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Growth differentiation factor 9 (GDF9) is a secreted protein belonging to the transforming growth factor beta superfamily and has been well characterized for its role during folliculogenesis in the ovary. Although previous studies in mice and sheep have shown that mutations in GDF9 disrupt follicular progression, the exact role of GDF9 in pigs has yet to be elucidated. The objective of this study was to understand the role of GDF9 in ovarian function by rapidly generating GDF9 knockout (GDF9−/−) pigs by using the CRISPR/Cas9 system. Three single-guide RNAs designed to disrupt porcine GDF9 were injected with Cas9 mRNA into zygotes, and blastocyst-stage embryos were transferred into surrogates. One pregnancy was sacrificed on day 100 of gestation to investigate the role of GDF9 during oogenesis. Four female fetuses were recovered with one predicted to be GDF9−/− and the others with in-frame mutations. All four had fully formed oocytes within primordial follicles, confirming that knockout of GDF9 does not disrupt oogenesis. Four GDF9 mutant gilts were generated and were grown past puberty. One gilt was predicted to completely lack functional GDF9 (GDF9−/−), and the gilt never demonstrated standing estrus and had a severely underdeveloped reproductive tract with large ovarian cysts. Further examination revealed that the follicles from the GDF9−/− gilt did not progress past preantral stages, and the uterine vasculature was less extensive than the control pigs. By using the CRISPR/Cas9 system, we demonstrated that GDF9 is a critical growth factor for proper ovarian development and function in pigs.

Enzymes of the ten-eleven translocation family are considered to play an important role in the regulation of DNA methylation patterns by converting 5-methylcytosine to 5-hydroxymethylcytosine. Known as a maternal transcript enriched in mature oocytes, ten-eleven translocation-3 (TET3) has been suggested to initiate DNA demethylation of the paternal genome in zygotes. Previous studies in mouse cells indicate that the N-terminal CXXC domain of TET3 is important in catalyzing the oxidation of 5-methylcytosine through its potential DNA binding ability; however, it is not clear whether the DNA binding capacity of CXXC domain is required for the 5-hydroxymethylcytosine conversion in mammalian embryos. Here, we identified TET3 isoforms in porcine oocytes and investigated the role of the oocyte specific TET3 isoform (pTET3L) in controlling postfertilization demethylation in porcine embryos. The pTET3L possessed sequences representing a known DNA binding domain, the CXXC, and injection of the TET3 CXXC fused with GFP into mature porcine oocytes resulted in exclusive localization of the GFP-CXXC in the pronuclei. The CXXC overexpression reduced the 5-methylcytosine level in zygotes and enhanced the DNA demethylation of the NANOG promoter in 2-cell stage embryos. Furthermore, there was an increase in the transcript abundance of NANOG and ESRRB in blastocysts developed from GFP-CXXC injected oocytes. Targeted knockdown of pTET3L resulted in the downregulation of pluripotency genes in subsequently developed blastocysts. The findings indicate that the CXXC domain of TET3 serves as a critical component for the postfertilization demethylation of porcine embryos and coordinates proper expression of pluripotency related genes in blastocysts.

New therapies to treat pancreatic cancer are direly needed. However, efficacious interventions lack a strong preclinical model that can recapitulate patients’ anatomy and physiology. Likewise, the availability of human primary malignant tissue for ex vivo studies is limited. These are significant limitations in the biomedical device field. We have developed RAG2/IL2RG deficient pigs using CRISPR/Cas9 as a large animal model with the novel application of cancer xenograft studies of human pancreatic adenocarcinoma. In this proof-of-concept study, these pigs were successfully generated using on-demand genetic modifications in embryos, circumventing the need for breeding and husbandry. Human Panc01 cells injected subcutaneously into the ears of RAG2/IL2RG deficient pigs demonstrated 100% engraftment with growth rates similar to those typically observed in mouse models. Histopathology revealed no immune cell infiltration and tumor morphology was highly consistent with the mouse models. The electrical properties and response to irreversible electroporation of the tumor tissue were found to be similar to excised human pancreatic cancer tumors. The ample tumor tissue produced enabled improved accuracy and modeling of the electrical properties of tumor tissue. Together, this suggests that this model will be useful and capable of bridging the gap of translating therapies from the bench to clinical application.

SummaryThe worldwide production of in vitro-produced embryos in livestock species continues to grow. The current gold standard for selecting quality oocytes and embryos is morphologic assessment, yet this method is subjective and varies based on experience. There is a need for a non-invasive, objective method of selecting viable oocytes and embryos. The aim of this study was to determine if ooplasm area, diameter including zona pellucida (ZP), and ZP thickness of artificially activated oocytes and in vitro fertilized (IVF) zygotes are indicative of development success in vitro and correlated with embryo quality, as assessed by total blastomere number. Diameter affected the probability of development to the blastocyst stage in activated oocytes on day 7 (P < 0.01) and day 8 (P < 0.001), and had a tendency to affect IVF zygotes on day 8 (P = 0.08). Zona pellucida thickness affected the probability of development on day 7 (P < 0.01) and day 8 (P < 0.001) in activated oocytes, and day 8 for IVF zygotes (P < 0.05). An interaction between ZP thickness and diameter was observed on days 7 and 8 (P < 0.05) in IVF zygotes. Area did not significantly affect the probability of development, but was positively correlated with blastomere number on day 8 for IVF zygotes (P = 0.01, conditional R2 = 0.09). Physical parameters of bovine zygotes have the potential for use as a non-invasive, objective selection method. Upon further development, methods used in this study could be integrated into embryo production systems to improve IVF success.

Direct injection of genome editing tools such as CRISPR/Cas9 system into developing embryos has been widely used to generate genetically engineered pigs. The approach allows us to produce pigs carrying targeted modifications at high efficiency without having to apply somatic cell nuclear transfer. However, the targeted modifications during embryogenesis often result in mosaicism, which causes issues in phenotyping founder animals and establishing a group of pigs carrying intended modifications. This study was aimed to establish a genomic PCR and sequencing system of a single blastomere in the four-cell embryos to detect potential mosaicism. We performed genomic PCR in four individual blastomeres from four-cell embryos. We successfully amplified target genomic region from single blastomeres of 4-cell stage embryo by PCR. Sanger sequencing of the PCR amplicons obtained from the blastomeres suggested that PCR-based genotyping of single blastomere was a feasible method to determine mutation type generated by genome editing technology such as CRISPR/Cas9 in early stage embryos. In conclusion, we successfully genotyped single blastomeres in a single 4-cell stage embryo to detect potential mosaicism in porcine embryos. Our approach offers a simple platform that can be used to screen the prevalence of mosaicism from designed CRISPR/Cas9 systems.

Immunocompromised mice are commonly utilized to study pancreatic cancer and other malignancies. The ability to xenograft tumors in either subcutaneous or orthotopic locations provides a robust model to study diverse biological features of human malignancies. However, there is a dire need for large animal models that better recapitulate human anatomy in terms of size and physiology. These models will be critical for biomedical device development, surgical optimization, and drug discovery. Here, we describe the generation and application of immunocompromised pigs lacking RAG2 and IL2RG as a novel model for human xenograft studies. These SCID-like pigs closely resemble NOD scid gamma mice and are receptive to human tumor tissue, cell lines, and organoid xenografts. However, due to their immunocompromised nature, these immunocompromised animals require housing and maintenance under germfree conditions. In this protocol, we describe the use of these pigs in a subcutaneous tumor injection study with human PANC1 cells. The tumors demonstrate a steady, linear growth curve, reaching 1.0 cm within 30 days post injection. The model described here is focused on subcutaneous injections behind the ear. However, it is readily adaptable for other locations and additional human cell types.

Oocyte activation occurs at the time of fertilization and is a series of cellular events initiated by intracellular Ca2+ increases. Consequently, oocytes are alleviated from their arrested state in meiotic metaphase II (MII), allowing for the completion of meiosis. Oocyte activation is also an essential step for somatic cell nuclear transfer and an important tool to overcome clinical infertility. Traditional artificial activation methods aim to mimic the intracellular Ca2+ changes which occur during fertilization. Recent studies emphasize the importance of cytoplasmic Zn2+ on oocyte maturation and the completion of meiosis, thus suggesting artificial oocyte activation approaches that are centered around the concentration of available Zn2+in oocytes. Depletion of intracellular Zn2+ in oocytes with heavy metal chelators leads to successful oocyte activation in the absence of cellular Ca2+ changes, indicating that successful oocyte activation does not always depends on intracellular Ca2+ increases. Current findings lead to new approaches to artificially activate mammalian oocytes by reducing available Zn2+ contents, and the approaches improve the outcome of oocyte activation when combined with existing Ca2+-based oocyte activation methods. Here, we review the important role of Ca2+ and Zn2+ in mammalian oocyte activation and development of novel oocyte activation approaches based on Zn2+ availability.

Artificial oocyte activation is an essential step in somatic cell nuclear transfer (SCNT) and can enhance viability of embryos as a form of assisted reproductive technology (ART) in clinics. Most artificial activation methods have been developed to increase cytosolic calcium (Ca2+) level in oocytes. Interestingly, recent studies have demonstrated that mammalian oocytes can be activated using N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), a Zn2+ chelator. Although effective, TPEN is also known to induce apoptosis and shows poor selectivity between free Zn2+ and protein-bound Zn2+. The aim of this study was to identify different Zn2+ chelators that can activate pig oocytes. Among five Zn2+ chelators examined, 1,10-phenanthroline (Phen), and tris(2-pyridylmethyl)amine (TPA) successfully activated pig oocytes. The level of available Zn2+ was reduced without any increase in Ca2+ in oocytes incubated with Phen or TPA, indicating that the oocyte activation occurred independently of Ca2+ signal. When various concentrations (100–500 μM) and incubation durations (10–120 min) of Phen and TPA were used to activate pig oocytes, 500 μM for 60 min and 100 μM for 60 min of Phen and TPA treatments, respectively, were found to be most effective in supporting embryo development. The frequency of blastocyst formation after the treatments was higher than 40% at day 7. When oocytes were incubated with TPEN, Phen, or TPA under their optimal treatment conditions, there was no significant difference in the frequencies of day 7 blastocyst formation among the three treatments. However, day 5 blastocyst formation was observed from the Phen- and TPA-treated oocytes, whereas no blastocyst was formed at day 5 in the TPEN-treated oocytes. The average total cell number in day 7 blastocysts was higher in the Phen treatment group than in the TPEN treatment (P

Colony-stimulating factor 2 (CSF2) functions in the reproductive tract to modulate the function of the preimplantation embryo. The β subunit of the CSF2 receptor (CSF2RB) is not expressed in the embryo, and signal transduction is therefore different than for myeloid cells where the receptor is composed of α (CSF2RA) and β subunits. Here, we produced embryos in which exons 5 and 6 of CSF2RA were disrupted using the CRISPR/Cas 9 system to test whether CSF2RA signaling was essential for actions of CSF2 in the bovine embryo. Wild-type and CSF2RA knockout embryos were treated with 10 ng/mL CSF2 or vehicle at day 5 of development. Blastocysts were harvested at day 8 to determine transcript abundance of 90 genes by real-time polymerase chain reaction (PCR). Responses in female blastocysts were examined separately from male blastocysts because actions of CSF2 are sex-dependent. For wild-type embryos, CSF2 altered expression of 10 genes in females and 20 in males. Only three genes were affected by CSF2 in a similar manner for both sexes. Disruption of CSF2RA prevented the effect of CSF2 on expression for 9 of 10 CSF2-regulated genes in females and 19 of 20 genes in males. The results confirm the importance of CSF2RA for regulation of gene expression by CSF2 in the blastocyst.Summary sentenceDisruption of CSF2RA blocked most CSF2-induced changes in gene expression in the blastocyst, indicating that, despite the absence of the β subunit of the receptor, the preimplantation embryo uses CSF2RA for signal transduction.

Historically, genetic engineering in livestock proved to be challenging. Without stable embryonic stem cell lines to utilize, somatic cell nuclear transfer (SCNT) had to be employed to produce many of the genetically engineered (GE) livestock models. Through the genetic engineering of somatic cells followed by SCNT, GE livestock models could be generated carrying site-specific modifications. Although successful, only a few GE livestock models were generated because of low efficiency and associated birth defects. Recently, there have been major strides in the development of genome editing tools: Zinc-Finger Nucleases (ZFNs), Transcription activator-like effector nucleases (TALENS), and Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated 9 (Cas9) system. These tools rely on the generation of a double strand DNA break, followed by one of two repair pathways: non-homologous end joining (NHEJ) or homology directed repair (HDR). Compared to the traditional approaches, these tools dramatically reduce time and effort needed to establish a GE animal. Another benefit of utilizing genome editing tools is the application of direct injection into developing embryos to induce targeted mutations, therefore, eliminating side effects associated with SCNT. Emerging technological advancements of genome editing systems have dramatically improved efficiency to generate GE livestock models for both biomedical and agricultural purposes. Although the efficiency of genome editing tools has revolutionized GE livestock production, improvements for safe and consistent application are desired. This review will provide an overview of genome editing techniques, as well as examples of GE livestock models for agricultural and biomedical purposes.

The ten-eleven translocation (TET) family (TET1/2/3) initiates conversion of 5-methylcytosine to 5-hydroxymethylcytosine, thereby orchestrating the DNA demethylation process and changes in epigenetic marks during early embryogenesis. In this study, CRISPR/Cas9 technology and a TET-specific inhibitor were applied to elucidate the role of TET family in regulating pluripotency in preimplantation embryos using porcine embryos as a model. Disruption of TET1 unexpectedly resulted in the upregulation of NANOG and ESRRB transcripts, although there was no change to the level of DNA methylation in the promoter of NANOG. Surprisingly, a threefold increase in the transcript level of TET3 was observed in blastocysts carrying modified TET1, which may explain the upregulation of NANOG and ESRRB. When the activity of TET enzymes was inhibited by dimethyloxalylglycine (DMOG) treatment, a dioxygenase inhibitor, to investigate the role of TET1 while eliminating the potential compensatory activation of TET3, reduced level of pluripotency genes including NANOG and ESRRB, and increased level of DNA methylation in the NANOG promoter was detected. Blastocysts treated with DMOG also presented a lower inner cell mass/TE ratio, implying the involvement of TET family in lineage specification in blastocysts. Our results indicate that the TET family modulates proper expression of NANOG, a key pluripotency marker, by controlling its DNA methylation profile in the promoter during embryogenesis. This study suggests that TET family is a critical component in pluripotency network of porcine embryos by regulating gene expression involved in pluripotency and early lineage specification.

Background The CRISPR/Cas9 system can effectively introduce site-specific modifications to the genome. The efficiency is high enough to induce targeted genome modifications during embryogenesis, thus increasing the efficiency of producing genetically modified animal models and having potential clinical applications as an assisted reproductive technology. Because most of the CRISPR/Cas9 systems introduce site-specific double-stranded breaks (DSBs) to induce site-specific modifications, a major concern is its potential off-targeting activity, which may hinder the application of the technology in clinics. In this study, we investigated off-targeting events in genome edited pigs/fetuses that were generated through direct injection of the CRISPR/Cas9 system into developing embryos; off-targeting activity of four different sgRNAs targeting RAG2, IL2RG, SCD5, and Ig Heavy chain were examined. Results First, bioinformatics analysis was applied to identify 27 potential off-targeting genes from the sgRNAs. Then, PCR amplification followed by sequencing analysis was used to verify the presence of off-targeting events. Off-targeting events were only identified from the sgRNA used to disrupt Ig Heavy chain in pigs; frequency of off-targeting was 80 and 70% on AR and RBFOX1 locus respectively. A potential PAM sequence was present in both of the off-targeting genes adjacent to probable sgRNA binding sites. Mismatches against sgRNA were present only on the 5′ side of AR, suggesting that off-targeting activities are systematic events. However, the mismatches on RBFOX1 were not limited to the 5′ side, indicating unpredictability of the events. Conclusions The prevalence of off-targeting is low via direct injection of CRISPR/Cas9 system into developing embryos, but the events cannot be accurately predicted. Off-targeting frequency of each CRISPR/Cas9 system should be deliberately assessed prior to its application in clinics. Electronic supplementary material The online version of this article (10.1186/s12896-019-0517-7) contains supplementary material, which is available to authorized users.

Table S1. Summary of off-targeting comparison for RAG2. Target gene (RAG2) guide sequence compared to potential off-target sequence. Location on chromosome, intron or exon region and presence of PAM site is indicated. Table S2. Summary of off-targeting comparison for IL2RG. Target gene (IL2RG) guide sequence compared to potential off-target sequence. Location on chromosome, intron or exon region and presence of PAM site is indicated. Table S3. Summary of off-targeting comparison for Ig heavy chain. Target gene (Ig heavy chain) guide sequence compared to potential off-target sequence. Location on chromosome, intron or exon region and presence of PAM site is indicated. Table S4. Summary of off-targeting comparison for SCD5. Target gene (SCD5) guide sequence compared to potential off-target sequence. Location on chromosome, intron or exon region and presence of PAM site is indicated. Table S5. Summary of off-targeting results for RAG2. Five pigs were tested for each potential off-target gene. No off-targeting events were found. Table S6. Summary of off-targeting results for IL2RG. Five pigs were tested for each potential off-target gene. No off-targeting events were found. Table S7. Summary of off-targeting results for SCD5. Six pigs were tested for each potential off-target gene. No off-targeting events were found. Table S8. Embryo transfer to generate SCD5 knockout fetuses. Day 5â 6 embryos were transferred into surrogates. A total of 6 fetus were obtained from one surrogate. Table S9. Genotype of SCD5 knockout fetuses. No wild-type allele was identified from the fetuses. Table S10. Summary of primers used to amplify genes tested for potential off-targeting sites as well as original RAG2 target gene. Table S11. Summary of primers used to amplify genes tested for potential off-targeting sites as well as original IL2RG target gene. Table S12. Summary of primers used to amplify genes tested for potential off-targeting sites as well as original Ig heavy chain target gene. Table S13. Summary of primers used to amplify genes tested for potential off-targeting sites as well as original SCD5 gene. (PDF 451 kb)

Traditionally, genetic engineering in the pig was a challenging task. Genetic engineering of somatic cells followed by somatic cell nuclear transfer (SCNT) could produce genetically engineered (GE) pigs carrying site-specific modifications. However, due to difficulties in engineering the genome of somatic cells and developmental defects associated with SCNT, a limited number of GE pig models were reported. Recent developments in genome-editing tools, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) 9 system, have markedly changed the effort and time required to produce GE pig models. The frequency of genetic engineering in somatic cells is now practical. In addition, SCNT is no longer essential in producing GE pigs carrying site-specific modifications, because direct injection of genome-editing systems into developing embryos introduces targeted modifications. To date, the CRISPR/Cas9 system is the most convenient, cost-effective, timely and commonly used genome-editing technology. Several applicable biomedical and agricultural pig models have been generated using the CRISPR/Cas9 system. Although the efficiency of genetic engineering has been markedly enhanced with the use of genome-editing systems, improvements are still needed to optimally use the emerging technology. Current and future advances in genome-editing strategies will have a monumental effect on pig models used in agriculture and biomedicine.

Hepatitis E virus (HEV), the causative agent of hepatitis E, is an important but incompletely understood pathogen causing high mortality during pregnancy and leading to chronic hepatitis in immunocompromised individuals. The underlying mechanisms leading to hepatic damage remain unknown; however, the humoral immune response is implicated. In this study, immunoglobulin (Ig) heavy chain JH -/- knockout gnotobiotic pigs were generated using CRISPR/Cas9 technology to deplete the B-lymphocyte population, resulting in an inability to generate a humoral immune response to genotype 3 HEV infection. Compared to wild-type gnotobiotic piglets, the frequencies of B lymphocytes in the Ig heavy chain JH -/- knockouts were significantly lower, despite similar levels of other innate and adaptive T-lymphocyte cell populations. The dynamic of acute HEV infection was subsequently determined in heavy chain JH -/- knockout and wild-type gnotobiotic pigs. The data showed that wild-type piglets had higher viral RNA loads in feces and sera compared to the JH -/- knockout pigs, suggesting that the Ig heavy chain JH -/- knockout in pigs actually decreased the level of HEV replication. Both HEV-infected wild-type and JH -/- knockout gnotobiotic piglets developed more pronounced lymphoplasmacytic hepatitis and hepatocellular necrosis lesions than other studies with conventional pigs. The HEV-infected JH -/- knockout pigs also had significantly enlarged livers both grossly and as a ratio of liver/body weight compared to phosphate-buffered saline-inoculated groups. This novel gnotobiotic pig model will aid in future studies into HEV pathogenicity, an aspect which has thus far been difficult to reproduce in the available animal model systems.IMPORTANCE According to the World Health Organization, approximately 20 million HEV infections occur annually, resulting in 3.3 million cases of hepatitis E and >44,000 deaths. The lack of an efficient animal model that can mimic the full-spectrum of infection outcomes hinders our ability to delineate the mechanism of HEV pathogenesis. Here, we successfully generated immunoglobulin heavy chain JH -/- knockout gnotobiotic pigs using CRISPR/Cas9 technology, established a novel JH -/- knockout and wild-type gnotobiotic pig model for HEV, and systematically determined the dynamic of acute HEV infection in gnotobiotic pigs. It was demonstrated that knockout of the Ig heavy chain in pigs decreased the level of HEV replication. Infected wild-type and JH -/- knockout gnotobiotic piglets developed more pronounced HEV-specific lesions than other studies using conventional pigs, and the infected JH -/- knockout pigs had significantly enlarged livers. The availability of this novel model will facilitate future studies of HEV pathogenicity.

Fertilized oocytes undergo genome-wide DNA demethylation with the exception of imprinted genes and certain repetitive elements. Ten-eleven translocation 3 (TET3) protein has been known to be responsible for the DNA demethylation process by catalysing oxidation of 5-methylcytosine. Recent studies in the mouse indicated that multiple Tet3 isoforms exist in oocytes, implying differential actions of the isoforms. Previously, we reported the sequence of TET3 in porcine oocytes (GenBank: KC137685). Here, we investigated the presence of TET3 isoforms in porcine oocytes and cumulus cells, and followed changes in the TET3 expression during oocyte maturation to further understand the mechanism of TET3 action in the DNA demethylation process. To identify porcine TET3 isoforms, 5′ RACE (rapid amplification of cDNA ends) was conducted using mRNAs isolated from oocytes and cumulus cells at both germinal vesicle (GV) and MII stages. Gene-specific primers for 5′RACE were designed to recognise conserved regions of TET3 that are present in all isoforms, based on EST databases and the cloned sequence in our previous study. The PCR-amplified 5′RACE products were cloned into a vector and subsequently sequenced. The 5′RACE revealed 3 different TET3 isoforms from GV and MII cumulus cells; no amplification was detected using oocytes, potentially due to a low amount of mRNA. Among the isoforms, the longest variant (TET3L) contained sequences for the CXXC domain, known to carry DNA binding properties. Then, RT-PCR was used to detect the presence of the isoforms in porcine oocytes. Interestingly, the expression of TET3 isoform containing the CXXC domain (TET3L) was only verified from the RT-PCR, suggesting that the isoform may be the predominant isoform in porcine oocytes. To characterise transcript abundance of porcine TET3L, RNAs were isolated from different cells and/or tissues including cumulus cells, oocytes, brain, spleen, and lung. The RT-qPCR was performed using the RNAs and ΔΔCT method was used to analyse the data; GAPDH was used as an internal control. Three biological replicates were used for analysis of RT-qPCR data and P-values of less than 0.05 from one-way analysis of variance were considered significant. The expression level of TET3L was much higher in MII oocytes compared with that in somatic tissues; MII oocytes expressed the TET3 isoform over 350-fold higher than MII cumulus cells and 18-fold higher than lung cells. Interestingly, the expression level of TET3L increased over 3-fold during oocyte maturation (i.e. from GV to MII stage oocytes), indicating that TET3L may have a significant role in DNA demethylation after fertilisation. In conclusion, the TET3 isoform containing CXXC domain (TET3L) is predominantly expressed in matured porcine oocytes, suggesting an important role of the TET3 CXXC domain in DNA demethylation in zygotes.

The objective of this study was to determine if physical parameters of bovine zygotes are correlated with invitro development success. We examined the relationship between a zygote's outer diameter (OD), cell area, and zona pellucida (ZP) thickness on blastocyst development and blastomere number. Bovine cumulus-oocyte complexes, from abattoir-derived ovaries, were matured in tissue culture medium 199-based maturation medium for 21-23h. Invitro fertilisation was performed with frozen thawed semen containing a pool of 4 Holstein bulls. After incubation with sperm for 16-18h, presumptive zygotes (n=875) were denuded and placed in 5µL droplets of synthetic oviductal fluid-bovine embryo 1 medium (SOF-BE1) under oil to be individually photographed using an inverted scope with a digital camera. Zygotes were then group-cultured in 50µL of synthetic oviductal fluid-BE1 droplets in a polyester micromesh to identify individuals. Development to the blastocyst stage was recorded on Day 7 and Day 8 of culture, and Day 8 embryos were fixed and stained with Hoechst 33342 (n=87). ImageJ was used to measure the OD, area, and ZP from images. Data were analysed with a logistic regression using the lme4 package in R with Day 7 or Day 8 blastocyst development as the response; OD, area, and ZP as predictors; and study replicate (n=11) and culture droplet as random effects. Residual estimates of area and ZP calculated from OD were used to account for collinearity. Average OD was 151.2µm, ranging 130.4-171.6µm. Average ZP was 11.8µm, ranging 7.2-17.5µm. Area averaged 9856.7µm2, ranging from 6421.9 to 13 814.8 µm2. There was a tendency for an effect of OD on probability of development on Day 8 (P=0.08) but not Day 7. There was an effect of ZP on the probability of development on Day 8 (P=0.04) but not Day 7. Blastocyst rates on Day 8 for zygotes with ZP 11.8µm were 21.7%. There was an interaction (P12.7µm and OD >155.8µm averaged 43.3%. The observed blastocyst rates for these two groups were 26.2% and 25%, respectively. Zygotes with OD between 146.9-155.8µm and ZP between 10.7-12.7µm had an estimated development probability of 22.2%. The obtained Day 8 blastocyst rate for this group was 22.5%. Area did not affect probability of development, but was positively correlated with total blastomere number at Day 8 (P=0.01; marginal R2=0.09). The observed interaction between ZP thickness and OD may be indicative of an optimum ooplasm area before maturation or fertilisation that may enhance development. Area assessment after fertilisation is not correlated with development success. In addition, these data in comparison with our previous data generated in parthenogenetic embryos indicate that artificial activation of oocytes is not an ideal model in place of IVF for studying development. We continue to demonstrate that physical parameters of zygotes may have potential as a noninvasive, objective selection tool of embryos.

Establishing proper levels of pluripotency is essential for normal development. The genome of gametes is remodelled upon fertilisation and pluripotency-related genes are expressed in blastocysts. Multiple pluripotency-related genes are involved in the well-orchestrated process; however, detailed mechanistic actions remain elusive. The PRDM family genes are reported to be closely related to the pluripotency. A previous report noted that PRDM14 plays an important role in the maintenance of pluripotency in human embryonic stem cells (ESCs) and potentially murine ESCs; loss of PRDM14 was found to cause abnormalities in genome-wide epigenetic status. Similarly, PRDM15 was found to be a key regulator of pluripotency in mouse ESCs. Structural similarities among the PRDM family suggest that other PRDM family genes may help to establish and maintain pluripotency in embryos. Unfortunately, little is known about the expression profile of PRDM family in porcine embryos. To expand our understanding of the role of PRDM family in porcine embryos, expression patterns of PRDM gene family were investigated using reverse transcription quantitative (RTq)-PCR. Candidate PRDM family genes were selected based on previous RNA-Seq data in porcine oocytes/embryos. To conduct this study, germinal vesicle (GV), MII, zygote, 4-cell, and blastocyst samples were collected. Complementary DNA synthesised from the samples was used for RT-qPCR to analyse the expression pattern of selected PRDM family genes: PRDM2, PRDM4, PRDM6, PRDM14, and PRDM15. The expression of target genes was normalized to the YWHAG level, an internal control. Then, GV stage was used as a control for ΔΔCT analysis. Two technical replications and three biological replications were performed. Analysis of variance was used for statistical analysis and P-values

Hepatitis E virus (HEV), the causative agent of hepatitis E, is an important but incompletely understood pathogen causing high mortality during pregnancy and leading to chronic hepatitis in immunocompromised individuals. The underlying mechanisms leading to hepatic damage remain unknown; however, the humoral immune response is implicated. In this study, immunoglobulin (Ig) heavy chain J(H)(−/−) knockout gnotobiotic pigs were generated using CRISPR/Cas9 technology to deplete the B-lymphocyte population, resulting in an inability to generate a humoral immune response to genotype 3 HEV infection. Compared to wild-type gnotobiotic piglets, the frequencies of B lymphocytes in the Ig heavy chain J(H)(−/−) knockouts were significantly lower, despite similar levels of other innate and adaptive T-lymphocyte cell populations. The dynamic of acute HEV infection was subsequently determined in heavy chain J(H)(−/−) knockout and wild-type gnotobiotic pigs. The data showed that wild-type piglets had higher viral RNA loads in feces and sera compared to the J(H)(−/−) knockout pigs, suggesting that the Ig heavy chain J(H)(−/−) knockout in pigs actually decreased the level of HEV replication. Both HEV-infected wild-type and J(H)(−/−) knockout gnotobiotic piglets developed more pronounced lymphoplasmacytic hepatitis and hepatocellular necrosis lesions than other studies with conventional pigs. The HEV-infected J(H)(−/−) knockout pigs also had significantly enlarged livers both grossly and as a ratio of liver/body weight compared to phosphate-buffered saline-inoculated groups. This novel gnotobiotic pig model will aid in future studies into HEV pathogenicity, an aspect which has thus far been difficult to reproduce in the available animal model systems. IMPORTANCE According to the World Health Organization, approximately 20 million HEV infections occur annually, resulting in 3.3 million cases of hepatitis E and >44,000 deaths. The lack of an efficient animal model that can mimic the full-spectrum of infection outcomes hinders our ability to delineate the mechanism of HEV pathogenesis. Here, we successfully generated immunoglobulin heavy chain J(H)(−/−) knockout gnotobiotic pigs using CRISPR/Cas9 technology, established a novel J(H)(−/−) knockout and wild-type gnotobiotic pig model for HEV, and systematically determined the dynamic of acute HEV infection in gnotobiotic pigs. It was demonstrated that knockout of the Ig heavy chain in pigs decreased the level of HEV replication. Infected wild-type and J(H)(−/−) knockout gnotobiotic piglets developed more pronounced HEV-specific lesions than other studies using conventional pigs, and the infected J(H)(−/−) knockout pigs had significantly enlarged livers. The availability of this novel model will facilitate future studies of HEV pathogenicity.

Artificial oocyte activation is an essential step in somatic cell nuclear transfer (SCNT) and can enhance viability of embryos as a form of assisted reproductive technology (ART) in clinics. Most artificial activation methods have been developed to increase cytosolic calcium (Ca

Maternal-to-zygotic transition is an event that developmental control of early embryos is switched from oocyte-derived factors to the zygotic genome. Ability to inhibit DNA replication, transcription, and translation is an important tool in studying events, such as zygotic genome activation, during embyogenesis. Here, we describe approaches to block DNA replication, transcription, and translation using chemical inhibitors. Then we also demonstrate how the transcript level of a maternally inherited gene, ten-eleven translocation methylcytosine dioxygenase 3, responses to the chemical treatments.

Maternal-to-zygotic transition is an event that developmental control of early embryos is switched from oocyte-derived factors to the zygotic genome. Ability to inhibit DNA replication, transcription, and translation is an important tool in studying events, such as zygotic genome activation, during embyogenesis. Here, we describe approaches to block DNA replication, transcription, and translation using chemical inhibitors. Then we also demonstrate how the transcript level of a maternally inherited gene, ten-eleven translocation methylcytosine dioxygenase 3, responses to the chemical treatments.

Ten-eleven translocation (TET) methylcytosine dioxygenases are considered to play an important role in regulation of DNA methylation patterns by converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). TET3 protein, a member of TET family, is enriched in mature oocytes and early stage embryos and contributes to DNA demethylation of the paternal genome in zygotes. N-terminal CXXC domain of TET3 is thought to be important in catalysing 5mC oxidation through its DNA binding potential. However, it is not clear whether specific DNA binding of CXXC domain is required for 5hmC conversion in mammalian embryos. Here, we investigated the role of TET3 CXXC domain in controlling 5hmC formation in fertilized pig embryos by injecting TET3 CXXC domain into mature pig oocytes as a dominant negative to inhibit the direct binding of TET3 to the genome through the CXXC domain. The CXXC domain of pig TET3 was identified through bioinformatics comparison of TET3 sequences among different species and cloned from mature pig oocyte-derived cDNA. To construct the green fluorescent protein (GFP)-CXXC fusion protein, CXXC sequence was subcloned into N-terminal GFP fusion vector, and then mRNA was synthesised by in vitro transcription. The GFP-CXXC mRNA (100 ng/µL) was injected into oocytes matured in vitro for 36 to 37h. Then, the oocytes were fertilized at 42h post-maturation. Water-injected oocytes served as a control. At 17h post-fertilization, zygotes were collected to analyse 5hmC level by immunocytochemistry. The level of 5hmC was analysed using ImageJ (https://imagej.nih.gov/ij/). Expression of pluripotency-related genes at Day 7 blastocysts was examined through quantitative RT-PCR; ΔΔCt method was used to analyse the quantitative RT-PCR data and Student’s t-test was used for statistical analysis. All experiments were conducted at least three times and P-values of less than 0.05 were considered significant. The GFP-CXXC was exclusively localised in pronuclei, indicating that the CXXC domain may lead to nuclear localization of TET3. The level of 5hmC in zygotes was not altered by the overexpression of GFP-CXXC. Interestingly, in 2-cell stage embryos, the 5hmC level was reduced in GFP-CXXC injected embryos compared with the control group, suggesting that CXXC domain is important for 5hmC formation post-DNA replication. There was an increase in transcript abundance of NANOG and ESRRB in blastocysts developed from GFP-CXXC injected oocytes compared with control blastocysts (P

Despite extensive efforts, cellular reprogramming in livestock species has had limited success. Induced pluripotent stem cells (iPSC) have been established; however, these cells often show incomplete reprogramming status, and constitutive expression of exogenous reprogramming factors is required due to inactivation of endogenous pluripotency-related genes. A previous study reported that overexpression of the Xenopus egg-derived WAVE1 gene assists reprogramming of murine somatic cells into the pluripotent state. The WAVE1 gene is also required for oocyte-mediated reprogramming by transcriptional activation of embryonic genes. In this study, we investigated the role of porcine WAVE1 in cellular reprogramming by inducing overexpression of WAVE1 in porcine fetal fibroblasts (PFF). Previously, we cloned the coding sequences (CDS) of porcine WAVE1 using porcine expressed sequence tags (EST) and predicted porcine WAVE1 sequences. The WAVE1 CDS, derived from porcine mature oocytes, was overexpressed in PFF by transfection using the Neon system. Then, G418-based antibiotic selection was performed to enrich cells constitutively overexpressing WAVE1. After cell culture for 4 weeks, RNA was extracted from the WAVE1 transfected and control PFF, and cDNA was synthesised from the RNA using random hexamers. The cDNAs were used for quantitative reverse transcription PCR to analyse the expression pattern of pluripotency- and reprogramming-related genes: POU5F1, NANOG, KLF2, SOX2, DPPA3, ZFP42, ESRRB, TET1, TET2, and TET3. The expression of target genes were normalized to GAPDH level and the ΔΔCt algorithm was used for analysis. Three technical replications and 4 biological replications were performed. Student’s t-test was used for the comparison and P-values

DNA methylation is one of the principal epigenetic modifications playing an essential role in regulating gene expression. The TET family (1-3) is implicated in initiating the demethylation process by converting 5-methylcytosine (5mC) to 5-hydroxymethyl cytosine (5hmC) during embryogenesis. Previous studies in mice suggest that TET1 is required for pluripotency and maintenance of embryonic stem cells by managing their epigenetic marks, specifically DNA methylation. This raises the possibility that TET1 is capable of establishing distinct epigenetic marks during embryo development, thus regulating pluripotency-related genes. However, this has not been demonstrated in any species. Previously we have demonstrated that the level of TET1 (mRNA and protein) was high in porcine blastocysts. In this study, we generated TET1 knockout embryos and analysed expression patterns of pluripotency-related genes in blastocysts to study the role of TET1 in maintaining pluripotency during porcine embryo development. The CRISPR/Cas9 system was applied to disrupt the TET1 gene during embryogenesis. Three single-guide RNAs (sgRNAs) were designed based on our previous cloning of the TET1 gene. In vitro-synthesised Cas9 mRNA (20 ng µL−1) and sgRNAs (10 ng µL−1 each) were injected into the cytoplasm of zygotes after IVF. A total of 605 zygotes were used for microinjection and subsequently 54 blastocysts were formed. As a control, 89 IVF blastocysts were developed from 240 embryos. Nine to ten blastocysts per group were collected on Day 7 to analyse gene expression patterns of TET family and pluripotency-related genes using quantitative RT-PCR. Three biological and 3 experimental replications were used. Differences in the gene expression were evaluated by ANOVA. As expected, there was a 2-fold decrease in the transcript level of TET1 in TET1-knockout blastocysts compared with that in control IVF blastocysts (P