Production of cloned pigs by using somatic cells as donors.
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Animals↗
Biomedical subjects
Randall S Prather
Publications and source records attributed to Randall S Prather.
Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning.
The presence of galactose alpha-1,3-galactose residues on the surface of pig cells is a major obstacle to successful xenotransplantation. Here, we report the production of four live pigs in which one allele of the alpha-1,3-galactosyltransferase locus has been knocked out. These pigs were produced by nuclear transfer technology; clonal fetal fibroblast cell lines were used as nuclear donors for embryos reconstructed with enucleated pig oocytes.
Alleles↗
Effect of elevated Ca(2+) concentration in fusion/activation medium on the fusion and development of porcine fetal fibroblast nuclear transfer embryos.
The present study examined the effect of elevated Ca(2+) concentration in fusion/activation medium on the fusion and development of fetal fibroblast nuclear transfer (NT) porcine embryos. Frozen-thawed and serum starved fetal fibroblasts were transferred into the perivitelline space of enucleated oocytes. Cell fusion and activation were induced simultaneously with electric pulses in 0.3 M mannitol-based medium containing 0.1 or 1.0 mM CaCl(2). Some fused embryos were further activated 1 hr after the fusion treatment by exposure to an electric pulse. The NT embryos were cultured in vitro for 6 days. Fusion and blastocyst formation rates were significantly (P<0.05) increased by increasing the Ca(2+) concentration from 0.1 mM (67.1 and 6.3%) to 1.0 mM (84.7 and 15.8%). However, no difference in the number of cells in blastocysts was observed between the two groups. A higher percentage of blastocyst was also observed when control oocytes were parthenogenetically activated in the presence of elevated Ca(2+) (19.3% vs. 32.4%, P<0.05). When the reconstituted oocytes were fused in the medium containing 1.0 mM CaCl(2), increasing the number of pulses from 2 to 3 or an additional activation treatment did not enhance the blastocyst formation rate or cell number in blastocysts. These results demonstrate that increasing the Ca(2+) concentration in the fusion/activation medium can enhance the fusion and blastocyst formation rates of fetal fibroblast NT porcine embryos without an additional activation treatment.
Animals↗
Transgenic pig expressing the enhanced green fluorescent protein produced by nuclear transfer using colchicine-treated fibroblasts as donor cells.
Fetal-derived fibroblast cells were transduced with replication defective vectors containing the enhanced green fluorescent protein (EGFP). The transgenic cells were treated with colchicine, which theoretically would synchronize the cells into G2/M stage, and then used as donor nuclei for nuclear transfer. The donor cells were transferred into the perivitalline space of enucleated in vitro matured porcine oocytes, and fused and activated with electrical pulses. A total of 8.3% and 28.6% of reconstructed oocytes showed nuclear envelope breakdown and premature chromosome condensation 0.5 and 2 hr after activation, respectively. Percentage of pronuclear formation was 62.5, 12 hr after activation. Most (91.4%) of the 1-cell embryos with pronuclei did not extrude a polar body. Most (77.2%) embryos on day 5 were diploid. Within 2 hr after fusion, strong fluorescence was detectable in most reconstructed oocytes (92.3%). The fluorescence in all NT embryos became weak 15 hr after fusion and disappeared when culture to 48 hr. But from day 3, cleaved embryos at the 2- to 4-cell stage started to express EGFP again. On day 7, 85.8% of cleaved embryos expressed EGFP. A total of 9.4% of reconstructed embryos developed to blastocyst stage and 71.5% of the blastoctysts expressed EGFP. After 200 reconstructed 1-cell stage embryos were transferred into four surrogate gilts, three recipients were found to be pregnant. One of them maintained to term and delivered a healthy transgenic piglet expressing EGFP. Our data suggest that the combination of transduction of somatic cells by a replication defective vector with the nuclear transfer of colchicine-treated donors is an alternative to produce transgenic pigs. Furthermore, the tissues expressing EGFP from descendents of this pig may be very useful in future studies using pigs that require genetically marked cells.
Animals↗
Cyclin B1 levels in the porcine 4-cell stage embryo.
The relative quantity of cyclin B1 was determined during the development of in vitro and in vivo derived porcine 4-cell embryos by western blotting and immunolocalised during the 4-cell stage. After cleavage to the 4-cell stage cyclin B1 localised to the cytoplasm at the 5, 10, 18 and 25 time points and localised to the nucleus 33 h post 4-cell cleavage (P4CC). The relative abundance of cyclin B1 was not significantly different in in vivo or in vitro derived 4-cell stage embryos cultured in the absence of the RNA polymerase inhibitor alpha-amanitin. Cyclin B1 protein was not detectable in embryos cultured in medium without alpha-amanitin for 5, 10, 18 or 25 h P4CC followed by culture in medium with alpha-amanitin to 33 P4CC. These results suggest that the maternal to zygotic transition of mRNA production that occurs at the 4-cell stage of the pig embryo does not result in an increase in cyclin B1 production. In addition, cyclin B1 protein levels remained constant in the absence of embryonic genome activation at the 4-cell stage.
Animals↗
Progress in producing knockout models for xenotransplantation by nuclear transfer.
The pig is thought to be the most suitable non-human organ source for xenotransplantation. However, one of the major constraints to using pig organs for xenotransplantation is human natural antibody-mediated hyperacute rejection (HAR). Elimination of alpha(1, 3) galactosyltransferase (GGTA1) from the pig is expected to be a solution to the problem of hyperacute and delayed vascular rejection. Based on the extensive efforts made in characterization of GGTA1 in structure and function, improvement in the technique of DNA transfection of somatic cells and optimization of the pig nuclear transfer (NT) procedures, a specific modification has been made to one copy of the GGTA1 gene thus rendering it non-functional. When homozygosity of the genetic modification is achieved, this will allow concentrated efforts to address the role of galalpha(1, 3) gal specific natural antibody in HAR, acute vascular rejection and to examine the efficacy of xenotransplantation in a non-human primate model without HAR confounding the experiments. Since swine are widely used as models of human disease, the ability to make specific genetic modifications to swine as described here is predicted to have a large impact not only on production agriculture but also on biomedicine.
Animals↗
Phenotyping of transgenic cloned piglets.
Numerous reports list the abnormalities obtained from cloning sheep and cattle. To date, few reports provide detailed information regarding the overall health status and performance data of cloned animals. This report follows three litters totaling 10 transgenic cloned piglets from birth through puberty. Significant findings from physical examinations and response to treatments are included, as well as necropsy data from five of the piglets that died during the study. The birth weights, placental weights, and growth rates for this group of piglets were not different from that of control animals raised in the same environment. Hematology and serum chemistry data were collected at 2 days of age, and at 2, 4, 8, 12, 16, 20, and 24 weeks of age. Results indicated a mild anemia and hypoproteinemia in the cloned piglets from birth through 4 weeks of age, but both conditions were corrected by 8 weeks of age. Echocardiography was performed on seven of the piglets. No anatomical defects were detected, but three of the piglets had decreased cardiac output values. However, both animals are growing and show no evidence of clinical disease. The immune system was evaluated by quantification of serum IgM and IgG levels and by determining the population of B-cells, macrophages, helper T-cells (CD4), cytotoxic T-cell (CD8), and double positive T-cells (CD4/CD8). With the exception of one animal, no abnormalities were detected with the immune system of the examined piglets. During the course of this study, five of the 10 piglets were euthanized or died, indicating there is a high mortality rate among cloned piglets, but the remaining five cloned piglets are free from detectable defects.
Animals↗
CRM1-mediated nuclear export is present during porcine embryogenesis, but is not required for early cleavage.
Regulated movement of cellular factors between the cytoplasm and nucleus is required for fundamental cellular processes ranging from cell cycle control to transcriptional regulation. CRM1 is a nuclear export factor whose function is to actively transport nuclear cargos that bear nuclear export sequences to the cytoplasm. Because CRM1 likely plays a role in the intracellular regulation of many cellular processes, we set out to characterize CRM1 function during early mammalian embryogenesis. A series of embryo culture experiments that employed a specific inhibitor of CRM1, leptomycin B, indicated that CRM1 function is not required for development until after the 4-cell stage of porcine embryo development. Immunolocalization of CRM1 in fixed embryos revealed that CRM1 is localized in a unique pattern during the period of time when the embryo does not have a developmental requirement for CRM1. Despite these findings, a microinjection assay showed that CRM1 function persists during this period of development. This demonstrates that although CRM1 is present in a functional form throughout mammalian embryo development, its function is not required for early cleavage.
Animals↗
Regulation of mitogen-activated protein kinase phosphorylation, microtubule organization, chromatin behavior, and cell cycle progression by protein phosphatases during pig oocyte maturation and fertilization in vitro.
We used okadaic acid (OA), a potent inhibitor of protein phosphatases 1 and 2A, to study the regulatory effects of protein phosphatases on mitogen-activated protein (MAP) kinase phosphorylation, morphological changes in the nucleus, and microtubule assembly during pig oocyte maturation and fertilization in vitro. When germinal vesicle (GV) stage oocytes were exposed to OA, MAP kinase phosphorylation was greatly accelerated, being fully activated at 10 min. However, MAP kinase was dephosphorylated by long-term (>20 h) exposure to OA. Correspondingly, premature chromosome condensation and GV breakdown were accelerated, whereas meiotic spindle assembly and meiotic progression beyond metaphase I stage were inhibited. OA also quickly reversed the inhibitory effects of butyrolactone I, a specific inhibitor of maturation-promoting factor (MPF), on MAP kinase phosphorylation and meiosis resumption. Treatment of metaphase II oocytes triggered metaphase II spindle elongation and disassembly as well as chromosome alignment disruption. OA treatment of fertilized eggs resulted in prompt phosphorylation of MAP kinase, disassembly of microtubules around the pronuclear area, chromatin condensation, and pronuclear membrane breakdown, but inhibited further cleavage. Our results suggest that inhibition of protein phosphatases promptly phosphorylates MAP kinase, induces premature chromosome condensation and meiosis resumption as well as pronucleus breakdown, but inhibits spindle organization and suppresses microtubule assembly by sperm centrosomes in pig oocytes and fertilized eggs.
4-Butyrolactone↗
Capacitative calcium entry mechanism in porcine oocytes.
The presence of the capacitative Ca(2+) entry mechanism was investigated in porcine oocytes. In vitro-matured oocytes were treated with thapsigargin in Ca(2+)-free medium for 3 h to deplete intracellular calcium stores. After restoring extracellular calcium, a large calcium influx was measured by using the calcium indicator dye fura-2, indicating capacitative Ca(2+) entry. A similar divalent cation influx could also be detected with the Mn(2+)-quench technique after inositol 1,4,5-triphosphate-induced Ca(2+) release. In both cases, lanthanum, the Ca(2+) permeable channel inhibitor, completely blocked the influx caused by store depletion. Heterologous expression of Drosophila trp in porcine oocytes enhanced the thapsigargin-induced Ca(2+) influx. Polymerase chain reaction cloning using primers that were designed based on mouse and human trp sequences revealed that porcine oocytes contain a trp homologue. As in other cell types, the capacitative Ca(2+) entry mechanism might help in refilling the intracellular stores after the release of Ca(2+) from the stores. Further investigation is needed to determine whether the trp channel serves as the capacitative Ca(2+) entry pathway in porcine oocytes or is simply activated by the endogenous capacitative Ca(2+) entry mechanism and thus contributes to Ca(2+) influx.
Animals↗
Mosaic gene expression in nuclear transfer-derived embryos and the production of cloned transgenic pigs from ear-derived fibroblasts.
Genetically modified domestic animals have many potential applications ranging from basic research to production agriculture. One of the goals in transgenic animal production schemes is to reliably predict the expression pattern of the foreign gene. Establishing a method to screen genetically modified embryos for transgene expression before transfer to surrogates may improve the likelihood of producing offspring with the desired expression pattern. In order to determine how transgene expression may be regulated in the early embryo, we generated porcine embryos from two distinct genetically modified cell lines by using the nuclear transfer (NT) technique. Both cell lines expressed the enhanced green fluorescent protein (eGFP); the first was a fibroblast cell line derived from the skin of a newborn pig that expressed eGFP, whereas the second was a fetal derived fibroblast cell line into which the eGFP gene was introduced by a retroviral vector. The reconstructed embryos were activated by electrical pulses and cultured in NCSU23. Although the in vitro developmental ability of each group of NT embryos was not different, the eGFP expression pattern was different. All embryos produced from the transduced fetal cell line fluoresced, but only 26% of the embryos generated from the newborn cell line fluoresced, and among those that did express eGFP, more than half had a mosaic expression pattern. This was unexpected because the fetal cell line was not clonally selected, and each cell had potentially different sites of integration. Embryos generated from the newborn cell line were surgically transferred to five surrogate gilts. One gilt delivered four female piglets, all of which expressed eGFP, and all had microsatellites identical to the donor. Here we demonstrate that transgene expression in all the blastomeres of an NT embryo is not uniform. In addition, transgene expression in a genetically manipulated embryo may not be an accurate indicator of expression in the resulting offspring.
Animals↗
High developmental competence of pig oocytes after meiotic inhibition with a specific M-phase promoting factor kinase inhibitor, butyrolactone I.
Butyrolactone I specifically inhibits M-phase promoting factor activation and prevents the resumption of meiosis. These experiments were conducted to examine effects of butyrolactone I on pig oocytes in a serum-free maturation system. The first experiment was conducted to determine the effect of butyrolactone I (0-100 microM) on nuclear maturation. At concentrations of > or =12.5 microM, germinal vesicle breakdown was prevented in >90% of the oocytes after 24 h of culture. In the second experiment, the kinetics of in vitro maturation of butyrolactone I-treated oocytes was investigated. Oocytes were treated with 0 or 12.5 microM butyrolactone I and FSH for 20 h and then cultured with LH in the absence of butyrolactone I for another 24 h. Fewer butyrolactone I-treated oocytes reached MII stage at 36 h compared with controls (5.8% vs. 62.4%, P < 0.01). However, by 44 h, 83.4% of butyrolactone I-treated oocytes reached MII compared with 88.6% of controls. In the third experiment, butyrolactone I-treated oocytes were fertilized and cultured in vitro. No differences (P > 0.05) were found between controls and treated groups in cleavage rate, blastocyst rate, or mean number of cells per blastocyst. Effects of butyrolactone I on mitogen-activated protein kinase activation and localization of microfilaments and active mitochondria were examined by Western blot analysis and laser scanning confocal microscopy, respectively. The results suggested that although butyrolactone I reversibly inhibited germinal vesicle breakdown and mitogen-activated protein kinase activation, it did not affect mitochondrial and microfilament dynamics. Butyrolactone I is a potent inhibitor of nuclear maturation of porcine oocytes, and the inhibition is fully reversible.
4-Butyrolactone↗
Na(+)/Ca(2+) exchanger in porcine oocytes.
The presence of the Na(+)/Ca(2+) exchange mechanism was investigated in porcine oocytes. Immature and in vitro-matured oocytes were loaded with the Ca(2+)-sensitive fluorescent dye fura 2 and changes in the intracellular free Ca(2+) concentration ([Ca(2+)](i)) were monitored after altering the Na(+) concentration gradient across the plasma membrane. Decreasing the extracellular Na(+) concentration induced an increase in [Ca(2+)](i) possibly by a Ca(2+) influx via the Na(+)/Ca(2+) exchanger. A similar Ca(2+) influx could also be triggered after increasing the intracellular Na(+) concentration by incubation in the presence of ouabain (0.4 mM), a Na(+)/K(+)-ATPase inhibitor. The increase in the [Ca(2+)](i) was due to Ca(2+) influx since it was abolished in the absence of extracellular Ca(2+), and the increase was mediated by the Na(+)/Ca(2+) exchanger since it was blocked by the application of amiloride or bepridil, inhibitors of Na(+)/Ca(2+) exchange. Verapamil (50 micro M) and pimozide (50 micro M), inhibitors of L- and T-type voltage-gated Ca(2+) channels, respectively, could not block the Ca(2+) influx. The Ca(2+) entry via the Na(+)/Ca(2+) exchanger could not induce the release of cortical granules and did not stimulate the resumption of meiosis. This was unexpected because Ca(2+) is thought to be a universal trigger for activation. Using antibodies raised against the exchanger, it was demonstrated that the Na(+)/Ca(2+) exchanger was localized predominantly in the plasma membrane. Reverse transcription-polymerase chain reaction revealed that porcine oocytes contain a transcript that shows 98.1% homology to the NACA3 isoform of the porcine Na(+)/Ca(2+) exchanger.
Amiloride↗
Effects of culture medium, serum type, and various concentrations of follicle-stimulating hormone on porcine preantral follicular development and antrum formation in vitro.
Developing a culture system for preantral follicles has important biotechnological implications due to the potential to produce large number of oocytes for embryo production and transfer. As an initial step toward accomplishing this long-term goal, a study was conducted to determine the effects of culture medium, serum type, and different concentrations of FSH on preantral follicular development in vitro. Specific endpoints included follicular growth rate, antrum formation, recovery rate of cumulus cell-oocyte complexes (COCs) from follicles, and oocyte meiotic competence. Compared with the North Carolina State University medium 23 (NCSU23), preantral follicles cultured in TCM199 medium for 4 days grew faster (P < 0.02). However, more follicles cultured in NCSU23 differentiated to form an antrum than in TCM199 (P < 0.01). For this reason, NCSU23 was chosen to investigate the role of FSH and serum type in regulating preantral follicular growth. Compared with the 0 mIU/ml FSH control, addition of 2 mIU/ml FSH to the medium stimulated follicular growth and antrum formation and suppressed apoptosis of granulosa cells (P < 0.05), supporting the essential role of FSH in preantral follicular growth and development. Another experiment compared fetal calf serum (FCS) with prepubertal gilt serum (PGS) and studied different concentrations of FSH in the culture medium (0.5, 1, and 2 mIU/ml). The best follicular growth rate was obtained with 2 mIU/ml compared with 0.5 or 1 mIU/ml FSH. Compared with PGS, FCS supplementation increased the cumulative percentage of antral follicles and COC recovery rate (P < 0.04). None of the oocytes recovered from any of these experiments reached metaphase II stage after maturation in vitro. In summary, culture medium, serum type, and FSH concentration in the medium interacted to affect follicular growth and antrum formation in vitro. These results suggest that a longer term culture of preantral follicles (>4 days) may be needed to produce oocytes capable of undergoing meiosis in vitro.
Animals↗
Cloning pigs as organ donors for humans.
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Alkyl and Aryl Transferases↗