Bovine Cloning Research Articles

Modification of alternative splicing in bovine somatic cell nuclear transfer embryos using engineered CRISPR-Cas13d.

Animal cloning can be achieved by somatic cell nuclear transfer (SCNT), but the resulting live birth rate is relatively low. We previously improved the efficiency of bovine SCNT by exogenous melatonin treatment or by overexpression of lysine-specific demethylase 4D (KDM4D) and 4E (KDM4E). In this study, we revealed abundant alternative splicing (AS) transitions during fertilization and embryonic genome activation, and demonstrated abnormal AS in bovine SCNT embryos compared with in vitro fertilized embryos. We used the CRISPR-Cas13d RNA-targeting system to target cis-elements of ABI2 and ZNF106 pre-mRNA to modify AS, thus reducing the ratio of abnormal-isoform SCNT embryos by nearly 50% and achieving a high survival rate (11%-19%). These results indicate that this system may provide an efficient method for bovine cloning, while also paving the way for further improvements in the efficiency of SCNT.

IMPROVING THE EFFICIENCY OF BOVINE SOMATIC CELL NUCLEAR TRANSFER PROTOCOL BY ESTABLISHMENT OF CULTURE MEDIUM CHANGING SYSTEM

Bovine cloning is expected as a promising technology for endangered genome conservation and the production of transgenic cloned cows to manufacture human recombinant proteins. However, cloned bovine embryos often resulted in poor development, thus reducing the success of producing cloned offspring. One of the reasons is based on culture medium, which directly relates to the development of cloned bovine embryos. This study aimed to establish culture medium changing system to improve the efficiency of bovine somatic cell nuclear transfer protocol. Our results showed that using the culture medium of Takahashi and First 1992 produced the cloned bovine blastocyst at the highest rate (9.4%, P < 0.05) among different modified synthetic oviduct fluid (mSOF) media. In addition, the rate of embryos developed to the hatching blastocyst was highest in the culture group within transfer embryos to the fresh medium with 5% FBS on day 3 and day 5 (18.5%) compared to the other treatments. Finally, we found that cloned embryos in the group of partial replacement presented significantly higher blastocyst formation rate and quality compared to the group of full replacement (27.6% and 15.6%, respectively, P < 0.05), and it also showed the highest number of cells in blastocyst (about 116 cells). In conclusion, using mSOF and changing the fresh medium supplemented with 5% FBS on day 3 following partial replacement on day 5 improved the preimplantation development of cloned bovine embryos and promoted the blastocyst quality.

Generation of Monogenetic Cattle by Different Techniques of Embryonic Cell and Somatic Cell Cloning – Their Application to Biotechnological, Agricultural, Nutritional, Biomedical and Transgenic Research – A Review

Abstract The development of effective approaches for not only the in vitro maturation (IVM) of heifer/cow oocytes and their extracorporeal fertilization (IVF) but also the non-surgical collection and transfer of bovine embryos has given rise to optimizing comprehensive in vitro embryo production (IVP) technology and improving other assisted reproductive technologies (ART s), such as cattle cloning by embryo bisection, embryonic cell nuclear transfer (ECNT) and somatic cell nuclear transfer (SCNT). The primary goal of the present paper is to demonstrate the progress and achievements in the strategies utilized for embryonic cell cloning and somatic cell cloning in cattle. Moreover, the current article is focused on recognizing and identifying the suitability and reliability of bovine cloning techniques for nutritional biotechnology, agri-food and biopharmaceutical industry, biomedical and transgenic research and for the genetic rescue of endangered or extinct breeds and species of domesticated or wild-living artiodactyl mammals (even-toed ungulates) originating from the family Bovidae.

Factors affecting early embryonic development in cattle: relevance for bovine cloning

Factors affecting early embryonic development in cattle: relevance for bovine cloning

Mitogen-Activated Protein Kinase Activity Is Not Essential for the First Step of Nuclear Reprogramming in Bovine Somatic Cell Nuclear Transfer.

For reprogramming a somatic nucleus during mammalian cloning, metaphase of the second meiotic division (MII) oocytes has been widely used as recipient cytoplasm. High activity of maturation-promoting factor (MPF) and mitogen-activated protein kinase (MAPK) is believed to accelerate the remodeling and/or reprogramming of a somatic nucleus introduced into the ooplasm by somatic cell nuclear transfer. We demonstrated previously that the first step in nuclear reprogramming is not directly regulated by MPF and MAPK because activated oocytes in which MPF activity is diminished and MAPK activity is maintained can develop to the blastocyst stage after receiving an M phase somatic nucleus in bovine cloning. In this study, our aim was to test whether MAPK activity is necessary for the first step in nuclear reprogramming and/or chromatin remodeling (phosphorylation of histone H3 at Ser3, trimethylation of histone H3 at Lys 9, and acetylation of histone H3 at Lys14) in bovine somatic cloning. We found that it was not necessary, and neither was MPF activity.

Melatonin significantly improves the developmental competence of bovine somatic cell nuclear transfer embryos.

Somatic cell nuclear transfer (SCNT) is a promising technology, but its application is hampered by its low efficiency. Hence, the majority of SCNT embryos fail to develop to term. In this study, the antioxidant melatonin reduced apoptosis and reactive oxygen species (ROS) in bovine SCNT embryos. It also increased cell number, inner cell mass (ICM) cell numbers, and the ratio of ICM to total cells while improving the development of bovine SCNT embryos in vitro and in vivo. Gene expression analysis showed that melatonin suppressed the expression of the pro-apoptotic genes p53 and Bax and stimulated the expression of the antioxidant genes SOD1 and Gpx4, the anti-apoptotic gene BCL2L1, and the pluripotency-related gene SOX2 in SCNT blastocysts. We also analyzed the epigenetic modifications in bovine in vitro fertilization, melatonin-treated, and untreated SCNT embryos. The global H3K9ac levels of melatonin-treated SCNT embryos at the four-cell stage were higher than those of the untreated SCNT embryos. We conclude that exogenous melatonin affects the expression of genes related to apoptosis, antioxidant function, and development. Moreover, melatonin reduced apoptosis and ROS in bovine SCNT embryos and enhanced blastocyst quality, thereby ultimately improving bovine cloning efficiency.

41 UPDATING THE ZONA-FREE METHOD FOR MOUSE CLONING USING HM1 EMBRYONIC STEM CELLS

The zona-free method of SCNT designed for bovine and pig cloning (Booth et al. 2001; Vajta et al. 2001; Oback et al. 2003) was successfully used for horse (Galli et al. 2003). Although simple and efficient in farm animals, its application in the mouse met several problems (Ribas et al. 2005, 2006). The aim of our work was to produce cloned mice using HM1 embryonic stem (ES)cells adapting a zona-free method. Seven- to 24-week-old superovulated B6D2F1 female mice were used as oocytes donors. Cumulus cells were removed by 0.3% hyaluronidase and the zona pellucida by 0.5% pronase in KSOM-HEPES (KSOM-H) 1 h later (Ribas et al. 2006) or immediately after hyaluronidase treatment at 37°C. The HM1 ES cells were cultured in KnockOut DMEM supplemented with leukemia inhibitory factor and 15% fetal bovine serum with or without 2i (Ying et al. 2008) and were synchronized at M phase by 3 ng mL–1 nocodazole for 3 h before fusion. Only spherical cells were selected for NT. Metaphase II chromosome spindle complexes were removed by micromanipulation in KSOM-H medium with 5 μg mL–1 cytochalasin B. Lectin-treated enucleated oocytes were attached to the donor cells in KSOM-H with nocodazole and fused by 2 pulses of 1.3 kV cm–1 DC for 30 μs in 0.3 M mannitol medium. Following 10- to 15-min incubation in KSOM-H, the fusion was assessed and repeated if the constructs were nonfused. Cloned embryos were activated in 1 mM SrCl2 in Ca2+-free KSOM medium for 2 to 2.5 or 5 to 6 h and cultured in 20-μL KSOM droplets using the well-of-the-well (WOW) method (Vajta et al. 2000) under mineral oil at 37°C and 5% CO2. Day 4 compacted morulae and blastocysts were surgically transferred into the uterus of Day-2.5 pseudopregnant recipients that were sacrificed on Day 19.5 to examine fetal development. The donor mice age was important for oocyte survival: ~16% of oocytes of 7- to 10-week-old mice lysed before or during fusion in 33% of experiments (n experiments = 15), whereas oocytes of older mice were not sensitive to enzymatic treatment and electric impulses even after 3 fusion rounds (n = 19). The time of pronase treatment did not affect oocyte survival, whereas extending the time between hyaluronidase treatment and enucleation revealed self-activation in ~25% of oocytes. The fusion efficiency of ES cells was significantly lower compared with serum-starved fibroblasts (61%, n = 623 v. 100%, n = 80). The duration of SrCl2 treatment did not affect embryo development (cleavage: 82% v. 84%; Day 4 blastocysts: 49% v. 52%). ES cell culture with 2i increased Day 4 blastocyst development (60.7% v. 50.4%; P = 0.07), and their ability to implant (52.6% v. 38.2%; P = 0.06). Moreover, only NT embryos derived from 2i-ES cells developed to term (8.2%, n = 5; P = 0.08), and produced live fetuses (4.9%, n = 3). In light of these results, the fusion of ES cells remains the critical step in the mouse zona-free protocol.Partially supported by grant Superpig from Regione Lombardia.

Efficiency of a SCNT bovine cloning program: the importance of the placentation time – preliminary data

Efficiency of a SCNT bovine cloning program: the importance of the placentation time – preliminary data

Production of a Healthy Farm-Born Calf by Modified Somatic Cell Nuclear Transfer

The primary intention of somatic cell nuclear transfer in cattle is commercially driven, notably to generate genetically superior animals with desired phenotypic traits as breeding stock. The widespread application of bovine cloning has been hampered by technical difficulties to which recently introduced zona pellucida-free procedures may offer a solution. The most radical approach is so-called ‘handmade cloning’ which does not require micromanipulators because enucleation and nucleus transfer are both performed manually. Handmade cloning is a rapid and efficient technique that requires less expertise and time than traditional nuclear transfer methods and the cost of equipment is significantly less. Here, we report the birth of the first farm-born calf from a modified handmade cloning process, with success achieved at only the second attempt. While caution is warranted, this represents potentially the first viable cloning method for large-scale improvement of cattle breeding.

Somatic Cell Nuclear Transfer Cloning: Practical Applications and Current Legislation

Somatic cloning is emerging as a new biotechnology by which the opportunities arising from the advances in molecular genetics and genome analysis can be implemented in animal breeding. Significant improvements have been made in SCNT protocols in the past years which now allow to embarking on practical applications. The main areas of application of SCNT are: Reproductive cloning, therapeutic cloning and basic research. A great application potential of SCNT based cloning is the production of genetically modified (transgenic) animals. Somatic cell nuclear transfer based transgenic animal production has significant advances over the previously employed microinjection of foreign DNA into pronuclei of zygotes. This cell based transgenesis is compatible with gene targeting and allows both, the addition of a specific gene and the deletion of an endogenous gene. Efficient transgenic animal production provides numerous opportunities for agriculture and biomedicine. Regulatory agencies around the world have agreed that food derived from cloned animals and their offspring is safe and there is no scientific basis for questioning this. Commercial application of somatic cloning within the EU is via the Novel Food regulation EC No. 258/97. Somatic cloning raises novel questions regarding the ethical and moral status of animals and their welfare which has prompted a controversial discussion in Europe which has not yet been resolved.

Aberrant Expression Patterns of Genes Involved in Segregation of Inner Cell Mass and Trophectoderm Lineages in Bovine Embryos Derived from Somatic Cell Nuclear Transfer

High rates of embryonic, fetal, or placental abnormalities have consistently been observed in bovine cloning. Segregation of inner cell mass (ICM) and trophectoderm (TE) lineages in early embryos is an important process for fetal and placental formation. In mouse embryos, differentiation of ICM and TE is regulated by various transcription factors, such as OCT-4, CDX2, and TEAD4, but molecular mechanisms that regulate differentiation in bovine embryos remain unknown. To clarify gene transcripts involved in segregation of ICM and TE lineages in bovine embryos, we examined the relative abundances of OCT-4, CDX2, TEAD4, GATA3, NANOG, and FGF4 transcripts in blastocyst embryos derived from in vitro fertilization (IVF). Furthermore, transcript levels of such genes in bovine embryos derived from somatic cell nuclear transfer (NT-SC) and in vivo (Vivo) were also compared. OCT-4, NANOG, and FGF4 transcript levels in IVF embryos were significantly higher in ICM than in TE. In contrast, the CDX2 transcript level was lower in ICM than in TE. In NT-SC embryos at the blastocyst stage, transcript levels of all genes except CDX2 were lower than that in Vivo embryos. In the elongated stage, expression levels of the six genes did not differ between NT-SC and Vivo embryos. We observed aberrant expression patterns of various genes involved in segregation of ICM and TE lineages in bovine NT-SC embryos. These results raise the possibility that abnormalities in the cloned fetus and placenta are related to the aberrant expression of genes involved in segregation and differentiation process in the early developmental stage.

Efficiency of AsynchronouslyIn Vitro-Matured Oocytes as Recipients for Nuclear Transfer and of Blind Enucleation in Zona-Free Bovine Cloning

We studied the dynamics of in vitro maturation of bovine oocytes, the efficiency of asynchronously matured oocytes as recipients for the generation of embryos produced by nuclear transfer, and the potential for using blind enucleation of zona-free bovine oocytes in bovine cloning. At 15 h after the initiation of maturation (hpm), oocytes were freed from both cumulus cells and the zona pellucida, and the dynamics of oocyte maturation were monitored every 30 min through the criterion of extrusion of the first polar body (PB1). More than 41% of bovine oocytes had extruded PB1 by 16.5 hpm, and were designated as representing a group of rapidly maturing oocytes. A second group, comprising about 25% of all oocytes, had extruded PB1 by 18.5-20.0 hpm. Examination of Hoechst 33342-stained samples demonstrated that PB1 on the surfaces of zona-free bovine oocytes were always located near the maternal chromosomes. Zona-free oocytes were enucleated by removing PB1 and about 3% of the adjacent oocyte cytoplasm without chromatin staining. Successful enucleation of zona-free bovine oocytes was achieved in 96.9% of cases. The rate of development to the blastocyst stage was significantly greater in embryos reconstructed from rapidly maturing oocytes (47.8%) than with oocytes maturing at 18.0-20.0 hpm (33.3%). Overall, two large groups of bovine oocytes could be distinguished during in vitro maturation by the time required to reach the second stage of metaphase. Bovine embryos reconstructed from rapidly maturing enucleated oocytes had a significantly greater rate of development to the blastocyst stage than did embryos derived from later-maturing oocytes. We conclude that blind enucleation is a simple and efficient method for preparing cytoplasts in zona-free bovine cloning.

48 TREATMENT OF CLONED BOVINE EMBRYOS WITH HISTONE DEACETYLASE INHIBITORS INCREASES IN VITRO DEVELOPMENT BUT NOT IN VIVO CLONING EFFICIENCY

Previous studies in the mouse have shown treatment of somatic cell nuclear transfer (SCNT) embryos with histone deacetylase inhibitors (HDACi) to significantly increase cloning efficiency (Kishigami S et al. 2006 BBRC 340, 183–189; van Thuan N 2007 Asian Reproductive Biology Society 4, 9 abst). Increasing histone acetylation may open donor chromatin allowing better access for oocyte cytoplasmic factors to facilitate reprogramming. Here, we determined the effect of two HDACi, Trichostatin A (TSA), and scriptaid (Sigma-Aldrich, Castle Hill, NSW, Australia), on bovine cloning efficiency. Zona-free SCNT was performed with serum starved fibroblasts fused to enucleated MII-arrested IVM oocytes. After 4 h, reconstructs were activated with 5 μm ionomycin and 2 mm 6-dimethylaminopurine (DMAP) and cultured individually in 5 μL drops of AgResearch synthetic oviduct fluid (SOF) medium. Treatment with HDACi commenced concomitant with the 4 h DMAP incubation and continued in SOF for the remainder of the treatment period; totalling either 18 or 48 h post activation (hpa). TSA concentrations examined were: 0, 5, 50, and 500 nm, with all treatments containing 0.5% DMSO (n = 1121). Following TSA treatment, increased histone (H) acetylation at lysine (K) of H4K5 was confirmed by semi-quantitative immunofluorescence at the eight-cell stage. Scriptaid concentrations examined were: 0, 5, 50, 250, and 1000 nm, with all treatments containing 0.5% DMSO during DMAP and 0.1% DMSO during IVC (n = 1059). In vitro development on Day 7 was expressed in terms of transferable quality embryos as a percentage of reconstructs cultured. Data were analyzed using a generalized linear model with binomial variation and logit link. Embryos from selected treatments were transferred singularly to recipient cows on Day 7 with pregnancy data analyzed using Fisher’s exact test. Day 7 in vitro development was significantly greater with 5 nm TSA treatment for 18 hpa compared to controls (47.1% v. 34.5%; P < 0.02). Treatment of embryos with TSA for 48 hpa had no effect at any concentration tested. In contrast, scriptaid treatment for 18 hpa had no effect in vitro, while exposure for 48 hpa at 1000 nm significantly increased the development of transferable quality embryos compared to 0 nm (44.0% v. 32.4%; P < 0.005). There was no significant difference in embryo survival rates at D150 of gestation between embryos treated with 0 or 5 nm TSA for 18 hpa (8/48 v. 10/48; 16.7% v. 20.8%). However, in vivo development at Day 150 of gestation following treatment of embryos with 1000 nm scriptaid for 48 hpa was significantly lower compared to controls (1/37 v. 6/31; 2.7% v. 19.4%; P < 0.05). Contrary to the mouse, TSA or scriptaid treatment as used in this study did not increase cloning efficiency in cattle. The use of various HDACi either alone or in combination with DNA demethylating agents may still prove beneficial for reprogramming following nuclear transfer. Supported by FRST C10X0303.

Improving the stages of bovine cloning technology with the use of oocytes without the zona pellucida

Bovine embryos are reconstructed with the use of fetal fibroblasts as donors of nuclei and enucleated oocytes without the zona pellucida as recipients. The stages of electrofusion and in vitro culturing of embryos without the zone pellucida up to the blastocyst stage is perfected.

Aberrant DNA methylation in 5′ regions of DNA methyltransferase genes in aborted bovine clones

High rate of abortion and developmental abnormalities is thought to be closely associated with inefficient epigenetic reprogramming of the transplanted nuclei during bovine cloning. It is known that one of the important mechanisms for epigenetic reprogramming is DNA methylation. DNA methylation is established and maintained by DNA methyltransferases (DNMTs), therefore, it is postulated that the inefficient epigenetic reprogramming of transplanted nuclei may be due to abnormal expression of DNMTs. Since DNA methylation can strongly inhibit gene expression, aberrant DNA methylation of DNMT genes may disturb gene expression. But presently, it is not clear whether the methylation abnormality of DNMT genes is related to developmental failure of somatic cell nuclear transfer embryos. In our study, we analyzed methylation patterns of the 5' regions of four DNMT genes including Dnmt3a, Dnmt3b, Dnmt1 and Dnmt2 in four aborted bovine clones. Using bisulfite sequencing method, we found that 3 out of 4 aborted bovine clones (AF1, AF2 and AF3) showed either hypermethylation or hypomethylation in the 5' regions of Dnmt3a and Dnmt3b, indicating that Dnmt3a and Dnmt3b genes are not properly reprogrammed. However, the individual AF4 exhibited similar methylation level and pattern to age-matched in vitro fertilized (IVF) fetuses. Besides, we found that the 5' regions of Dnmt1 and Dnmt2 were nearly completely unmethylated in all normal adults, IVF fetuses, sperm and aborted clones. Together, our results suggest that the aberrant methylation of Dnmt3a and Dnmt3b 5' regions is probably associated with the high abortion of bovine clones.

Aberrant DNA methylation imprints in aborted bovine clones

Genomic imprinting plays a very important role during development and its abnormality may heavily undermine the developmental potential of bovine embryos. Because of limited resources of the cow genome, bovine genomic imprinting, both in normal development and in somatic cell nuclear transfer (SCNT) cloning, is not well documented. DNA methylation is thought to be a major factor for the establishment of genomic imprinting. In our study, we determined the methylation status of differential methylated regions (DMRs) of four imprinted genes in four spontaneously aborted SCNT-cloned fetuses (AF). Firstly, abnormal methylation imprints were observed in each individual to different extents. In particular, Peg3 and MAOA were either seriously demethylated or showed aberrant methylation patterns in four aborted clones we tested, but Xist and Peg10 exhibited relatively better maintained methylation status in AF1 and AF4. Secondly, two aborted fetuses, AF2 and AF3 exhibited severe aberrant methylation imprints of four imprinted genes. Finally, MAOA showed strong heterogeneous methylation patterns of its DMR in normal somatic adult tissue, but largely variable methylation levels and relatively homogeneous methylation patterns in aborted cloned fetuses. Our data indicate that the aborted cloned fetuses exhibited abnormal methylation imprints, to different extent, in aborted clones, which partially account for the higher abortion and developmental abnormalities during bovine cloning.

Optimized combined electrical–chemical parthenogenetic activation for in vitro matured bovine oocytes

Sperm-mediated oocyte activation is a complex procedure, both in steps and duration, not yet been completely mimicked during in vitro studies, e.g., parthenogenesis or somatic cell nuclear transfer. Furthermore, parthenogenetic studies have been recognized as a suitable model for studying activation efficiency for nuclear transfer cloning. This study, therefore, was conducted to develop an optimized artificial activation method, based on bovine cloning. In vitro matured bovine oocytes were initially exposed to electrical pulse, used for cell fusion during cloning, and then treated with 15 temporal sequential combinations of 3 chemical activators [calcium ionophore (CI), strontium (SR) and ethanol (ET)], followed by exposure to a protein kinase inhibitor or used for in vitro fertilization as control group. Treated and naturally fertilized oocytes were further cultured for up to 8 days. Embryo development was scored daily and blastocyst cell counting was carried out using differential staining at day 8 of culture. Among 15 temporal sequential combinations of three chemical activators, the best cleavage rates were associated with double (SR–CI, 84.4%), triple (CI–SR–ET, 79.4%) and single (CI, 73.7%) compounds, respectively, which were not significantly different with each other and with in vitro fertilized (IVF) (85.5%). The highest blastocyst rates were gained with ET–SR (24.5%), SR–CI–ET (20.4%) and CI (24.5%) accordingly which were not significantly different with each other but significantly lower than IVF (47%). Embryo cell counting further confirmed reasonably better quality of blastocysts produced using double, triple and single compounds. Although most of the sequential artificial activation compounds induced high cleavage rate, close to IVF, but this did not assure comparable further embryo development to the blastocyst stage. Nevertheless, the results suggest exposure of in vitro matured bovine oocytes to electrical pulse, followed by exposure to CI–6-dimethylaminopurine (6-DMAP) or ET–SR–6-DMAP could be regarded as the optimal artificial activation protocol for in vitro development of parthenogenic bovine oocytes or as a step for activation protocol in cloning procedure.

Bovine Oocytes with the Potential to Reprogram Somatic Cell Nuclei Have a Unique 23-kDa Protein, Phosphorylated Transcriptionally Controlled Tumor Protein (TCTP)

Despite the long-held assumption that reprogramming factors are present in mammalian oocytes at the second metaphase stage, the molecular nature of these factors is not known. Here, we demonstrated that oocytes with the potential to reprogram somatic cell nuclei have a unique 23-kDa protein, phosphorylated transcriptionally controlled tumor protein (TCTP). Injection of TCTP double-stranded RNA into germinal vesicle oocytes decreased the potential of nuclear-transferred (NT) oocytes, but not in vitro fertilized oocytes, to develop into blastocysts. Phosphorylated TCTP is considered to facilitate the first step of somatic cell reprogramming. After transfer of blastocysts that developed from NT oocytes fused with cumulus cells in which phosphorylated TCTP peptide was previously incorporated, the recipient pregnancy rate (47%) increased and the abortion rate (13%) decreased. Moreover, all seven cloned calves survived for at least 1 month after parturition, and had no morphologic abnormalities. The present study demonstrated that pretreatment of donor cells with phosphorylated TCTP peptide has a beneficial effect on the potential of bovine somatic cell nuclei to develop into normal cloned calves. Before widespread application of TCTP for bovine cloning, however, a large-scale embryo transfer study using different donor cell lines of various origins is necessary.

97 CLONED CALF PRODUCTION BY AGGREGATION OF SOMATIC CELL NUCLEAR-TRANSFERRED EMBRYOS

A low pregnancy rate and high frequency of abortion are observed in bovine somatic cell cloning. It is suggested that one of the factors is the low cell number of nuclear-transferred (NT) embryos at the blastocyst stage compared with that of in vivo-derived embryos. We reported that aggregation of bovine NT embryos can develop to blastocysts with cell numbers that are equivalent to in vivo-derived embryos (Akagi et al. 2005 Reprod. Fertil. Dev. 17, 162 abst). In this study, we examined the in vivo development of aggregates of NT embryos after embryo transfer (ET). Following culture in serum-starved medium for 5 to 7 days, fibroblast cells were used as donor cells for NT. NT was performed as previously described (Akagi et al. 2003 Mol. Reprod. Dev. 66, 264–272). Eight-cell stage embryos on Day 2 or 16- to 32-cell stage embryos on Day 4 were used for embryo aggregation after removal of the zona pellucida. Three NT embryos were placed into the depression in a drop of TCM-199 with 50 µg mL−1 phytohemagglutinin for 20 min, and NT aggregates were then moved into the depression in a drop of IVD-101 (Akagi et al. 2005). On Day 7 after NT, ET was performed nonsurgically. Results are summarized in Table 1. Pregnancy rates obtained with the aggregated NT embryos tended to be high compared with the single NT embryos. All pregnant cows in the single NT embryos and 6 of 8 pregnant cows in the aggregated NT embryos were aborted within 90 days. One cow carrying an aggregate of 3 Day 2 NT embryos went to term, and one carrying an aggregate of 3 Day 4 NT embryos is still pregnant at more than 7 months. These results demonstrate that aggregates of 3 NT embryos can develop to term in cattle. Table 1.In vivo development of aggregated NT embryos

82 EFFECT OF DONOR CELL TRANSFECTION EVENTS ON EMBRYO AND FETAL SURVIVAL IN CLONING

In a large-scale bovine cloning program intended to obtain transgenic animals, it is important to maximize the number of calves produced. The present experiment was designed to test the hypothesis that different transfection events of the same somatic cell line can affect embryo and/or fetal survival. A fetal cell line was established from a 75-day-old Jersey female fetus. It was used as control and was also transfected 3 times with the same protocol. They were named Transfection 1, 2, and 3. Genetically modified cells were produced and isolated after selection with geneticin for 10–15 days following liposome transfection with a DNA construct containing a selectable neomycin resistance gene. Oocytes were aspirated from slaughterhouse ovaries and matured in TCM-199 + 5% FCS at 39°C for 24 h. Matured oocytes were denuded by vortexing for 3 min in TL-HEPES with 1 mg mL−1 bovine testis hyaluronidase. Metaphases were assessed, and oocytes were enucleated by visualization with Hoechst 33342 (5 µg mL−1) under UV light (<6 s). Donor cells from different treatments were used for nuclear transfer at G0/G1 cell cycle stages and were fused to enucleated oocytes by an electrical pulse. After 3 h, activation was induced by incubation in TL-HEPES with 5 µM ionomycin for 4 min and 2 mM 6-DMAP for 3 h. The oocytes were then washed with TL-HEPES and cultured in SOF medium with an atmosphere of 5% CO2 + 5% O2 + 90% N2. Development to blastocysts (Days 7 to 9) was recorded. Two blastocysts were transferred nonsurgically per recipient cow, and pregnancies at 30 days were determined by ultrasonography. All data were analyzed by chi-square test. In vitro development to blastocysts was similar in all treatment groups. One birth was obtained from the control. Four and 7 births were obtained from Transfections 1 and 3, respectively. Although Transfection 2 had good in vitro development, this treatment did not produce any pregnancy. This fact demonstrated that the transfection event provides an additional source of variability in obtaining live transgenic animals. Our results pointed out the necessity to monitor fetal survival by ultrasonography in order to detect as soon as possible any deficiencies in development introduced by transfection. Table 1.Effect of different transfection events of same line on embryo and fetal survival