C6
Nexia Biotechnologies, Inc.,5 Vaudreuil-Dorion, Quebec, Canada J7V 8P5
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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assisted reproductive technology, embryo, gene regulation, in vitro fertilization, sperm
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Successful cloning by nuclear transfer using somatic cells depends on the appropriate combination of cell-cycle stages between the donor and host cell. It has been proposed that better results are obtained with donor cells arrested at the G0/G1 phase following serum starvation and host cytoplasts arrested at metaphase II [4, 17, 18]. However, our preliminary data have shown that transgene transfection and selection procedures require prolonged culture periods, leading to slower and fewer cycling cells, which interferes with proper G0/G1 synchronization of donor cells for nuclear transfer. Thus, cycling cells are often erroneously chosen and fused to metaphase-arrested enucleated oocytes, leading to chromatin condensation, pulverization, and abnormal spindle assembly [19, 20], with dramatic effects on embryonic ploidy and developmental outcome [21]. On the other hand, preactivated cytoplasts have been successfully used in nuclear transfer experiments with interphase blastomere nuclei [22, 23], suggesting that activated host oocytes could provide an alternative for somatic cell cloning when donor nuclei synchronization cannot be efficiently attained.
In addition to improved nuclear transfer efficiencies, more accurate methods for screening both genetically modified nuclear donor cells and reconstructed embryos before transfer to surrogate females are needed. In mice, green fluorescent protein (GFP) reporter vectors have been effectively used to select transgenic embryos produced by pronuclear injection [24]. Furthermore, GFP selection of donor cells has been used to produce transgenic offspring by nuclear transfer in mice [25], pigs [15], and goats [12]. Finally, cattle GFP-expressing blastocysts have been produced after nuclear transfer from transfected somatic cells [26, 27], but to our knowledge, no GFP expression of offspring or germ line transmission has yet been reported. In the present study, our prime objective was to determine the developmental potential of transgenic embryos produced by nuclear transfer using nonsynchronous donor cells with oocytes at the telophase stage. Secondarily, we assessed the benefits of using a selectable marker, the GFP-expression transgene, to screen transgenic cloned embryos before embryo transfer and the resulting offspring for somatic and germ line transmission.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fibroblast cell cultures were established from a slaughterhouse-derived male bovine fetus at approximately 50 days of gestation. Fetal tissues were minced and digested with 0.25% trypsin and 0.02% EDTA (Gibco BRL, Burlington, ON, Canada) at 37°C for 10 min. Isolated cells were washed with Dulbecco modified eagle medium (DMEM; Gibco BRL) supplemented with 10% fetal bovine serum (FBS; Gibco BRL) and 0.5% antibiotics (penicillin, 10 000 U ml-1; streptomycin, 10 mg ml-1; Gibco BRL) at 37°C in 5% CO2. Once confluent, cells were frozen in 10% dimethyl sulfoxide in DMEM supplemented with 10% FBS.
Frozen aliquots were thawed and transfected with the CEEGFP plasmid (kindly provided by Dr. Tatsuyuki Takada, National Children's Medical Research Centre, Tokyo, Japan) containing the enhanced, humanized version of the GFP-reporter gene driven by the human elongation factor-1
promoter and cytomegalovirus (CMV) enhancer and the neomycin-resistance cassette under the control of SV-40 promoter [12, 24, 28]. Cells were transfected using Lipofectamine (Gibco BRL) according to the manufacturer's instructions [12]. A total of 14 GFP-expression clones were generated either by selection under G418 or by low-density plating and selection of GFP-positive colonies under blue light. The clones were expanded and frozen at the concentration of 0.5–1 x 106 ml-1 for subsequent use as donor nuclei. Two clonal lines, which had been selected by visual assessment, were used for nuclear transfer (see Fig. 1). Normal chromosomal number (2n = 60XY) was determined before use of cells for nuclear transfer. Slides for cytogenetic analysis were prepared by standard techniques [29]. Briefly, fetal fibroblasts were cultured with colcemid (Sigma, St. Louis, MO) to increase the number of cells in mitotic metaphase, spread onto slides, stained with Giemsa, and assessed for chromosome number and sex.
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Production of Host Cytoplasts
Ovaries were collected from a local abattoir and transported in saline at 30–35°C to the laboratory within 2 h of slaughter. Follicles with diameters between 2 and 8 mm were punctured with a 19-gauge needle, and cumulus-oocyte complexes (COCs) with several layers of cumulus cells and homogeneous oocyte cytoplasm were washed in Hepes-buffered tissue-culture medium (TCM-199; Gibco BRL) supplemented with 10% (v/v) FBS. Groups of 20 COCs were placed in 100 µl of bicarbonate-buffered TCM-199 supplemented with 10% FBS, 50 µg ml-1 of LH (Ayerst, London, ON, Canada), 0.5 µg ml-1 of FSH (Folltropin-V; Vetrepharm, St-Laurent, PQ, Canada), 1 µg ml-1 of estradiol-17ß (Sigma), 22 µg ml-1 of pyruvate (Sigma), and 50 µg ml-1 of gentamicin (Sigma). After 20–22 h of in vitro maturation (IVM), host oocytes were vigorously shaken in a 0.2% hyaluronidase (Sigma) solution to remove the cumulus cells, selected for the presence of the first polar body, and used for nuclear transfer.
Reconstruction of Embryos by Nuclear Transfer
Transfected fibroblasts were thawed at 37°C, washed with -DMEM containing 10% FBS, and seeded into a 60-mm Petri dish using the same medium. After 4–6 h in culture, the dish was rinsed with PBS to remove floating, unattached cells, and trypsin was added to isolate the attached fibroblasts, which were used as donor cells for nuclear transfer. Two protocols of oocyte enucleation were used to determine the role of the cell-cycle stage of the host cytoplasm on developmental outcome.
In the first protocol (metaphase enucleation), cumulus-denuded oocytes were placed in PBS containing 7.5 µg ml-1 of cytochalasin B (Sigma), and approximately 30% of the cytoplasm adjacent to the first polar body was removed. After microsurgery, oocytes were placed in medium containing 5 µg ml-1 of Hoechst 33342 (Sigma) for 15 min and then subjected briefly to ultraviolet irradiation to confirm the removal of chromatin. A GFP-positive donor cell was introduced into the perivitelline space of the enucleated oocyte, and the resulting couplet was placed in a 0.3 M mannitol solution containing 0.1 mM MgSO4 and 0.05 mM CaCl2 and subjected to a 1.5-kV electric pulse lasting 70 µsec. Previous experiments have indicated that exposure to an electric pulse at 24–26 h of IVM causes low rates of activation. After electrical stimulation, couplets were washed and cultured in 50 µl drops of Menezo B2 medium (MB2; Pharmascience, Paris, France) supplemented with 10% FBS in the presence of bovine oviduct epithelial cells (BOEC) under equilibrated mineral oil at 39°C in a humidified atmosphere of 5% CO2 in air. After 1–2 h in culture, couplets were examined to determine fusion and then exposed to 5 µM ionomycin (Sigma) for 4 min to induce parthenogenetic activation. Reconstructed oocytes were cultured in MB2 and BOEC for 7 days.
In the second protocol (telophase enucleation), denuded and selected oocytes were cultured for an additional 4 h (from 24 to 28 h) in IVM drops, activated with ionomycin, and placed for 2 h in the incubator to allow for extrusion of the second polar body. During or immediately after polar body extrusion, telophase II-stage oocytes were exposed for 15 min to medium containing cytochalasin B, and approximately one-tenth of the cytoplasm adjacent to the second polar body was removed [30]. A GFP-positive fibroblast cell was injected into the perivitelline space and fused to the host cytoplast at approximately 2.5 h after activation. Electrofusion parameters and culture conditions of reconstructed embryos were equal to those described above.
Synchronization of Recipient Heifers and Embryo Transfer
Heat synchronization of the recipient heifers was induced by injecting 500 µg of the prostaglandin F2-analogue cloprostenol (Estrumate; Schering Canada, Inc., Montreal, PQ, Canada). Six to 7 days after the standing heat, one or two fresh blastocysts were transferred to the uterine horn ipsilaterally to the presence of the ovary with a corpus luteum. Embryos were washed with TCM-199 Hepes-buffered medium supplemented with 20% FBS, loaded into a 250-µl straw, and transferred. After embryo transfer, heifers were monitored daily for heat behavior and examined by ultrasound after 30 days from embryo transfer to confirm the pregnancy.
Polymerase Chain Reaction and Fluorescent In Situ Hybridization Analysis of Cloned Animals
Genomic DNA from the cloned calves was analyzed by polymerase chain reaction (PCR) to determine the presence of the transgene using GFP-specific primers. The primers (forward: 5'-AGA CTG AAG TTA GGC CAG CTT GG-3'; reverse: 5'-GTC TTG TAG TTG CCC GTC GTC CTT-3') amplified a 490-base pair (bp) fragment, which spanned the elongation factor-1 promoter and the GFP-reporter gene. The PCR amplification consisted of 35 cycles with annealing at 55°C for 45 sec and extension at 72°C for 45 sec. Chromosomal integration of the transgene was demonstrated by fluorescent in situ hybridization (FISH) in fibroblast cells derived from skin biopsy specimens taken from the cloned calves. The transgene hybridization signal was visualized using a biotin-labeled probe containing the entire CEEGFP construct and fluorescent antibodies, followed by staining with propidium iodide [12, 31, 32].
GFP Presence and Expression in Embryos and Calves
During the period of in vitro culture, part of the reconstructed embryos were briefly exposed to blue light to determine expression of the GFP at different stages of development. At Day 7 after reconstruction, embryos that attained the blastocyst stage were examined to certify the expression of GFP. Some embryos were fixed with 10% formalin for 10 min, placed onto slides in a mounting solution containing 5 µg ml-1 of Hoechst 33342, and examined by epifluorescence to determine the number of nuclei per blastocyst.
Skin biopsies were performed on two cloned calves at 48 h after birth to establish fibroblast cell cultures. After a few passages, fibroblasts were treated with trypsin, fixed in 2% paraformaldehyde, and analyzed by fluorescence-activated cell sorting (FACS) to determine the percentage of cells expressing GFP. Some GFP-positive and -negative fibroblast cells were used in nuclear transfer. Cell cultures were also established from the placental tissue, and expression of GFP in these cells was determined by exposure to blue light. Different tissues were recovered after slaughter, snap-frozen at -70°C, and examined. Tissues recovered included brain, hypophysis, heart, lung, liver, kidney, pancreas, spleen, bladder, intestine, testis, muscle, tongue, and skin. Tissues were cryosectioned, mounted onto slides, and analyzed under blue light to determine the expression of GFP.
Semen samples were obtained from one of the cloned calves at 15 mo of age by means of an artificial vagina and were frozen in liquid nitrogen using a sodium citrate, egg yolk, and glycerol extender [33]. Slaughterhouse-derived oocytes were in vitro fertilized and cultured as described previously [34]. Cleaved embryos at different stages of development were briefly exposed to blue light to determine both the proportion and the stage when GFP was first expressed. At Day 9 of culture, DNA was extracted from GFP-positive and -negative embryos using the DNeasy Tissue Kit (Qiagen, Mississauga, ON, Canada). Then, PCR reactions from each embryo were made to verify the presence of GFP sequences, using the primers described above, of bovine-specific satellite DNA (forward: 5'-TGG AAG CAA AGA ACC CCG CT-3'; reverse: 5'-TGC TCA GAA ACC GCA CAC TG-3'), which produces a 212-bp amplicon, and to verify the sex of the embryo through Y chromosome-specific sequences (forward: 5'-GAT CAC TAT ACA TAC ACC ACT-3'; reverse: 5'-GCT ATG CTA ACA CAA ATT CTG-3'), which produces an 167-bp amplicon [35]. The PCR amplification consisted of 20 or 40 cycles of annealing at 60°C for 45 sec and extension at 72°C for 45 sec for the autosomic and Y chromosome-specific sequence, respectively.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The cell-cycle stage of donor cells was characterized by flow cytometry at 5 h after thaw (i.e., the expected time of embryo reconstruction). Results demonstrated that donor cells were not synchronized at any specific stage of the cell cycle (Fig. 1). Although a fair proportion of the cells (76%) were at the G0/G1 stage, the remaining cells were equally distributed at the S (12.5%) and G2/M (11.5%) stages, a typical cell-phase pattern found in cycling fibroblasts. Moreover, as determined by 5-bromodeoxyuridine incorporation, approximately two-thirds of the cells were undergoing DNA synthesis during a 2-h period preceding oocyte reconstruction (data not shown).
To verify whether actively cycling cells require a specific host cytoplasm stage to complete nuclear reprogramming and support development, donor cells were electrofused with host cytoplasts either at 1–2 h before (metaphase) or at 2.5–3 h after (telophase) activation with ionomycin. The rate of development to the blastocyst stage after 7 days of in vitro culture and the quality of the blastocysts produced, as evidenced by morphological evaluation and the number of nuclei, were not significantly different between metaphase and telophase host oocytes, demonstrating that both cytoplasts have similar potential to produce transgenic blastocysts after somatic nuclear transfer (Table 1).
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Some of the embryos that developed to the blastocyst stage at Day 7 were transferred into surrogate heifers synchronized with prostaglandin. A total of 15 metaphase- and 11 telophase-derived embryos were transferred to eight and six heifers, respectively, resulting in four and one positive gestations, respectively, detected by ultrasound on Day 55 after embryo transfer. All five recipients were pregnant at 280 days of gestation, and four produced live offspring after cesarean section (Fig. 2). One recipient heifer from the metaphase group showed signs of dystocia and produced a stillborn fetus showing accumulation of liquids in the internal cavities and severe liver fibrosis. One liveborn calf derived from the metaphase group developed respiratory distress within 24 h from birth and was treated with oxygen and antibiotics. The calf was killed 1 wk after birth because of increased respiratory distress caused by pulmonary hypertension accompanied by circulatory insufficiency from abnormal heart formation. Macroscopic evaluation at autopsy showed abnormalities of the myocardium, aorta, and pulmonary artery typical of Eisenmenger syndrome. The heart showed a patent ductus arteriosus, myocardium hypertrophy, dilation of the right atrium and ventricle, and a large ventricular septal defect (3.5 x 2 cm). Moreover, the aorta exited the heart from the right ventricle, and the pulmonary trunk was severely dilated and tortuous. Although no macroscopic defects were identified in the remaining cloned calves, all three displayed symptoms of respiratory disorders between 24 and 48 h after birth. Calves were treated using antibiotics and provided with oxygen support whenever required, and although the calves recovered after 2–3 wk of intensive care, health standards remained fragile, with occasional relapses of pneumonia.
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Transgene Expression During Preimplantation Development
To verify whether the pattern of control of GFP expression was affected by the cytoplast stage, embryos reconstructed with metaphase and telophase host cytoplasts were analyzed during preimplantation development (Fig. 3). In both cytoplast groups, GFP disappeared immediately after embryo reconstruction and remained undetected up to the 8-cell stage at 60 h after reconstruction. Independently of the cytoplast stage, GFP was observed in nuclear transfer embryos at the late 8- to 16-cell stage at 80 h postreconstruction, which corresponds with the period of major embryonic genome activation in bovine embryos [36]. After 80 h, embryos that continued development to the blastocyst stage at Day 7 and beyond were consistently GFP positive in both metaphase- and telophase-reconstructed groups, indicating a similar pattern of remodeling of the somatic chromatin activity after nuclear transfer both before and after oocyte activation.
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Transgene Identification and Expression in Cloned Offspring
The presence and activity of the GFP transgene in cloned calves was confirmed by PCR, FISH, and GFP expression in tissues and primary cell cultures derived from offspring and their placental membranes. The FISH analysis of cells derived from the clones showed three sites of transgene integration (Fig. 4). The FACS analysis of fibroblast cultures established from a metaphase- and the telophase-derived cloned calves revealed approximately 50% GFP-positive cells (Fig. 5). However, although the patterns of GFP intensity were normally distributed in the metaphase-derived clone, fibroblast samples from the telophase-derived clone contained a bimodal distribution, with a population of more intense GFP expression. Tissues obtained from cloned calves were cryosectioned and examined under blue light (Fig. 6). Although all clones expressed GFP, patterns of expression were variable within most tissues analyzed (i.e. positive and negative cell groups), having no clear correlation with any particular cell type.
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To determine whether nuclear transfer could reprogram somatic cells in which GFP expression was silenced, GFP-negative and -positive skin-derived fibroblasts obtained from cloned offspring were fused to metaphase-enucleated oocytes and examined at Days 1, 5, and 8 of development in vitro. As reported above with GFP-positive fetal-derived fibroblasts, reconstructed embryos derived from GFP-positive skin fibroblasts were negative at Day 1 but became positive (17 of 17) at Days 5 and 8 after nuclear transfer. However, cloned-derived skin fibroblast donor cells that were GFP-negative at the time of reconstruction remained mostly negative (18 of 19) throughout the period of in vitro culture to the blastocyst stage, indicating that the oocytes' cytoplasm was unable to reprogram or "activate" the silenced transgene after nuclear transfer. Nonetheless, one morula derived from negative donor fibroblasts was weakly positive, suggesting that some GFP-inactive nuclei may be partially reprogrammed after passage through the cytoplasm of early embryos.
Transgene Transmission Through the Germ Line
Semen was collected from a metaphase-derived clone and used for in vitro fertilization (IVF) experiments to determine transmission of the GFP transgene in the germ line. Presumptive zygotes fixed at 15 h post-IVF revealed an 80% (141 of 175) fertilization rate, of which 20% (29 of 141) were polyspermic, as evidenced by the presence of more than two pronuclei. Thirty-three percent of oocytes fertilized developed to the blastocyst stage, and approximately half (46%, 139 of 303) expressed GFP, suggesting that one or more of the three integrated transgenes observed by FISH were inactive. As observed with reconstructed embryos, GFP expression during development of IVF embryos was initiated at the late 8-cell stage. To confirm the presence of the transgene in the IVF-derived embryos, DNA from GFP-positive (n = 31) and GFP-negative (n = 29) embryos was amplified by PCR (Fig. 7). The transgene was amplified in 29 (94%) GFP-positive and in 4 (14%) GFP-negative embryos (Table 2), supporting the presence of silent transgene sequences. To control for proper DNA extraction and to determine the sex of the embryo, samples from each embryo were amplified using sets of primers directed toward autosomal and Y-specific sequences. A higher proportion of males were detected in the GFP-expressing (76%) than in the GFP-nonexpressing (44%) embryos, indicating that the presence of at least one sperm haplotype carrying a Y chromosome in polyspermic embryos was directly related with a transgene haplotype, leading to GFP expression. Twenty percent (29 of 141) of the fertilized oocytes analyzed at 18 h after IVF contained more than two pronuclei, indicating that polyspermy might have caused the higher frequency of males in the GFP-positive group. The FISH analysis of several GFP-negative embryos indicated the presence of the transgene, further supporting the hypothesis of the silencing of transgenes during passage through the germ line.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recent reports have shown that animals generated by nuclear transfer from genetically transformed cells are mostly also transgenic, which greatly improves the low efficiency levels obtained with traditional pronuclear microinjection-based protocols [7–9, 11, 12, 14]. Nonetheless, improvements are needed both in more accurately selecting nuclear donor cells that adequately express the transgene and in obtaining more consistent, full-term gestations and liveborn offspring after nuclear transfer. Indeed, some offspring derived by nuclear transfer from transfected donor cells are not transgenic because of difficulties in selecting a pure clonal population of transfected somatic cells. Identified in jellyfish (Aequorea victoria), GFP is a single peptide of 238 amino acids that absorbs blue light and emits green light without any substrate or cofactor. Expression of GFP has been shown in several species, and it has been widely used as an expression marker to study mechanisms of gene regulation [37]. The GFP-reporter vectors have been inserted in somatic cells and embryos [15, 26, 27], and offspring [12, 38] have been produced after nuclear transfer. In the present study, we demonstrate that the GFP gene, under the control of the human elongation factor 1- promoter and the CMV-immediate early enhancer, can be used as a selection marker in bovine transgenesis, not only for the nuclear donor cells before nuclear transfer and for selecting reconstructed embryos before embryo transfer but also for selecting germ line transmission by the identification of transgene-expressing embryos generated from founder transgenic sires.
The extended period of culture and the harsh treatments required to transfect and select expressing clones from primary somatic cell cultures is detrimental to their capacity to replicate through mitosis, which tends to shorten their normal life span and to interfere with standard protocols used for cell-cycle synchronization (i.e., serum starvation and in vitro cell confluence) [39]. Asynchronous transfer of cycling donor nuclei into metaphase-arrested oocytes results in detrimental effects to the chromatin [40]. Postactivation, telophase-enucleated oocytes contain the capacity to reprogram transplanted chromatin without chromatin damage and, consequently, represent a viable alternative for cloning with use of such nonsynchronized somatic cells. Telophase-enucleated host cytoplasts have been used successfully to generate somatic clones in goats [11] and have been shown to support proper remodeling of somatic components of the chromatin after nuclear transfer in cattle and mice [41, 42]. Our results demonstrate that metaphase- and telophase-reconstructed oocytes did not differ in embryonic development rates or cell numbers, indicating that the protocols are equivalent both quantitatively and qualitatively. Moreover, timing and intensity of transgene expression during early embryogenesis was equal among metaphase- and telophase-reconstructed groups, suggesting equivalent functional remodeling of chromatin activity in embryos reconstructed with both types of cytoplasts. Initially, GFP was detected at the 8- to 16-cell stage of reconstructed embryos, which corresponds to the timing of major embryonic genome activation in cattle and indicates that the transgene was "switched on" at the appropriate time in the reconstructed embryos. Such normalcy contrasts with the results of previous studies in pigs and cattle, in which reconstructed embryos either began GFP expression before the respective species-specific timing of transition to a transcriptionally active embryonic genome or, as concluded by the authors, resulted from carryover protein [26, 43, 44]. These contrasting results may have resulted from differences in the stability of GFP, differences in the promoters utilized in each study, or transgene positional effects. Although previous results in pigs have identified a mosaic expression of GFP in reconstructed embryos [15], no such effect was observed in the present study. However, some abnormally developing embryos did present variable patterns of expression among blastomeres (data not shown), which may be related to the presence of apoptotic or anucleate blastomeres.
Four live and one stillborn cloned calves were produced from the transfer of 26 reconstructed embryos, a gestational outcome similar to those in previous reports using embryos reconstructed with transfected [7, 8] and nontransfected [6, 8, 18] fibroblast donor cells. A major problem associated with the use of somatic cell-derived cloned embryos is the high incidence of fetal mortality and abortions [6, 45]. Our results contrast with these previous studies, because all five pregnancies confirmed at Day 45 of gestation continued to term. However, as reported in other studies [6, 46, 47], all cloned calves were unhealthy at or within a few hours after birth, either because of a general respiratory insufficiency, circulatory malformations, or widespread abdominal fluid accumulation with hepatic fibrosis. Even though problems associated with somatic cell cloning may be attributed to abnormal resetting of epigenetic factors involved in the control of gene expression, a possible detrimental effect of other factors, such as culture conditions or transgene insertion site, should not be discarded. Indeed, it has been observed that the presence of a transgene can interfere with the development of embryos reconstructed with somatic nuclei [8, 48]. Moreover, GFP expression has been associated with congenital heart defects in transgenic offspring produced through pronuclear microinjection [49], suggesting that the cardiac defects observed here may not have been caused by the cloning procedure itself. Use of the sperm from the transgenic clones to generate offspring may determine whether positional effects of the transgene or abnormalities caused by the GFP protein during development are the causes of problems with the circulatory system.
The FACS analysis of fibroblast cultures established from metaphase- and telophase-reconstructed clones, as well as the in situ assessment of several tissues from cloned offspring, demonstrated that GFP was not expressed in all of the cells. It is possible that expression of the transgene is affected by epigenetic modification during development and/or culture in vitro, such as methylation, as previously demonstrated [12, 50, 51]. This epigenetic silencing of GFP expression seems to be fairly stable, because the transfer of nuclei from GFP-negative cells did not lead to reprogramming of the silenced transgene. Moreover, PCR amplification analysis performed in embryos produced by IVF using sperm from a transgenic cloned bull suggest that one or two of the three inserted transgenes identified by FISH were silenced after passage through the germ line.
In summary, we demonstrate in the present study that GFP transgenic calves can be obtained from transfected somatic cell nuclei transplanted into either metaphase- or telophase-enucleated oocytes. Moreover, the GFP transgene is transmitted in the germ line and can therefore be used to select embryos derived from transgenic animals.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Lawrence C. Smith, University of Montreal, CRRA/Faculté de Médecine Vétérinaire, Université de Montréal, 3200 Sicotte, Ste-Hyacinthe, PQ, Canada J2S 7C6. FAX: 450 778 8103; smithl{at}medvet.umontreal.ca
Received: 6 August 2002.
First decision: 29 August 2002.
Accepted: 30 December 2002.
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