Advances in genome editing technologies, such as the CRISPR/Cas9 system, have facilitated gene manipulation and the generation of pigs resistant to disease, models for studying human diseases and for xenotransplantation. However, double-strand breaks generated by the CRISPR/Cas9 system are preferentially repaired by the non-homologous end joining (NHEJ) pathway compared with high-fidelity homology-directed repair (HDR). All reports of pigs created by zygote injection of the CRISPR/Cas9 system result from NHEJ rather than HDR. The molecule known as RS-1 was found to stimulate RAD51 and thus enhance HDR. Thus, our goals are to understand this pathway by evaluating the response, as well as dosage and temporal effects, of RS-1 on porcine embryo development, and to determine a safe concentration to achieve high HDR rates without affecting embryo development. As RS-1 was added to culture medium from a 7.5mM stock solution in DMSO, we also evaluated whether RS-1 in solution could have affinity and migrate to the mineral oil overlay placed in the media during embryo culture. After IVF, embryos were cultured without RS-1 (control groups) or in the presence of RS-1 at 7.5 and 15 µM. At 7.5 µM, RS-1 improved HDR in rabbits created by zygote injection of the CRISPR/Cas9. Based on that, we designed five experimental groups to evaluate short- (A, 20h) and long-term (B, 144 h) effects of RS-1: two groups in the presence of RS-1 at 7.5 µM (7.5 µM A and 7.5 µM B), two control groups (control A and control B), and a group in the presence of RS-1 at 15 µM A for short-term exposure only. First, embryos in the groups containing (7.5 µM A, 7.5 µM B, and 15 µM A) and without RS-1 (control A and control B) were cultured for 20h without the mineral oil overlay. Then, embryos from control A, 7.5 µM A, and 15 µM A groups were washed and cultured without RS-1 until Day 6 in the presence of the mineral oil overlay. To evaluate long-term effects of RS-1, embryos from control B and 7.5 µM B groups were washed and transferred to medium without or with RS-1 at 7.5 µM, respectively, and cultured until Day 6 with no mineral oil overlay. We report the mean values for each set of data±s.e.m., and the degree of statistical significance in all analyses was defined at P<0.05. We observed that the presence of RS-1 at 15 µM decreased cleavage rates (control A 79.1±2.7%; 7.5 µM A 83.6±1.6%; 15 µM 70.9±4.4%; control B 80.8±1.4%; 7.5 µM B 80.0±2.8%), and long-term exposure to RS-1 decreased development to the blastocyst stage (control A 46.5±3.0%; 7.5 µM A 42.7±3.4%; 15 µM 41.8±4.7%; control B 44.9±3.3%; 7.5 µM B 28.8±4.9%). In addition, RS-1 exposure decreased the total cell number compared with the controls (control A 61.4±2.8; 7.5 µM A 49.2±3.0; 15 µM 48.5±2.3; control B 59.9±3.4; 7.5 µM B 41.5±2.5), however no differences in apoptosis rates were observed between the treatments. Our work will serve as a basis to understand the effects of RS-1 and RAD51 during embryonic development and to improve the applicability of customizable nucleases for the production of genetically modified pigs.

Oocyte competence is one of the key factors determining the proportion of embryos that develop to the blastocyst stage. There is vast evidence that IVM oocytes exhibit less developmental potential than their invivo counterparts. Here, we tested whether supplementation of three cytokines [FGF2 (40 ngmL−1), LIF (20ngmL−1), and IGF1 (20 ngmL−1), termed FLI] improved oocyte maturation, and as a consequence, preimplantation development of bovine embryos invitro. In the first experiment, cumulus-oocyte complexes (COCs) were collected from abattoir-derived ovaries and placed in maturation medium,±FLI, for 18 to 22h. At the end of maturation, COCs were fertilized with sperm from a single Holstein bull known to have high fertility. After an 18- to 20-h fertilization period, putative zygotes were cultured in synthetic oviductal fluid for 8 days. The number of embryos that underwent at least one cellular division (cleavage) and the number of embryos that developed to the blastocyst stage was recorded on Days 3 and 8 after insemination, respectively. The COCs supplemented with FLI (n=554) and controls (n=534) were evaluated across 5 replicates. There was no difference in the cleavage rate (P>0.05) between the two treatments. Development to the blastocyst stage was higher (P=0.05) for FLI-treated COCs (34.9%±1.96) than for the control group (23.9%±1.96). In a second experiment, COCs (n=204) supplemented±FLI were collected and fixed at 6, 12, 18, and 24h after placement in maturation medium. The number of transzonal projections in the COCs was determined by localization of actin filaments by using confocal microscopy. Data were analysed by ANOVA using the GLM procedure of SAS software (version 9.4; SAS Institute Inc.). The model included treatment, time, and the interaction of treatment×time as fixed effects. There was no difference (P>0.05) in the number of transzonal projections at 6h (166.3±8.6 vs. 143.9±8.8) and 12h (107.8±8.4 vs. 128.3±7.7) between FLI-treated and control COCs. However, FLI-treated COCs had fewer (P<0.05) transzonal projections at 18h (67.9±7.8 vs. 100.1±7.7) and 24h (56.4±7.2 vs. 80.6±7.4) compared with the controls. There was a significant treatment×time interaction (P=0.006). In a third experiment, we tested whether the timing of transzonal projection disassociation affected lipid accumulation in the embryo. Blastocysts (n=59) on Day 8 produced from COCs matured±FLI were collected and lipid content was determined by using Nile Red staining. There was no difference (P>0.05) in lipid content between treatments. Thus, supplementation of maturation medium with FLI accelerates the disassociation of transzonal projections in COCs and improves subsequent embryonic development to the blastocyst stage while having no detectable effect on lipid content. Further research is necessary to understand how these cytokines modulate IVM of bovine oocytes. This project was supported by Food for the 21st Century and the Clifton Murphy scholarship fund.

Supplementation of glutamine to porcine embryo culture medium improves blastocyst development, increases leucine consumption, and enhances mitochondrial activity. In cancer cells, glutamine has been implicated in the phosphorylation and activation of mechanistic target of rapamycin (MTOR) to support rapid cellular proliferation. The objective of this study was to determine if phosphorylation of MTOR in porcine blastocysts was dependent upon the concentration of glutamine in the medium and presence of leucine, another activator of MTOR. Presumptive zygotes (n=732 per treatment across 6 replicates) were split into four groups and cultured in 0, 1, 3.75, or 10mM GlutaMAX, an L-glutamine alternative, with or without 0.2mM leucine. On Day 6, percentages of embryos developed to the blastocyst stage were recorded. Blastocyst-stage embryos (n=100 per treatment) were collected for immunoblotting to detect total MTOR and phosphorylated (Ser2448) MTOR (pMTOR) with 3 replicates per target. To assess MTOR and lysosomal colocalization as another indicator of MTOR activation, blastocyst-stage embryos (n=15 per treatment) were fixed, probed with antibodies against MTOR and lysosomal associated membrane protein 1 (LAMP1), and imaged on a confocal microscope. Data were analysed by using the GLM procedure in SAS 9.4 (SAS Institute Inc.), and differences between means were detected by using one-way ANOVA followed by a least significant difference test with P<0.05 declared significant. Compared with the other treatments, culture in 0mM GlutaMAX resulted in decreased blastocyst development with (26.2±4.8%) or without (23.2±3.0%) leucine. Embryos cultured in 3.75mM GlutaMAX had increased development to the blastocyst stage (+Leu: 49.7±4.2%; −Leu: 46.2±2.6%) compared with culture in 1mM (+Leu: 39.5±3.5%; −Leu: 37.8±2.1%). In the presence of leucine, culture in 0mM GlutaMAX resulted in decreased pMTOR abundance compared with 3.75 or 10mM GlutaMAX. In the absence of leucine, embryos cultured in 0mM GlutaMAX had decreased abundance of total MTOR compared with all other groups, and pMTOR abundance was decreased in embryos cultured in 0 or 1mM compared with 3.75 or 10mM GlutaMAX. Furthermore, embryos cultured in 0mM GlutaMAX had decreased colocalization of MTOR and LAMP1 compared with those cultured in 3.75 or 10mM GlutaMAX. Therefore, glutamine supplementation is sufficient to prompt MTOR phosphorylation in porcine embryos. Further analyses are examining the phosphorylation status of MTOR downstream targets and effects of inhibiting enzymes involved in glutaminolysis. Funding was provided by the United States Department of Agriculture, National Institute of Food and Agriculture (2019-67011-29543) and Food for the 21st Century at the University of Missouri.

Recently, our laboratory has identified that supplementation of 3 cytokines, 40ng mL−1 FGF2, 20ng mL−1 LIF, and 20ng mL−1 IGF1 (termed FLI), to porcine oocyte maturation medium leads to an increase in maturation percentage and embryo development to blastocyst. Similarly, addition of FLI to porcine embryo culture at Day 2 after fertilization increased the number of embryos that became blastocyst. The objective of this study was to determine whether the effect of FLI on embryonic development was maintained in the bovine. Two variations of the bovine culture medium, basic SOF and SOF-BE2 (containing 0.4mM trisodium citrate and 2.56mM myo-inositol), were supplemented with FLI at the beginning of culture, and development to the blastocyst stage on Day 8 was determined. Embryos were produced from abattoir-derived oocytes from Bos taurus breeds and fertilized with sperm from a single Holstein bull proven of high fertility under in vitro conditions. All statistical analyses were completed by logistic regression using the Genmod procedure of SAS v. 9.4 (SAS Institute Inc., Cary, NC, USA). In the first experiment, zygotes (oocytes exposed to sperm) were placed in culture in basic SOF with or without supplementation with FLI. After 4 replicates containing 90 control embryos and 93 FLI-treated embryos we found an increase (P<0.05) in blastocyst percentages of 21.1±0.2% for the control compared with 40.9±0.2% for the FLI-treated embryos. In a second experiment, zygotes were cultured in SOF-BE2 with or without supplementation with FLI. A total of 679 zygotes (332 control and 347 FLI-treated zygotes) across 4 replicates were evaluated. Percentage of embryos developing to the blastocyst stage increased (P<0.05) from 33.2±0.07% in the control group to 42.6±0.07% in the FLI-supplemented group. Next, blastocysts derived from the second experiment were vitrified for subsequent transfer to synchronized Angus recipient heifers. Re-expansion 16h after thawing was recorded before transfer. For the control group, 10 out of 24 embryos (41.7%), and 28 out of 41 embryos (68.3%) of the FLI-treated embryos, re-expanded. Three re-expanded blastocysts were then nonsurgically transferred to the uterine horn of each synchronized heifer (N=3 control embryo containing heifers, 9 FLI embryo containing heifers). The number of viable fetuses (presence of a heartbeat) per heifer will be determined via ultrasound around Day 35. Although more vitrification and transfer experiments are being completed, preliminary data suggest that supplementation of FLI during culture might improve of the embryos to freezing. Furthermore, supplementation of FLI improves development to the blastocyst stage in bovine embryos under different conditions. This work was supported in part by funds from the Agriculture and Food Research Initiative Competitive Grant no. 2013-68004-20365 from the United States Department of Agriculture National Institute of Food and Agriculture and National Institutes of Health Grant R01 HD072898, Food for the 21st Century, and the Clifton Murphy Scholarship Fund.

One of the key regulators of gene expression in mammals is DNA methylation. The Tet family (Tet1–3) is suggested to be involved in regulating the level of methylation by hydroxylating a methyl group from 5-methylcytosine to form 5-hydroxymethylcystosine. This hydroxylation alters the 3-dimensional structure of the DNA and results in altered gene expression. Previous studies conducted in the mouse have shown that Tet1 is important for inner cell mass specification by regulating the apparent level of methylation on a specific promoter region in blastocysts and Tet3 is related to the apparent paternal DNA demethylation after fertilization by hydroxylating the paternal genome. The objective of this study was to investigate the expression profile of the Tet family in porcine oocytes and pre-implantation-stage embryos derived from IVF and somatic cell nuclear transfer (SCNT). The RNA was isolated from donor cells, germinal vesicle (GV), MII and 2-cell and blastocyst stage embryos (20 oocytes or embryos per group). Levels of mRNA for each Tet gene were measured by quantitative real-time RT-PCR. The levels of each mRNA transcript were compared to YWHAG, a housekeeping gene that shows a constant level of expression throughout pre-implantation embryo development and normalized to the GV stage. The analysis was repeated with 3 biological replications and 2 experimental replications. Differences in gene expression were compared by ANOVA and P < 0.05 was considered significant. No difference was found in the levels of the Tet family members between GV and MII stage oocytes. Compared with GV stage oocytes, up-regulation of Tet3 at the 2-cell stage was detected in both IVF and SCNT embryos, 4.7 and 6.2 fold, respectively. A dramatic increase in Tet1 was also observed at the blastocyst stage in IVF and SCNT embryos when compared with the GV stage, 65.7 and 79.7 fold increases, respectively. Interestingly, the level of Tet3 was down-regulated in blastocyst embryos at a 25 or more fold decrease compared with GV. The level of Tet2 remained constant throughout embryo development. Embryos (2-cell and blastocyst) compared from IVF and SCNT showed no difference in Tet expression levels. Donor cells had significantly lower levels of Tet2 and Tet3 when compared with GV. Our results indicate that the Tet family shows a dynamic expression profile during porcine pre-implantation embryo development. High expression of Tet3 in 2-cell stage embryos suggests its importance during the post-activation demethylation process. The increase of Tet1 transcript in blastocysts suggests that Tet1 is involved in regulating the type of methylation at the blastocyst stage. These results are consistent with results from previous mouse studies. There was no misregulated expression of the Tet family in SCNT embryos compared with IVF embryos, thus indicating successful reprogramming of the Tet family after SCNT. Lower levels of Tet2 and Tet3 would indicate that Tet1 is important for maintaining type of methylation in donor cells. This is the first report on the profile of the Tet family during porcine pre-implantation embryo development and further studies are needed to clarify their role during this stage.

Skin-derived progenitors (SKP) are capable of generating both neural and mesodermal progeny in vitro: neurons, Schwann cells, adipocytes, osteocytes and chondrocytes, thus exhibiting characteristics similar to embryonic neural crest stem cells. SKP show distinct transcriptional profiles when compared with neurospheres/neural stem cells in the central nervous system (CNS) and skin-derived fibroblasts, indicating a novel type of multipotent stem cell derived from the dermis of the skin. However, it remains unclear whether SKP cells can produce ectoderm and mesoderm lineages or other germ layers in vivo, although oocyte-like structures can be induced from porcine SKP in vitro. Embryonic chimeras are a well-established tool for investigating cell lineage determination and cell potency through normal embryonic development. Thus the purpose of this study was to investigate the in vivo developmental potential of porcine SKP by chimera production. Porcine SKP cells and fibroblasts were isolated from the back skin of Day 35 to 50 GFP transgenic fetuses. Individual cells or clusters of male GFP transgenic SKP and skin-derived GFP-expressing fibroblasts were injected into pre-compact in vitro-fertilized (IVF) embryos, respectively and then transferred into corresponding surrogates 24 h post-injection. Additional injected embryos were cultured in PZM3 medium for another 2 days until the blastocyst stage and subsequently stained with Hoechst 33342. Interestingly, in some of the chimeras the injected SKP cells migrated and dispersed into different locations of the host blastocysts, whereas in others they remained as a cluster of cells within the chimeric blastocysts. In contrast, the fibroblast cells were not observed to spread around the host blastocysts. Two chimeric fetuses were recovered at the middle of gestation and a litter of viable piglets was born. Genomic DNA was extracted from various tissues of chimeric piglets and subjected to PCR amplification. Two chimeric fetuses and 2 out of 6 piglets carried the GFP transgene in SKP-derived chimeras, but GFP was not present in the fibroblast-derived chimeric fetuses (n = 6). Surprisingly, the GFP transgene was present in various tissues of two SKP-derived chimeric piglets, including lung, heart, liver, artery, kidney, brain, skin, muscle, gut, ovary, pancreas and stomach, thus representing the 3 germ layers (ectoderm, mesoderm and endoderm). In addition, SRY was detected in several tissues of the two GFP-positive female chimeric piglets, confirming the chimerism of these piglets. Therefore, it appears that porcine SKP can contribute to various cell types of the 3 germ layers and have a broader developmental potency than previously expected. Alternatively, pre-compact (4-cell and 8-cell stage) embryos may provide a unique environment for reprogramming skin-derived progenitors into a more primitive state by the process of embryonic compaction. This study was funded by NIH National Center for Research Resources (R01RR013438) and Food for the 21st Century at the University of Missouri.

There have been significant improvements in the culture of porcine embryos in vitro; however, it is still suboptimal. Improvements in porcine embryo culture would benefit utilisation of porcine embryos for a variety of purposes. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is known to be expressed in the female reproductive tract and the level of its expression is high between conception and implantation. Previous studies show supplementing GM-CSF in embryo culture promotes embryonic development in human and bovine embryos. The aim of this study was to investigate the effect of GM-CSF on the culture of porcine embryos derived from somatic cell nuclear transfer (SCNT) and IVF. Different concentrations of recombinant porcine GM-CSF (0, 2, 10 ng mL–1) were introduced into Porcine Zygote Medium 3 from Day 1 to 6. Frequencies of cleaved embryos and blastocyst formation were recorded and analysed by using ANOVA following arcsin transformation. Total cell number in blastocysts from each group were counted and compared by using the Student's t-test. Differences at P < 0.05 were considered significant. A total of 563 SCNT embryos from 6 different donor cell lines on 11 different days were produced for the study. Incubation of SCNT embryos with GM-CSF did not affect the frequency of cleaved embryos. Frequencies of cleaved embryos in control (0 ng mL–1), 2 ng mL–1 GM-CSF and 10 ng mL–1 GM-CSF were 64.2%, 68.1% and 65.0%, respectively. Interestingly, both concentrations of GM-CSF significantly increased the frequency of blastocyst formation as compared with the control. In 2 ng mL–1 and 10 ng mL–1 of GM-CSF groups, 30.8% and 32.3% of embryos reached blastocyst respectively, whereas only 22.4% of embryos reached blastocyst in the control group. A significant increase in total cell number in blastocysts was observed when GM-CSF was introduced into embryo culture. An average of 28.8 ± 0.9 cells was recorded in the control group, whereas 31.9 ± 1.1 and 31.8 ± 1.1 were observed in 2 ng mL–1 and 10 ng mL–1 of GM-CSF groups, respectively. Similar effects were observed when GM-CSF was introduced to the culture of IVF embryos. For IVF study, 525 embryos were generated on 10 different days and embryos cultured in the presence of GM-CSF tended to show higher blastocyst formation (P = 0.1). Frequencies of blastocyst per cleaved in the 3 groups were 55.7% (control), 65.7% (2 ng mL–1 GM-CSF) and 66.7% (10 ng mL–1 GM-CSF). In addition, culture of IVF embryos with GM-CSF significantly increased total cell number in Day 6 blastocysts. Total cell number in blastocysts in 2 ng mL–1 GM-CSF (34.2 ± 0.8) and 10 ng mL–1 GM-CSF (34.4 ± 1.2) were significantly higher compared with control (27.3 ± 1.2). Our results indicate that introducing GM-CSF into embryo culture media can increase the quality of blastocyst stage embryos. An increase in the frequency of blastocyst formation and total cell number in blastocysts suggests that GM-CSF can be used to produce better-quality embryos in vitro. Currently, effects of GM-CSF on implantation of SCNT embryos are under investigation. Further studies would elucidate the specific mechanism of GM-CSF on porcine embryos.

Transcriptional deep sequencing analysis by Bauer et al. (2010) revealed a significant increase in expression of the arginine transporter SLC7A1 in in vitro–cultured porcine blastocysts compared with those cultured in vivo and this was corrected through supplemental arginine. This indicates an important role for arginine during porcine embryo development. Arginine is the precursor for nitric oxide (NO) production and previous work in mice and cattle has shown decreased development when embryos were cultured with a nitric oxide synthase (NOS) inhibitor. The NOS activity is inhibited by monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA) that are released during degradation of proteins methylated by protein arginine methyltransferases (PRMT). The enzyme dimethylarginine dimethylaminohydrolase (DDAH) is responsible for degrading MMA and ADMA in the cell. Therefore, the goal of this study was to investigate whether this PRMT-DDAH-NO axis exists in pre-implantation porcine embryos. To this end, expression of PRMT1, PRMT3, PRMT5, DDAH1 and endothelial NOS (NOS3) was analysed at different stages of embryonic development using real-time quantitative RT-PCR. In addition, the effect of supplemental arginine (1.69 mM) on the expression of the aforementioned genes was investigated. Production of NO in porcine embryos was also visualised using 4-amino-5-methylamino-2,7-difluorofluorescein diacetate (DAF-FM-DA). In vitro–fertilized porcine embryos were collected at the 4-cell and blastocyst stages. The RNA was isolated from pools of 18 to 20 embryos and cDNA, was synthesised using Superscript III (Invitrogen, Carlsbad, CA, USA). Real-time PCR analysis was performed and the mean fold change in gene expression from the reference gene YWHAG was analysed by t-test after a log transformation. Expression of PRMT3 and PRMT5 was significantly higher (P < 0.05) in blastocysts versus 4-cell embryos. Expression of PRMT1, however, was higher in 4-cell embryos (P < 0.05). The expression of DDAH1 was detected in 4-cell embryos, but DDAH1 became undetectable by the blastocyst stage. Previous microarray analysis in our laboratory by Whitworth et al. (2005 Biol. Reprod. 72(6), 1437–1451) also revealed a significant up-regulation of DDAH2 expression at the 4-cell stage versus blastocysts. Expression of NOS3 was undetectable in the 4-cell and blastocyst; however, NO was detected in 4-cell and blastocyst stage embryos by using DAF-FM-DA. This suggests that a different NOS may be acting in the porcine embryo. Addition of arginine did not have a significant effect on expression of the analysed genes. These results suggest that PRMT-DDAH regulated NO production may play a role during porcine embryo development. Understanding the PRMT-DDAH-NO axis and its regulation during embryonic development will further our ability to tailor in vitro culture so that it more appropriately mimics that of an in vivo environment. Funding was provided by NIH U42 RR18877.

As the importance of swine models in biomedical research increases, it is essential to develop low-cost, high-throughput systems to cryopreserve swine germplasm for maintenance of these models. However, porcine embryos are exceedingly sensitive to low temperature and successful cryopreservation is generally limited to the use of vitrification in open systems that allow direct contact of the embryos with liquid nitrogen (LN2). This creates a high risk of pathogen transmission. Therefore, cryopreservation of porcine embryos in a “closed” system is of very high importance. In this study, in vitro-produced (IVP) porcine embryos were used to investigate cryosurvival and developmental potential of embryos cryopreserved in a closed system. Optimal centrifugal forces to completely disassociate intracellular lipids from blastomeres were investigated using Day-4 embryos. Cryosurvival of delipidated embryos was investigated by vitrifying the embryos immediately after centrifugation, or after development to blastocysts. In this study, centrifugation for 30 min at 13,000 g was adequate to completely delipidate the embryos; furthermore, these embryos were able to survive cryopreservation at a rate comparable to those centrifuged for only 12 min. When delipidated embryos were vitrified at the blastocyst stage, there was no difference in survival between embryos vitrified using OPS and 0.25 mL straws. Some embryos vitrified by each method developed to term. These experiments demonstrated that porcine embryos can be cryopreserved in a closed system after externalizing their intracellular lipids. This has important implications for banking swine models of human health and disease.

Coagulation Factor IX is a vital protein that plays an important role in the blood coagulation cascade. Hereditary deficiency in the Factor IX gene can result in hemophilia type B, the second most common hemophilia. The milk of transgenic livestock can be an efficient vehicle for producing complex, post-translationally modified blood proteins. The advantages include decreased pathogen risk as well as 100-fold or more production efficiency over blood fractionation and cell culture biotechnology. Although the mammary gland can make many of the complex post-translational modifications necessary for biological function of blood proteins, improvements in propeptide cleavage and decreased proteolytic degradation are desirable. To explore the possibility of producing bioactive human Factor IX protein in pig milk, male and female Landrace fetal fibroblast cells were co-transfected by electroporation with 3 different transgene constructs, Factor IX (FIX), Furin, and SERPINA1. The SERPINA1 construct (containing a Neo selectable marker) was delivered into fetal fibroblasts at a 10-fold-lower molar concentration than the other 2 constructs. Following selection in Geneticin, the presence of all 3 genes was verified by PCR and then cells were used as donors for somatic cell nuclear transfer. Thirteen F0 female piglets from 3 potential different integrations were delivered, and 6 piglets were validated by PCR to be positive for all 3 genes. Among the 6 transgenic pigs, 3 are healthy and able to reach puberty. Milk was collected by induced lactation from 2 gilts. A short murine whey acidic protein promoter-Furin gene was used to limit Furin to the lowest levels needed for pro-FIX processing. Furin was expressed to increase propeptide cleavage efficiency, with the result being complete processing of pro-FIX to FIX at ∼0.3 g L–1 pro-FIX. Total FIX levels were ∼1 g L–1. SERPINA1 was also co-expressed at ∼1 g L–1 or more and this serine protease inhibitor did not seem to inhibit furin processing of the pro-FIX. Fifteen F0 male piglets from 3 potential different integration sites were delivered and all of them were positive for all 3 genes. Four F0 males were chosen to breed with wild-type females, and 5 litters of F1 piglets were born. Of 63 F1 piglets, 22 were tri-transgenic and 3 were di-transgenic (only carrying FIX and Furin). Two F1 females were mated with wild-type males, are confirmed to be pregnant, and will be used to determine the expression level and bioactivity of the Factor IX protein in the milk. Funded by the NIH NCRR (RR018877) and R01 HL078944.

Nuclear transfer efficiency in pigs and other large animal species is low. Previous studies have shown that histone deacetylase inhibitor (Scriptaid) and proteasomal inhibitor (MG132) treatment of somatic cell nuclear transfer (SCNT) pig embryos enhances blastocyst formation and pregnancy. The current experiment was carried out to determine the effects of combined MG132 and Scriptaid treatment on early development of cloned pig embryos reconstituted by SCNT. A total of 328 sow oocytes procured from ART (Madison, WI, USA) were reconstructed using α-1,3-Galactosyltransferase knockout hDAF transgenic pig fetal-derived fibroblast cells. Immediately after electrofusion and activation, SCNT oocytes were treated with 0, 1, or 10 μM MG132 for 2 h and then treated with 500 nM Scriptaid for another 16 h. The SCNT embryos were washed and cultured in Porcine Zygote Medium 3 for 7 days. Percent cleavage was determined on Day 2, and blastocyst formation and cell number were determined on Day 7. The experiment was repreated 8 times. There was no difference (P > 0.05) in percent cleavage (57.9 to 66.7%), or cell number (25.5 to 30.6) among the 3 groups. Interestingly, while there was no difference in the percent blastocyst between the 1 μM and 0 μM MG132 treatment groups, more oocytes from the 1 μM MG132 group developed into blastocysts than in the 10 μM MG132 group (25.1 ± 4.6% v. 12.9 ± 3.3%; P = 0.045). Further research will be conducted to transfer these embryos to surrogate gilts to determine the true developmental competence of these embryos. Supported by the National Institutes of Health National Center for Research Resources (RR018877 and RR013438), and Food for the 21st Century.

The physiological role of cumulus cells (CC) surrounding oocytes is particularly important for normal cytoplasmic maturation of oocytes. However, removal of CC from oocytes is inevitable for some embryo manipulation techniques, such as germinal vesicle (GV) transfer, somatic cell haploidization, and oocyte cryopreservation. The present study was designed to determine an optimal method to culture porcine denuded oocytes (DO). The results indicated CC from cumulus-oocyte complexes at the GV stage (GVCC) or at the metaphase II stage, and mural granulosa cells could not improve the maturation of DO. However, GVCC could enhance the development of matured porcine DO after fertilization; the percentage of blastocysts was increased from 1.1 to 17.2% (P < 0.05), and the relative value of the x-axis and y-axis of spindles was also increased (P < 0.05). Coculture with GVCC had no effect on the distribution of mitochondria and cortical granules. The results contribute to our understanding of the mechanisms by which CC promote oocyte maturation and contribute to optimization of protocols for in vitro maturation of DO.

Somatic cell nuclear transfer (SCNT) in pigs relies primarily on the utilization of fetal-derived fibroblast cells, and the resultant clones tend to exhibit a significant level of phenotypic instability, which may be due to epigenetic reprogramming and/or genomic damage in the donor cells. In addition to the compromised phenotypic stability, production of transgenic clones through SCNT is inefficient, because the restricted lifespan of somatic donor cells in culture can be limiting when the genetic modification requires selection. In contrast, stem cells proliferate rapidly and do not undergo senescence at a high rate, so the selection process can be extended. Since there is no report of an embryonic stem cell line derived in the pig that could contribute to the germ line, we decided to investigate the utility of porcine skin-derived stem cells (SSCs). Porcine SSCs were isolated from the skin on the back of day 35 to 50 Yorkshire fetuses. The SSCs were cultured continually in SSCs medium (DMEM/F12 containing B-27, 20 ng mL–1 of epidermal growth factor, and 40 ng mL–1 of basic fibroblast growth factor) at 37.8°C, 5% CO2, 95% air. The SSCs expressed the neural progenitor marker nestin, as well as genes that are critical for pluripotency, such as Oct4 and Stat3. The SSCs proliferated actively in vitro and retained a normal karyotype after long-term culture. Electron microscopy revealed 2 distinct cell types within the spheres; elongated cells at the sphere periphery had invaginated nuclear envelopes and prominent nucleoli, and these cells displayed few, but large elongated mitochondria with transversal cristae as well as large cisternae of rough endoplasmic reticulum. In contrast, the cells in the center of the spheres were predominantly round-shaped, with a large round nucleus or cuboidal. The SSCs can be genetically modified with long-term positive selection, and 50 μg mL–1 G418 appeared to be an appropriate dose of G418 for selection of the transfected SSCs. Finally, NT embryos reconstructed with SSCs showed high rates of pre- and post-implantation development.The cell number in the blastocyst stage embryos derived from cloning with the SSC was significantly higher than those of the blastocysts derived from IVF (28.5 ± 1.9, 16.8 ± 4.0, respectively, P < 0.05), although there was no significant difference in blastocyst formation rates between these groups (21 to 25%). Three of the animals became pregnant in 4 surrogate gilts which received cloned embryos and reached to term. Two healthy male cloned piglets and 1 healthy female cloned piglet are genetically identical to the SSCs. Funding for this study was provided by the National Institutes of Health.

Although transgenic animals have been successfully cloned, the process is still inefficient. One of the limitations is the use of somatic donor cells that have a limited lifespan. If a genetic modification is made, the selection process must be initiated and completed rapidly or the cells will undergo senescence. Identification of a stem cell that would proliferate rapidly and not undergo senescence would prove to be very valuable. Here we report attempts at cloning by using porcine skin-derived sphere stem cells to determine if they are a suitable donor cell type. Skin-derived stem cells were isolated from fetal skin and express the neural progenitor marker NES, as well as genes that may be critical for pluripotency such as POU5F1 and STAT3. The skin-derived stem cells proliferate rapidly in vitro and retain a normal karyotype after long-term culture. In the present study, skin-derived stem cells were cultured and frozen in liquid nitrogen from passage 1 to passage 8. To investigate the developmental potential of the skin-derived stem cells, we performed nuclear transfer (NT) and compared their preimplantation developmental efficiency to that of the embryos derived from in vitro fertilization (IVF). Cumulus–oocyte complexes (COCs) were aspirated from antral follicles of ovaries from prepubertal gilts. Approximately, groups of 50-70 COCs were matured in vitro in 500 µL TCM-199 per culture well for 40–44 h at 38.5C, in a humidified atmosphere of 5% CO2 in air. The donor cells were thawed and cultured one day before NT; skin-derived stem cells were pipetted vigorously in PBS-EDTA to isolate individual cells. For IVF, cryopreserved ejaculated spermatozoa were thawed and washed and then resuspended with fertilization medium (mTBM). The MII oocytes were co-incubated with sperm for 6 h, and then transferred to PZM3 and cultured. For NT and IVF, respectively, the percent cleavage at 48 h in PZM3 was 64.9 8.2% (169/208) and 62.1 3.1% (94/184) (P > 0.05), the percent blastocysts after 6 days was 21.5 5.8% (53/208) and 25.2 3.4% (46/184) (P > 0.05), and the number of nuclei per blastocyst was 28.5 1.9 (NT, maximum was 58) and 16.8 4.0 (IVF, maximum was 31) (P < 0.05). To determine development post-implantation, some cloned embryos were cultured in PZM3 for 15.5 h and an average of 112 cloned embryos were transferred to the oviducts of four naturally cycling gilts on Day 0–1 of standing estrus. Three of the animals were pregnant: one of them farrowed two male piglets on August 14th, with the other two due on September 8th and 9th. Future studies will involve performing NT and ET on skin-derived stem cells from a higher passage number to determine if they would be suitable for genetic modification prior to NT.

Identification of transcripts produced during bovine embryogenesis is the first step in describing the normal developmental program. To that end, mRNA was isolated from in vitro-matured metaphase II oocytes (MPII), in vitro-produced 2-cell-stage (2-Cell), in vitro-produced precompact morula-stage (PCM), in vitro-produced blastocyst-stage (BL), and in vitro-produced nuclear transfer blastocyst-stage (NTBL) embryos. The mRNA was isolated by using Dynabeads® (Dynal, Inc., Lake Success, NY, USA), and amplified by using the SMART system. PCR products were purified and ligated into pSPORT1 and electroporated into E. coli. Random clones were selected for DNA sequencing. Sequence data were evaluated for quality and clustered by sequence similarity with sequences generated from a larger expressed sequence tag (EST) project (http://genome.rnet.missouri.edu/Bovine/) by using the tlcluster program from the University of Iowa. Sequences over 100 bp in length with average Phred scores of over 20 for the entire sequence were submitted to GenBank (NIH genetic sequence database). Sequences were compared to the bovine TIGR (The Institute for Genomic Research) and human databases to gather annotation. The best comparison is listed below by using the HUGO Gene Nomenclature Committee standards (http://www.gene.ucl.ac.uk/nomenclature/) when possible. The number of unique clusters, i.e. no match in GenBank, was 53, 120, 109, 115, and 135, for MPII, 2-Cell, PCM, BL, and NTBL, respectively. The total number of clusters per tissue ranged from 224 to 992. The percent of clusters (number of clusters per total number of ESTs) per library was 12% (224/1762), 42% (746/1771), 48% (819/1715), 49% (900/1818) and 53% (992/1876) for MPII, 2-Cell, PCM, BL, and NTBL, respectively. Either the quality of the MPII library was lower or the complexity of the MPII mRNA was less than mRNA in the other tissues. Examples of mRNA that were in different abundance are shown in Table 1. Clearly, as in other species, there are significant changes in mRNA abundance during early embryogenesis. Furthermore, NTBL embryos, even though they are morphologically similar to BL, possess a population of mRNA that is distinct from that in BL. Table 1. Comparison of mRNA Abundance During Bovine Embryogenesis This work was funded by the USDA NRI 2003–35205–12812 and Food for the 21st Century.