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Regular Article |
a Department of Animal and Dairy Science, University of Georgia, Athens, Georgia 30602
b Research Institute for Genetic Engineering and Biotechnology, TUBITAK, Gebze, Kocaeli 41470, Turkey
c Prolinia Inc., Athens, Georgia 30602
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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assisted reproductive technology, developmental biology, embryo
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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An important aspect of using NT technology to produce transgenic and gene-targeted livestock is the source of donor cells and the in vitro techniques used to obtain viable donor nuclei for NT. Genetically modified cells can be selected in vitro, and only cells with stable, integrated transgenes are used as donor cells. In cattle, sheep, pigs, and goats, fetal cells have been used to produce transgenic livestock because of their rapid growth and potential for multiple cell divisions before senescence in culture [2–5], whereas adult somatic cell cloning primarily has been used to replicate a particular female [6, 7] or male [8, 9] genotype. In order to conduct gene targeting in adult phenotypes, it is desirable to obtain sufficient colonies of confirmed transgenic cells derived from the adult animal for use as donor cells in the NT process. Subsequently, cells derived from a fetal NT of that same transgenic genotype will be required in order to target the other allele.
To date, there are limited studies on using adult transgenic donor cells in the NT procedure. Recently, adult somatic cells remained totipotent (cloned offspring) after long-term culture, suggesting that adult cells might also be viable in producing cloned transgenic offspring [10]. In a previous study we demonstrated that genetically modified granulosa cells could be used to produce transgenic NT embryos after long-term culture [11]. However, to date it has not been shown that transgenic adult cells can complete clonal propagation, a requirement for future gene targeting via homologous recombination.
Enhanced green fluorescence protein (EGFP) is known as a useful reporter system because gene expression can be detected in living cells [12, 13]. This visual indication may be used in gene insertion or targeting detection systems, including the use of EGFP as part of a fusion gene or internal ribosomal entry sequence (IRES) construct. Recently, several reports have described the use of EGFP variants in producing transgenic NT offspring or embryos in goats [14], pigs [15, 16], and cattle [11, 17]. Although several studies have indicated that EGFP may reduce viability of transfected cells containing this gene [11, 18], no deleterious effect of EGFP on embryo development has been reported.
In this study we examined whether adult and NT fetal cells derived from the adult phenotype can complete transgenic clonal selection and propagation, and then used to produce viable NT transgenic embryos. We believe this is the first report to demonstrate the use of clonal lines from transgenic adult and fetal fibroblast cells of the same genotype in the production of transgenic NT bovine embryos.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adult skin fibroblast Tissue biopsy was obtained from the ear of a 13-yr-old cow and washed several times in PBS containing 10% (v:v) penicillin/streptomycin (10 000 U/ml penicillin G, 10 000 µg/ml streptomycin; Sigma Chemical Company, St. Louis, MO). The tissue biopsy was cut into small pieces, and tissue explants were cultured in Dulbecco modified Eagle medium (DMEM) F-12 (Sigma) supplemented with 10% fetal bovine serum (FBS, Bio Whitaker Inc., Walkersville, MD) and 1% (v:v) penicillin/streptomycin (10 000 U/ml penicillin G, 10 000 µg/ml streptomycin, Sigma) at 37°C in 35-mm tissue culture plates in a humidified atmosphere of 5% CO2 in air. After 10 days in culture the explants were removed, and cells were harvested by trypsinization, counted, and seeded in 75 cm2 tissue culture flasks. When the cells reached confluency, they were collected by trypsinization and frozen in DMEM-F12 supplemented with 40% FBS and 10% dimethyl sulfoxide (DMSO; Sigma).
Cloned fetus fibroblast A Day 70 fetus cloned from the same donor cow [19] was surgically removed from the recipient's uterus and cells were obtained from the lung of the fetus as described previously in a study on gene targeting in human fibroblast cells [20]. A piece of the lung was cut into small pieces, and these small explants were cultured in 35-mm tissue culture plates. When the cells reached confluency they were expanded and frozen as described above.
Genetic Transformation of Cells and Establishment of Transgenic Cell Lines
Adult and fetal cells were transfected with a plasmid-containing EGFP gene under control of the cytomegalovirus promoter and neomycin resistant gene, which allows selection using geneticin under control of an SV40 promoter (pEGFP-N1, Clontech Laboratories, Inc., Palo Alto, CA) using a polyamine transfection reagent (GeneJammer, Stratagene, La Jolla, CA) according to the manufacturer's instructions. Briefly, 2 x 105 cells at passage 4 were seeded in a 35-mm culture plate 1 day before transfection. Cells were transfected with 2 µg of linearized pEGFP-N1. After transfection, the cells were exposed to 600 µg/ml of geneticin (G418, Sigma) for 20 days. After 20 days, 300 µg/ml of geneticin was used during the selection process to obtain stable expression, and single colonies were isolated in the presence of G418 and expanded. Transfected cell colonies were picked up by using cloning discs (Fisher Scientific, Springfield, NJ) and placed in 24-well plates. Cells in each well were examined for EGFP expression under fluorescent light using a standard fluorescein isothiocyanate (FITC) filter set and marked as positive and negative cell lines. In the adult cell group, when confluence was achieved, cells were passaged to 6-well plates and then to 100-mm plates. A portion of the cells from each EGFP-positive and EGFP-negative cell lines were plated for chromosome analysis, immunofluorescence cell staining, and DNA isolation. The remaining cells were frozen in DMEM-F12 supplemented with 40% FBS and 10% (v:v) DMSO. In the fetal cell group, only EGFP-positive cell lines were expanded, examined for chromosome number by immunofluorescence detection and for the presence of the transgene, and frozen. After transfection and selection, the total culture duration of cells was approximately 54 days.
Polymerase Chain Reaction Analysis of Clonal Lines
Genomic DNA was isolated from each cell line using a DNA purification kit (Wizard Genomic DNA kit, Promega purification; Promega, Madison, WI) and analyzed by polymerase chain reaction (PCR) for the presence of EGFP and Neo genes. Primers used to amplify the 555-base pair (bp) fragment of the neomycin-resistance gene were 5'-GTGTTC-CGGCTGTCAGCGCA-3' and 5'-GTCCTGATAGCGGTCCGCCA-3'. The second set of primers that amplified a 495-bp fragment of the EGFP reporter gene were 5'-ACGGCAAGCTGACCCTGAA-3' and 5'-GGGT-GCTCAGGTAGTGGTT-3'. After denaturation at 94°C for 1 min, 30 cycles of PCR amplification were performed at 94°C for 1 min, 60°C for 1 min, and 70°C for 1 min. PCR products were applied to a 2.0% agarose gel containing 0.5 µg/ml of ethidium bromide, and then visualized under UV light.
Expression of p53 Protein
We examined the donor cells for possible immortalization based on p53 expression [21]. To detect p53 expression in cells by immunofluorescence cell staining, cells from transgenic adult and fetal fibroblast lines, and from nontransfected adult and fetal fibroblast and positive control HEP-G2 cells (American Type Culture Collection, Manassas, VA) were plated on chamber slides (Lab-Tek II chamber slide, Nalgen Nunc International, Naperville, IL). After reaching 75%–80% confluency, cells were fixed with 3.7% paraformaldehyde in PBS for 15 min at 4°C, rinsed with PBS, and then permeabilized with 1% Triton X-100 in PBS for 10 min at room temperature and blocked with 10% normal goat serum (NGS) in PBS with 0.5% Triton X-100 for 30 min. Slides were incubated with primary antibody for p53 (p53 [DO-1], Santa Cruz Biotechnology, Inc., Santa Cruz, CA) diluted 1:100 in PBS with 1.5% NGS for 1 h, and washed with PBS. Goat anti-mouse antibody conjugated with rhodamine diluted 1:200 in PBS with 1.5% NGS was applied to the slides for 1 h at room temperature. Slides were mounted with 90% glycerol in PBS and examined under UV light.
Chromosome Analysis and Determination of Population Doublings of Donor Cell Lines
The ploidy of the transfected and expanded adult and fetal fibroblast cell lines, and of nontransfected cells was examined using a standard preparation of metaphase spreads [10]. Briefly, 24 h after plating, cells were exposed to 0.08 µg/ml of democolcine solution (Sigma) for 1 h and then were treated with KCl (0.075 M) for 15 min at 37°C. The cells were then fixed in acetic acid:methanol (1:3 v:v), and drops of cell suspension were spread on clean microscope slides. The chromosomes were stained with 5% Giemsa for 5–10 min. The numbers of chromosomes were counted under a light microscope at 1000x magnification. At least 60 metaphase spreads/lines were examined. Population doublings of clonal lines was estimated using the log 10(N/N0) x 3.33 formula (where N is the number of cells harvested and N0 is the number of cells plated).
Recipient Cytoplasm Preparation
In vitro maturation of bovine oocytes and enucleation were performed as described previously [2, 7, 11]. Briefly, bovine cumulus oocyte complexes (COCs) were recovered by aspiration of antral follicles (3–8 mm in diameter) on ovaries obtained from a slaughterhouse. Only COCs with a compact, nonatretic cumulus oophorus-corona radiata and a homogenous ooplasm were selected. Oocytes were matured in TCM 199 (Gibco Inc., Grand Island, NY) supplemented with 10% FBS, 50 µg/ml sodium pyruvate, 1% v:v penicillin/streptomycin (10 000 U/ml penicillin G, 10 000 µg/ml streptomycin), 1 ng/ml rIGF-1 (Sigma), 0.01 U/ml bovine LH, and 0.01 U/ml bovine FSH (Sioux Biochemicals, Sioux Center, IA). Maturation was performed in 4-well plates overlaid with mineral oil at 39°C in humidified 5% CO2 in air for 16–18 h. After maturation, the cumulus-corona was removed by vortexing COCs in TL Hepes medium containing 100 U/ml hyaluronidase (Sigma). Maturated oocytes were enucleated with a 15 µm (internal diameter) glass pipette (Eppendorf, Westburg, NY) by aspirating the first polar body and MII plate in a small volume of surrounding cytoplasm. The oocytes were previously stained in TL Hepes containing 2 µg/ml Hoechst 33342 and 7.5 µg/ml cytochalasin B (Sigma) for 10–15 min and then kept in TL Hepes supplemented with 7.5 µg/ml Cytochalasin B during enucleation. Enucleation was performed under ultraviolet light to ensure removal of oocyte chromatin.
Donor Cell Preparation and NT
In the first experimental group, two different EGFP-positive cell lines (AF40-gfp, AF1-gfp) and two different EGFP-negative cell lines (AF47-neo, AF4-neo) from transfected adult fibroblast cells, and nontransfected adult fibroblast cells at passage 4 were used as donors. In the second experimental group, one EGFP-positive cell line (FF3-gfp) from transfected cloned fetal fibroblast cells, and nontransfected cloned fetal fibroblast cells at passage 4 were used as donors. In both experimental groups, the donor cells were cultured with 10% FBS and allowed to grow to confluency (G1/G0). Immediately before donor cells were transferred into the enucleated oocytes, the cells were dissociated by trypsinization with 0.25% trypsin-ethylenediamine tetraacetic acid solution (Sigma). The cells were pelleted and resuspended in DMEM/F-12 + 10% FBS. A single cell was inserted into the perivitelline space of the enucleated oocyte by using a 15-µm (internal diameter) glass pipette [2]. For transfer, the brightest cells in each transgenic cell line were selected under UV light using the FITC filter set. Oocyte-cell complexes were placed in TCM 199 containing 10% FCS at 39°C in 5% CO2 in air until fusion.
Fusion and Activation of Oocyte-Cell Complexes
Oocyte-cell complexes were fused by using a needle-type electrode [11, 15] in Zimmermann fusion medium [22]. The single cell-oocyte couple was sandwiched between two wires arranged in a straight line and attached to micromanipulators. The contact surface between the cytoplast and the donor cell was perpendicular to the electrodes. The distance between the electrodes was approximately 150 µm (the diameter of the oocyte). A single, direct current pulse of 40 V of 20 µsec duration was applied using an LF 101 Fusion Machine (TR Tech Co., Tokyo, Japan). Following the pulse the complexes were cultured in TCM 199 supplemented with 10% FBS for 2 h, and fusion rates were determined. Activation of NT units was performed as described previously [11, 23] after modification. Briefly, 2 h after fusion, NT oocytes were exposed to 5 µM calcium ionophore for 10 min (A23187, Sigma) followed by incubation in TCM 199 supplemented with 10% FBS, 2.5 µg/ml cytochalasin D (Sigma), and 10 µg/ml cycloheximide (Sigma) for 1 h at 39°C in 5% CO2 in air, and in TCM 199 with 10% supplemented FBS and 10 µg/ml cycloheximide for 5 h at 39°C in 5% CO2, 5% O2, and 90% N2.
In Vitro Culture of NT Embryos
After activation, NT oocytes were cultured in 50-µl culture drops of Beltsville Agricultural Reseach Center (BARC) medium (a kind gift of Dr. Kevin Wells, USDA, Beltsville, MD) containing BSA [11, 24]; placed into a 60-mm culture plate overlaid with mineral oil at 39°C in 5% CO2, 5% O2, and 90% N2 for 5 days; and cleaved NT embryos were transferred into 50-µl culture drops of BARC + BSA medium containing 5% FBS and cultured for an additional 2 days.
Examination of Ploidy and Cell Number of Blastocysts
Blastocysts at Day 7 were cultured in medium containing 0.04 µg/ml democolcine (Sigma) and 200 µg/ml heparin (Sigma) for 2.5 h. After this incubation period, blastocysts were treated with 0.5% sodium citrate (38°C) in distilled H2O for 4 min, and then treated with cold methanol, acetic acid, and water (v:v, 3:2:1) for 15–30 sec, and placed on a slide. The slides were dried at room temperature for 1 h and stained with 5% Giemsa solution for 5 min. At least 6 metaphase spreads per blastocyst were counted under a light microscope at 1000x magnification. To examine cell number, 5–10 blastocysts per cell line were counted. Blastocyst stage embryo nuclei were stained on slides in a PBS solution and 10% glycerol containing 1 mg/ml of Hoechst 33342. A drop (
20 µl) of staining solution containing 1–3 embryos was placed in the center of a slide, a coverslip was placed over the drop, and the edges were sealed. Nuclei were visualized and counted using UV light.
Statistical Analysis
Experiments were repeated 3 times. Differences among groups were analyzed by one-way ANOVA. Data were analyzed after arcsin square transformation (SigmaStat, Jandel Scientific, San Rafael, CA). A probability of P < 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Following transfection and selection, 49 colonies from transfected adult cells were picked up and expanded. When examined under a UV/FITC filter, 35 (71.4%) colonies expressed the EGFP. Visual observations suggested that the expression level of EGFP differed among clonal lines. Clonal lines expressing EGFP were classified as 16 (Fig. 1A, 45.7%) high-expressing, and 19 (Fig. 1B, 54.2%) low-expressing clones. At the time of NT, all cells from EGFP transgenic adult and fetal lines expressed EGFP (Fig. 1C). Each cell line was observed to behave differently in culture, and generally, lines expressing EGFP proliferated more slowly than lines that did not express EGFP. Each clonal line had 1.3–1.8 x 106 cells (an estimated total of 20.3–20.8 population doublings) when they were frozen. Thirty-five colonies from transfected fetal cells were counted and 30 (85.7%) of the colonies expressed EGFP. Each clonal line had 1.5–2 x 106 cells (an estimated 20.5–20.9 population doublings) when they were frozen. After at least 20 population doublings, two adult cell lines (AF1-gfp and AF4-neo) and one fetal cell line (FF3-gfp) used for NT were able to grow and were passaged three times during NT experiments (one experiment per week). After completing the NT experiment, cell lines were further cultured and passaged several times. However, two other adult lines (AF40-gfp and AF47-neo) were cultured and passaged during the NT experiment, but they failed to propagate following the NT experiments.
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Characterization of Donor Cells and Integration of Transgenes
A majority of the cells showed a normal chromosomal complement (60 chromosomes including XX chromosomes) in nontransfected adult fibroblasts at passage 4 (94.8%) and in nontransfected fetal fibroblasts at passage 4 (95.2%). Adult and fetal fibroblasts (at passage 4) were used for transfection, and all transgenic adult and fetal cell lines were examined for chromosome numbers. A majority of the cells from each line (in the adult line, 78.4%–90%; and in fetal line group, 70.5%–94.3%) showed a normal chromosome number (60, including XX chromosomes). Chromosome numbers of the lines used for NT are presented in Table 1. The presence of both genes (Neo and EGFP) was examined by PCR in all lines. The cell lines from adult and fetal fibroblasts that expressed EGFP had both genes, but lines that did not express EGFP had the neogene only. After immunofluorescence staining of the cells, p53-positive nuclei were not observed in any cell lines, whereas all control cells were positive for p53 nuclear staining, as expected.
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EGFP Expression and Ploidy of NT Embryos
Fusion rates for NT units were determined 2 h after the fusion pulse, and green fluorescence was observed in all fused NT units when examined under a UV/FITC filter. There was no evidence of mosaicism in any cleaved embryos expressing EGFP. EGFP expression was observed in all blastocysts from both adult lines and the fetal line. A variation in EGFP expression was not observed in blastocysts (Fig. 1E). Samples of blastocysts from all groups were processed for chromosome analysis. In the adult cell group, 25%–100% of blastocysts from transfected cells and 88% of blastocysts from nontransfected cells were diploid (Table 2). In the fetal cell group, 75% of blastocysts from transfected fetal cells and 77.7% of blastocysts from nontransfected fetal cells were diploid (Table 2). Only 12.5% of blastocysts from the AF47-neo cell line were tetraploid. Most of mixoploid blastocysts were diploid/tetraploid (Fig. 1, F and G.). The percentage of polyploid cells was less than 15% in mixoploid blastocyst.
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Developmental Rates of Embryos
There was no significant difference in blastocyst developmental rates between NT units from nontransfected adult and fetal cells of the same genotype (Table 3, 20.1% vs. 18.3%, respectively). Developmental rates of NT units derived from adult EGFP-positive (11.1% vs. 11.3%) and EGFP-negative (17.9% vs. 21.5%) lines were similar. However, when data were combined, there was a significant difference between NT units from EGFP-positive and EGFP-negative lines (Table 3; 11.3% vs. 19.8%, respectively, P < 0.05). Developmental rates of NT units from adult negative and nontransfected cells were similar (Table 3, 19.8% vs. 20.1%). In the fetal cell group, development rates of NT units from transfected and nontransfected fetal fibroblast cells were significantly different (Table 3; 6.4% vs. 18.3%, respectively, P < 0.05). There was a significant difference in developmental rates between NT units from adult EGFP-positive cells and fetal EGFP-positive cells (Table 3; 11.3% vs. 6.4%, respectively). Cell numbers and morphology of blastocysts from all experimental groups were similar.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Generally, an early passage of donor cells are used for NT [2–4, 7, 8, 26, 27]. Although live clones have been obtained from adult somatic cells in sheep [6], cattle [7–10, 28], and pigs [27], production of transgenic animals by cloning has been limited to fetal-derived transfected somatic donor cells [2, 4, 14, 29]. Kasinathan et al. [30] found that cells from a 15-yr-old animal had a life span of about 18 population doublings, and suggested that adult cells are likely to become senescent before selection for transgenic clonal lines would be completed. However, cells from a 13-yr-old animal could complete clonal propagation in this study. The lines had about 20 population doublings when they were used for NT. The cells from 2 adult lines were able to divide following NT. Two studies [2, 30] reported that fetal fibroblast cells had 30 population doublings; however, another study [31] indicated that fibroblast cells of the same age had 60 population doublings. Therefore, population doublings for adult cells might be different among animals or culture conditions. Although population doublings were not examined, two recent studies demonstrated that developmental rates of NT embryos derived from adult cells after long-term culture (10–15 passages) were higher than embryos derived from shorter-term culture (5 passages) [10, 11]. The extended life span of the dividing cells might be related to mutations or allelic loss of genes involved in senescence, such as the p53 gene [21, 32]. It was observed that immortalized cells had p53-positive nuclei, but fetal fibroblast cells after 94 population doublings were negative for p53 [21]. When we examined the cell lines for possible immortality, abnormal p53 expression was not determined in fetal or adult cell lines. In addition, 1 previous study indicated that late passage immortalized epithelial cells used for NT did not result in blastocysts [33]. In the present study, all transgenic lines resulted in transgenic blastocysts. The percentage of cells with normal chromosome numbers in all experimental groups was similar to those obtained by Kubota [10], who used adult fibroblast cells in a late passage.
A recent study showed that fibroblasts obtained from cloned calves have an extended cell culture life span [31]. Cells derived from cloned fetuses had an approximately 50% longer proliferative life span than those obtained from a nonmanipulated fetus of similar age [31]. Another approach could be the use of recloned fetuses to benefit transgenic animal production by increasing the number of population doublings. Therefore, we also used fibroblasts from a cloned fetus of the same genome in this study. In a previous study [8], as well as in the current study, there was no significant difference in developmental rates between NT embryos from nontransfected adult and fetal fibroblasts of the same genotype. However, in the current study, the EGFP transgenic fetal fibroblast cells had the lowest embryo developmental rate among all lines (adult and fetal). Because other EGFP-positive clonal adult lines also had lower developmental rates than negative and nontransfected adult cells, the low embryo developmental rate might be related to the site of integration or to the negative effect of EGFP on the donor cells, and the development of transgenic embryos. Although green fluorescent protein is believed to be a nontoxic biological marker, Hanazono and coworkers [18] reported that high-expressing cells (the brightest cells) died within a matter of days after transfection. They hypothesized that this was due to a deleterious effect of the gene product. Transgenic murine and bovine embryos expressing the green fluorescent protein gene have been reported [34–37], but systematic examination for toxicity of the gene product was not performed. However, 2 recent studies reported that EGFP did not reduce the development of transgenic NT embryos [16, 17]. In this study, another reason for the lower developmental rate of EGFP transgenic embryos might be the use of UV light in order to visually identify the cells that express the same level of EGFP during NT. In a previous study, a deleterious effect of EGFP was observed on granulosa cells, but not on embryo development following NT [11]. In a recent study [38], clonal lines of transgenic fibroblast cells derived from the same fetus resulted in different embryo development when used for NT in pigs. In the present study, there was not a significant difference in developmental rates between embryos from AF1-gfp and AF40-gfp (EGFP-positive adult lines) and between embryos from AF4-neo and AF47-neo (EGFP-negative adult lines). However, developmental rates of embryos from EGFP-positive cells were lower than those from EGFP-negative cells. In the fetal cell group, it could not be determined whether there was any difference between transgenic clonal lines, because only one line was used for NT in this study.
We also examined the ploidy of the NT embryos from adult and fetal clonal lines in this study. Chromosomal analysis of developing mammalian embryos has shown that a considerable proportion of morphologically normal embryos are chromosomally abnormal [39, 40]. Results from diploid-tetraploid mouse chimeras have confirmed that even high proportions of tetraploid cells are tolerated, but the ability to trace the tetraploid cells has also shown that they are eliminated in the developing embryos and are preferentially allocated to the extraembryonic membranes [41, 42]. Embryonic mixoploidy is also a common phenomenon in domestic animal species [43–46]. An early study of bovine embryos developed in vivo reported that 41.5% of morphologically normal blastocysts were diploid-tetraploid mosaics [43]. One study indicated that 72% of the in vitro-produced blastocysts were mixoploid [44], and another study reported that 50% of NT embryos were normal ploidy when they were analyzed at the 2-cell stage [45]. In this study, mixoploidy was 11%–62.5%. The percentage of polyploid cells in mixoploid embryos was less than 15%. One blastocyst from only 1 adult cell line was polyploid (12.5%). Polyploid embryos from NT or in vitro fertilization have been reported previously [45, 46]. The present study demonstrates that NT blastocysts derived from adult and cloned fetal fibroblasts, after long-term culture and transfection, do not show a higher percentage of chromosomal abnormalities than those reported in previous studies. However, relatively few metaphase spreads could be counted, and with greater numbers, the percentage of the abnormalities might have been closer to those reported previously.
In summary, a majority of the cells had the normal chromosomal number after clonal propagation, and p53 expression was normal in adult and fetal clonal lines. Development of NT embryos derived from adult fibroblasts was not affected by transfection and antibiotic selection of donor cells because developmental rates for nontransfected and transfected (EGFP-nonexpressing) derived embryos were similar. The lower developmental rate of embryos produced from cells (adult and fetal) expressing EGFP may indicate a deleterious effect of EGFP. Therefore, adult cells can complete transgenic clonal propagation required in gene targeting and so may be used to produce transgenic cloned calves. Although transgenic fetal cells gave low embryo development in this study, they could complete clonal propagation and resulted in transgenic embryos. Embryos from adult and fetal transgenic lines had similar morphology, cell number, and ploidy. Therefore, cells from a cloned fetus can also be used to produce transgenic embryos, and it is likely that so too can animals with targeted genetic manipulations such as knockouts or other types of genetically engineered cloned animals. Although it was not tested here, it is likely that a cloned fetus from a transgenic adult cell line may serve as a cell source for a second round of transgenic clonal propagation to target the other allele for gene knockout. However, it should be considered that EGFP may be toxic and reduce embryo development in future gene targeting studies.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by AT Children's Project and Prolinia Inc. 
2 Correspondence: Steven L. Stice, Department of Animal and Dairy Science, University of Georgia, 425 River Road, Athens, GA 30602. FAX: 706 542 7925; sstice{at}arches.uga.edu
Accepted: December 27, 2001.
Received: November 7, 2001.
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