Hee-Tae Cheong

PRODUCTION OF NUCLEAR TRANSFER-DERIVED SWINE THAT EXPRESS THE ENHANCED GREEN FLUORESCENT PROTEIN

The ability to add or delete specific genes in swine will likely provide considerable benefits not just to agriculture but also to medicine, where pigs have potential as models for human disease and as organ donors. Here we have transferred nuclei from a genetically modified fibroblast cell line to porcine oocytes, matured in vitro under defined culture conditions, to create piglets expressing enhanced green fluorescent protein. The nuclear transfer-derived piglets were of normal size, although some mild symptoms of “large offspring syndrome” were evident. These experiments represent a next step towards creating swine with more useful genetic modifications.

Mosaic Gene Expression in Nuclear Transfer-Derived Embryos and the Production of Cloned Transgenic Pigs from Ear-Derived Fibroblasts

Abstract 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.

Developmental Potential of Porcine Nuclear Transfer Embryos Derived from Transgenic Fetal Fibroblasts Infected with the Gene for the Green Fluorescent Protein: Comparison of Different Fusion/Activation Conditions

Abstract The in vitro developmental potential of porcine nuclear transfer (NT) embryos was evaluated. Oocytes were matured for 42–44 h, and metaphase II-oocytes were enucleated. Fetal fibroblasts infected with the enhanced green fluorescent protein (EGFP) gene were serum-starved for 3–5 days. A single cell was injected into the perivitelline space of the enucleated oocytes. The reconstructed oocytes were allocated to different fusion and activation conditions. In experiment 1, two different fusion/activation conditions were compared: two pulses of 1.2 kV/cm for 30 μsec (group A), or one pulse of 1.6 kV/cm for 30 μsec followed in 30 min by one pulse of 1.2 kV/cm for 30 μsec (group B). Parthenogenetic controls were created by using the group A parameter. The fusion rate in group A (mean ± SEM, 68.4% ± 3.9%) was higher (P < 0.05) than in group B (59.4% ± 2.3%). The rates of cleavage (50.1% ± 4.6% to 62.8% ± 5.5%) were not different among control and treatment groups. However, the rate of parthenogenetic control embryos developing to the blastocyst stage (18.1% ± 3.1%) was higher (P < 0.05) than the rate of NT embryos (5.9% ± 1.7% and 4.9% ± 2.5%). In experiment 2, we compared two pulses of 1.2 kV/cm (group C) versus two pulses of 1.3 kV/cm (group D). For two control groups, the same pulses as those given to group C or D, respectively, were supplied. The fusion rate in group D (70.6% ± 4.2%) was higher (P < 0.05) than in group C (58.9% ± 2.7%). The cleavage rates were not different among control and treatment groups (58.1% ± 8.1% to 73.6% ± 6.0%). However, the rate of embryos developing to the blastocyst stage in group D (3.5% ± 1.7%) was lower (P < 0.05) than in controls and group C (11.4% ± 2.0% to 16.4% ± 1.1%). In experiment 3, we examined whether the presence of cytochalasin B (CB) during donor cell injection affects the development of NT embryos. The fusion rate of oocytes in the group with CB (78.4% ± 1.4%) was higher (P < 0.05) than in the group without CB (70.9% ± 0.2%). The cleavage rate of the control group (85.5% ± 4.9%) was higher (P < 0.05) than those of the treatment groups (61.6% ± 2.7% and 63.9% ± 4.3%). However, the rates of embryos developing to the blastocyst stage (8.1% ± 2.5% to 19.1% ± 6.0%) and the mean cell number of blastocysts (29.4 ± 5.2 to 45.7 ± 6.4) were not different among control and treatment groups. Green fluorescence was observed at all stages in NT embryos. These results indicate that two pulses of 1.2 kV/cm are enough for fusion/activation of NT embryos to develop to the blastocyst stage, and that the presence of CB during donor cell injection is not necessary for early development of NT embryos.

Development of reconstituted pig embryos by nuclear transfer of cultured cumulus cells

This study tested the effects of oocyte collection method, activation protocol and maturational age of recipient oocytes on the in vitro development of nuclear transfer embryos reconstructed with cultured cumulus cells. Cumulus cells synchronized in G0/G1 phase by serum-starvation culture were transferred into enucleated oocytes that were collected by aspiration or dissection method and cultured for 33 or 44 h. Reconstituted embryos were activated with a combination of calcium ionophore A23187 or electric pulse and cycloheximide (CHXM), and cultured for 6 days. Oocyte collection methods, activation treatment in the presence of cytochalasin B and activation protocols did not affect the developmental rate of embryos reconstituted with 44-h-matured recipients. However, the development of embryos reconstituted with 33-h-matured recipients was significantly improved (P<0.05) by activation with the combination of electric pulse and CHXM. The present study shows that reconstituted porcine embryos derived from cultured cumulus cells can develop to the blastocyst stage, and that their development can be improved by reconstruction with young oocyte cytoplasts following activation with a combination of electric pulse and CHXM.

Ameliorative effects of melatonin against hydrogen peroxide-induced oxidative stress on boar sperm characteristics and subsequent in vitro embryo development.

Melatonin, the major secretory product of the pineal gland, scavenges a variety of reactive oxygen and nitrogen species in vivo and in vitro, indicating that melatonin is a potent function as an antioxidant. The objective of this study was to investigate the effect of melatonin in the presence or absence of hydrogen peroxide (H(2)O(2)) on sperm characteristics (motility, viability, survival rate, membrane integrity, lipid peroxidation (LPO) and mitochondria activity) and also to examine the developmental rates to the blastocysts stage of porcine oocytes fertilized in vitro with semen treated with or without melatonin (100 nM) in the presence or absence of H(2)O(2) (250 μM). The sperm were treated with melatonin in the presence or absence of H(2)O(2) for 3, 6, 9 and 12 h at 37°C and then analysed for the sperm characteristics. The porcine embryos were produced by in vitro maturation and in vitro fertilization (IVM/IVF) using semen treated with or without melatonin (100 nM) in the presence or absence of H(2)O(2) (250 μM) for 6 h. The semen characteristics, including motility, viability, survival rate, membrane integrity and mitochondria activity, were higher in the groups that were treated with melatonin in comparison to other groups, irrespective of incubation periods. Malondialdehyde levels in control, melatonin and melatonin + H(2)O(2) groups were lower than H(2)O(2) only group. A positive correlation was shown among motility, viability, survival rate and membrane integrity, but a negative correlation was observed between LPO and the other evaluation methods. The developmental rates to blastocysts of IVM/IVF porcine oocytes fertilized by semen treated with melatonin were significantly increased compared with any other groups, with the cell number of blastocysts shown to have a similar trend to the developmental rates. These results demonstrate that melatonin can improve the semen characteristics during in vitro storage and support the developmental ability of IVM/IVF embryos in pigs.

Control of nuclear remodelling and subsequent in vitro development and methylation status of porcine nuclear transfer embryos

We attempted to control the nuclear remodelling of somatic cell nuclear transfer embryos (NTs) and examined their subsequent development and DNA methylation patterns in pigs. Porcine foetal fibroblasts were fused to enucleated oocytes treated with either 5 mM caffeine for 2.5 h or 0.5 mM vanadate for 0.5 h. After activation, NTs were cultured in vitro for 6 days to examine their development. The nuclear remodelling type of the reconstituted embryos was evaluated 1 h after fusion. Methylated DNA of in vitro-fertilised (IVF) embryos and NTs at various developmental stages and of donor cells was detected using a 5-methylcytosine (5-MeC) antibody. Caffeine-treated NTs induced premature chromosome condensation at a high rate (P<0.05), whereas most vanadate-treated NTs formed a pronucleus-like structure. Although cleavage rates to the two-cell stage did not differ among groups, delayed cleavage was observed in the vanadate-treated group. The blastocyst formation rate was significantly reduced by vanadate treatment compared with caffeine-treated and non-treated (control) NT groups (P<0.05). The apoptotic cell index of NT blastocysts was lower in the caffeine-treated group than in other groups (P<0.05). The methylation patterns were similar among NTs, but more hypermethylated DNA was observed at the four-cell stage of control and vanadate-treated NTs when compared with that in IVF embryos (P<0.05). Thus, the nuclear remodelling type controlled by caffeine or vanadate treatment can affect in vitro development and the methylation status of NTs in relation to nuclear reprogramming.

Antioxidative effects of astaxanthin against nitric oxide-induced oxidative stress on cell viability and gene expression in bovine oviduct epithelial cell and the developmental competence of bovine IVM/IVF embryos.

The aim of the present study was to elucidate the fundamental mechanism of bovine oviduct epithelial cell (BOEC) co-culture on developmental capacity of bovine in vitro oocyte maturation/in vitro fertilization (IVM/IVF) embryos. We examined the effects of astaxanthin against nitric oxide-induced oxidative stress on cell viability by MTT assay, lipid peroxidation (LPO) by using thiobarbituric acid (TBA) reaction for malondialdehyde (MDA) and the expression of antioxidant genes (CuZnSOD, MnSOD and Catalase) or apoptosis genes (Bcl-2, Caspase-3 and Bax) by RT-PCR in BOEC. We also evaluated the developmental rates of bovine IVM/IVF embryos co-cultured with BOEC pre-treated with astaxanthin (500 μM) in the presence or absence of sodium nitroprusside (SNP, 1000 μM) for 24 h. Cell viability in BOEC treated with SNP (50-2000 μM) lowered, while astaxanthin addition (50-500 μM) increased it in a dose-dependent manner. Cell viability in astaxanthin plus SNP (1000 μM) gradually recovered according to the increase in astaxanthin additions (100-500 mM). The LPO in astaxanthin group (50-500 μM) gradually decreased in a dose dependent manner and among SNP or astaxanthin plus SNP group, SNP alone and astaxanthin (50 μM) plus SNP shown a significant increase than other groups (p < 0.05). Expression of apoptosis or antioxidant genes was detected by RT-PCR. Bcl-2 and antioxidant genes were detected in astaxanthin or astaxanthin plus SNP group, and Caspase-3 and Bax genes were only found in SNP group. When bovine IVM/IVF embryos were cultured for 6-7 days under co-culture system such as BOEC treated with astaxanthin in the presence or absence of SNP, the developmental ability to blastocysts in 500 μM astaxanthin group was the highest of all groups. These results suggest that astaxanthin has a antioxidative effect on cell viability and LPO of BOEC, and development of bovine IVM/IVF embryos due to the induction of antioxidant genes and suppression of apoptosis genes.

CORRELATION OF DEVELOPMENTAL DIFFERENCES OF NUCLEAR TRANSFER EMBRYOS CELLS TO THE METHYLATION PROFILES OF NUCLEAR TRANSFER DONOR CELLS IN SWINE

Methylation of DNA is the most commonly studied epigenetic mechanism of developmental competence and somatic cell nuclear transfer (SCNT). Previous studies of epigenetics and the SCNT procedures have examined the effects of different culture media on donor cells and reconstructed embryos, and the methylation status of specific genes in the fetus or live offspring. Here we used a microarray based approach to identify the methylation profiles of SCNT donor cells including three clonal porcine fetal fibroblast-like cell sublines and adult somatic cells selected from kidney and mammary tissues. The methylation profiles of the donor cells were then analyzed with respect to their ability to direct development to the blastocyst stage after nuclear transfer. Clonal cell lines A2, A7, and A8 had blastocyst rates of 11.7%a, 16.7%ab, and 20.0%b, respectively (ab P

Effects of various electric fields on the fusion and in vitro development of mouse two-cell embryos.

This study was undertaken to examine the effects of various electric fields such as alternating current (a.c.) voltage, fusion pulse strength, pulse duration, pulse number and electrode geometry on blastomere fusion and developmental rates of mouse two-cell embryos. The a.c. voltages (6 and 12 V/mm) did not affect the fusion and developmental rates. High fusion and developmental rates were obtained when pulse strengths of 1.0 to 2.5 kV/cm, pulse durations of 30 to 90 mu sec and pulse numbers of 1 to 6 were applied using a wire chamber. Comparison of electrode geometries showed that fusion rates were similarly high (93 to 98%) when pulse strengths of 1.0 to 2.5 kV/cm were applied, regardless of the electrode geometry. However, significantly lower developmental rates were observed in a rectangular chamber compared with those in a wire chamber, except when the pulse strength was 1.0 kV/cm. It was further observed that in a rectangular chamber, the developmental rate decreased with increasing pulse strength from 1.0 to 2.0 and 2.5 kV/cm. The results of this study indicate that by using a wire chamber, electric fields can be successfully applied across a relatively wide range of pulse strength, duration and number to provide sufficiently high fusion and subsequent developmental rates. The fusion conditions did, however, vary with chambers of different electrode geometries.

Effects of Activation Timing on the Fertilization Rate and Early Embryo Development in Porcine ROSI Procedure

AbstractPurpose: This study was undertaken to evaluate the optimal exposure time for nuclear envelope breakdown (NEBD) after injection of a round spermatid and to investigate the effect of oocyte activation timing on the fertilization rate and early embryo development in porcine round spermatid injection procedure. Methods: Injected oocytes were fixed at 0.5, 1, 2, 3, and 4 h before activation, and NEBD state was examined. The three groups of oocytes were activated before and after the injection of spermatid using a single direct current pulse (100 V/mm, 50 μs): group 1) at 2 h before the injection (pre), group 2) within 0.5 h after the injection (immediate), and group 3) at 2 h after the injection (post). Activated oocytes were cultured and pronucleus formation and blastocyst development was evaluated at 15–18 h and 7–8 days after the injection, respectively. Results: The proportion of oocytes with NEBD significantly increased in the groups with over 2 h of exposure time (p<0.05) and oocyte with premature chromosome condensation began to appear 3 h after the injection. Normal fertilization and development rate to the blastocyst stage were significantly higher in the post group than in those of the pre or immediate group (p<0.05). Conclusion: The optimal exposure time for NEBD is 2 h after the injection, and activation in 2 h after round spermatid injection improved the normal fertilization and early embryo development rate.