Identification of the Porcine Vascular Endothelial Cell-Specific Promoter ESAM1.0 Using Transcriptome Analysis.

The vascular endothelium of xenografted pig organs represents the initial site of rejection after exposure to recipient immune cells. In this study, we aimed to develop a promoter specific to porcine vascular endothelial cells as a step toward overcoming xenograft rejection. Transcriptome analysis was performed on porcine aortic endothelial cells (PAECs), ear skin fibroblasts isolated from GGTA knockout (GTKO) pigs, and the porcine renal epithelial cell line pk-15. RNA sequencing confirmed 243 differentially expressed genes with expression changes of more than 10-fold among the three cell types. Employing the Human Protein Atlas database as a reference, we identified 34 genes exclusive to GTKO PAECs. The endothelial cell-specific adhesion molecule (ESAM) was selected via qPCR validation and showed high endothelial cell specificity and stable expression across tissues. We selected 1.0 kb upstream sequences of the translation start site of the gene as the promoter ESAM1.0. A luciferase assay revealed that ESAM1.0 promoter transcriptional activity was significant in PAECs, leading to a 2.8-fold higher level of expression than that of the porcine intercellular adhesion molecule 2 (ICAM2) promoter, which is frequently used to target endothelial cells in transgenic pigs. Consequently, ESAM1.0 will enable the generation of genetically modified pigs with endothelium-specific target genes to reduce xenograft rejection.

Application of Enzyme-Linked Fluorescence Assay (ELFA) to Obtain In Vivo Matured Dog Oocytes through the Assessment of Progesterone Level.

Successful dog cloning requires a sufficient number of in vivo matured oocytes as recipient oocytes for reconstructing embryos. The accurate prediction of the ovulation day in estrus bitches is critical for collecting mature oocytes. Traditionally, a specific serum progesterone (P4) range in the radioimmunoassay (RIA) system has been used for the prediction of ovulation. In this study, we investigated the use of an enzyme-linked fluorescence assay (ELFA) system for the measurement of P4. Serum samples of estrus bitches were analyzed using both RIA and ELFA, and the measured P4 values of ELFA were sorted into 11 groups based on the standard concentration measured in RIA and compared. In addition, to examine the tendency of changes in the P4 values in each system, the P4 values on ovulation day (from D - 6 to D + 1) in both systems were compared. The ELFA range of 5.0-12.0 ng/mL was derived from the RIA standard range of 4.0-8.0 ng/mL. The rates of acquired matured oocytes in RIA and ELFA were 55.47% and 65.19%, respectively. The ELFA system successfully produced cloned puppies after the transfer of the reconstructed cloned oocytes. Our findings suggest that the ELFA system is suitable for obtaining in vivo matured oocytes for dog cloning.

Mortality, growth, and egg production do not differ between nontransgenic and transgenic female chickens with ubiquitous expression of the 3D8 single chain variable fragment gene.

To date, many transgenic (TG) chicken lines have been developed, but few studies have performed a comparative analysis of their mortality, growth, and egg productivity. Previously, we reported the production of 3D8 scFv TG chickens showing antiviral activity. Here, we performed a biometric characterization of TG offspring female chickens. We selected 40 TG and 40 non-TG offspring female chicks among newly hatched chicks produced via artificial insemination of semen from heterotypic 3D8 scFv males into wild-type female chickens. Serum was collected at 14 wk of age, and serum concentrations of biochemical parameters, cytokines, and sex hormones were analyzed. Mortality and growth were monitored daily from 1 to 34 wk, egg productivity was monitored daily from 20 to 34 wk, and the weekly average values were used for analyses. Some serum parameters and cytokines were significantly different between non-TG and TG offspring female chickens. The levels of phosphorus (PHOS), total protein (TP), albumin (ALB), globulin (GLOB), and alanine aminotransferase (ALT) were significantly higher in non-TG chickens (P < 0.05). The levels of alkaline phosphatase (ALP) and gamma-glutamyltransferase (GGT) were significantly higher in TG chickens (P < 0.05). The levels of insulin growth factor-1 (IGF-1), interferon-gamma (INF-γ), interleukin-4 (IL-4), and IL-8 were significantly lower in TG chickens (P < 0.05). Despite these differences, the mortality rates, body weight, egg production rates, and egg weight were not significantly different in the experimental groups of non-TG and TG offspring female chickens (P > 0.05). In conclusion, ubiquitous expression of the 3D8 scFv gene in TG offspring female chickens does not affect some biometric characteristics, including mortality, growth, and egg productivity.

Autophagic activation in porcine oocytes is independent of meiotic progression.

In this study, we built on our previous research that discovered that autophagy activated the metaphase I stage during porcine oocytes in vitro maturation. We investigated the relationship between autophagy and oocyte maturation. First, we confirmed whether autophagy was activated differently by different media (TCM199 and NCSU-23) during maturation. Then, we investigated whether oocyte maturation affected autophagic activation. In addition, we examined whether the inhibition of autophagy affected the nuclear maturation rate of porcine oocytes. As for the main experiment, we measured LC3-II levels using western blotting after inhibition of nuclear maturation via cAMP treatment in an in vitro culture to clarify whether nuclear maturation affected autophagy. After autophagy inhibition, we also counted matured oocytes by treating them with wortmannin or a E64d and pepstatin A mixture. Both groups, which had different treatment times of cAMP, showed the same levels of LC3-II, while the maturation rates were about four times higher after cAMP 22 h treatment than that of the 42 h treatment group. This indicated that neither cAMP nor nuclear status affected autophagy. Autophagy inhibition during in vitro oocyte maturation with wortmannin treatment reduced oocyte maturation rates by about half, while autophagy inhibition by the E64d and pepstatin A mixture treatment did not significantly affect the oocyte maturation. Therefore, wortmannin itself, or the autophagy induction step, but not the degradation step, is involved in the oocyte maturation of porcine oocytes. Overall, we propose that oocyte maturation does not stand upstream of autophagy activation, but autophagy may exist upstream of oocyte maturation.

Research Note: Embryonic viability by weight difference between donor and surrogate eggs in a surrogate eggshell incubation system.

A surrogate eggshell incubation system is a well-defined method to apply to avian genetic modification. In this study, we tried to investigate whether the egg weight differences between donor and surrogate eggs have an effect on donor viability. The groups were divided by egg weight differences between the donor and surrogate eggs into 4 in each system. The viability at d 4 was evaluated at the end of System II, the embryos alive were transferred into the second surrogate eggshells, and the viability at d 5, 6 was evaluated at early phase of System III. Then, the viability of System III was evaluated at different incubation period: d 6-12, d 13-18, d 19-21, and hatching rate was evaluated at d 22. Although the effect of egg weight differences between the donor and surrogate eggs was not observed, a specific group in System III showed higher survival and hatching rate than other group (P > 0.05).

Brachygnathia Inferior in Cloned Dogs Is Possibly Correlated with Variants of Wnt Signaling Pathway Initiators.

Abnormalities in animals cloned via somatic cell nuclear transfer (SCNT) have been reported. In this study, to produce bomb-sniffing dogs, we successfully cloned four healthy dogs through SCNT using the same donor genome from the skin of a male German shepherd old dog. Veterinary diagnosis (X-ray/3D-CT imaging) revealed that two cloned dogs showed normal phenotypes, whereas the others showed abnormal shortening of the mandible (brachygnathia inferior) at 1 month after birth, even though they were cloned under the same conditions except for the oocyte source. Therefore, we aimed to determine the genetic cause of brachygnathia inferior in these cloned dogs. To determine the genetic defects related to brachygnathia inferior, we performed karyotyping and whole-genome sequencing (WGS) for identifying small genetic alterations in the genome, such as single-nucleotide variations or frameshifts. There were no chromosomal numerical abnormalities in all cloned dogs. However, WGS analysis revealed variants of Wnt signaling pathway initiators (WNT5B, DVL2, DACT1, ARRB2, FZD 4/8) and cadherin (CDH11, CDH1like) in cloned dogs with brachygnathia inferior. In conclusion, this study proposes that brachygnathia inferior in cloned dogs may be associated with variants in initiators and/or regulators of the Wnt/cadherin signaling pathway.

Development and in vitro evaluation of recombinant chicken promoters to efficiently drive transgene expression in chicken oviduct cells.

Virus injection into EGK-X embryos is a well-defined approach in avian transgenesis. This system uses a chicken ovalbumin gene promoter to induce transgene expression in the chicken oviduct. Although a reconstructed chicken ovalbumin promoter that links an ovalbumin promoter and estrogen-responsive enhancer element (ERE) is useful, a large viral vector containing the ovalbumin promoter and a target gene restricts viral packaging capacity and produces low-titer virus particles. We newly developed recombinant chicken promoters by linking regulatory regions of ovalbumin and other oviduct-specific genes. Putative enhancer fragments of the genes, such as ovotransferrin (TF), ovomucin alpha subunit (OVOA), and ovalbumin-related protein X (OVALX), were placed at the 5`-flanking region of the 2.8-kb ovalbumin promoter. Basal promoter fragments of the genes, namely, pTF, lysozyme (pLYZ), and ovomucoid (pOVM), were placed at the 3`-flanking region of the 1.6-kb ovalbumin ERE. The recombinant promoters cloned into each reporter vector were evaluated using a dual luciferase assay in human and chicken somatic cells, and LMH/2A cells treated with 0-1,000 nM estrogen, and cultured primary chicken oviduct cells. The recombinant promoters with linking ovalbumin and TF, OVOA, pOVM, and pLYZ regulatory regions had 2.1- to 19.5-fold (P < 0.05) higher luciferase activity than the reconstructed ovalbumin promoter in chicken oviduct cells. Therefore, recombinant promoters may be used to efficiently drive transgene expression in transgenic chickens.

Immunosuppression-enhancing effect of the administration of allogeneic canine adipose-derived mesenchymal stem cells (cA-MSCs) compared with autologous cA-MSCs in vitro.

BACKGROUND: Recently, mesenchymal stem cells therapy has been performed in dogs, although the outcome is not always favorable. OBJECTIVES: To investigate the therapeutic efficacy of mesenchymal stem cells (MSCs) using dog leukocyte antigen (DLA) matching between the donor and recipient in vitro. METHODS: Canine adipose-derived MSCs (cA-MSCs) isolated from the subcutaneous tissue of Dog 1 underwent characterization. For major DLA genotyping (DQA1, DQB1, and DRB1), peripheral blood mononuclear cells (PBMCs) from two dogs (Dogs 1 and 2) were analyzed by direct sequencing of polymerase chain reaction (PCR) products. The cA-MSCs were co-cultured at a 1:10 ratio with activated PBMCs (DLA matching or mismatching) for 3 days and analyzed for immunosuppressive (IDO, PTGS2, and PTGES), inflammatory (IL6 and IL10), and apoptotic genes (CASP8, BAX, TP53, and BCL2) by quantitative real-time reverse transcriptase-PCR. RESULTS: cA-MSCs were expressed cell surface markers such as CD90+/44+/29+/45- and differentiated into osteocytes, chondrocytes, and adipocytes in vitro. According to the Immuno Polymorphism Database, DLA genotyping comparisons of Dogs 1 and 2 revealed complete differences in genes DQA1, DQB1, and DRB1. In the co-culturing of cA-MSCs and PBMCs, DLA mismatch between the two cell types induced a significant increase in the expression of immunosuppressive (IDO/PTGS2) and apoptotic (CASP8/BAX) genes. CONCLUSIONS: The administration of cA-MSCs matching the recipient DLA type can alleviate the need to regulate excessive immunosuppressive responses associated with genes, such as IDO and PTGES. Furthermore, easy and reliable DLA genotyping technology is required because of the high degree of genetic polymorphisms of DQA1, DQB1, and DRB1 and the low readability of DLA 88.

Isolation and characterization of cultured chicken oviduct epithelial cells and in vitro validation of constructed ovalbumin promoter in these cells.

OBJECTIVE: Transgenic hens hold a great promise to produce various valuable proteins. Through virus transduction into stage X embryo, the transgene expression under the control of constructed chicken ovalbumin promoters has been successfully achieved. However, a validation system that can evaluate differently developed ovalbumin promoters in in vitro, remains to be developed. METHODS: In the present study, chicken oviduct epithelial cells (cOECs) were isolated from oviduct tissue and shortly cultured with keratinocyte complete medium supplemented with chicken serum. The isolated cells were characterized with immunofluorescence, western blot, and flow cytometry using oviduct-specific marker. Chicken mutated ovalbumin promoter (Mut-4.4-kb-pOV) was validated in these cells using luciferase reporter analysis. RESULTS: The isolated cOECs revealed that the oviduct-specific marker, ovalbumin protein, was clearly detected by immunofluorescence, western blot, and flow cytometry analysis revealed that approximately 79.40% of the cells contained this protein. Also, luciferase reporter analysis showed that the constructed Mut-4.4-kb-pOV exhibited 7.1-fold (p<0.001) higher activity in the cOECs. CONCLUSION: Collectively, these results demonstrate the efficient isolation and characterization of cOECs and validate the activity of the constructed ovalbumin promoter in the cultured cOECs. The in vitro validation of the recombinant promoter activity in cOECs can facilitate the production of efficient transgenic chickens for potential use as bioreactors.

Enhanced in vitro maturation of canine oocytes by oviduct epithelial cell co-culture.

Canine-assisted reproductive techniques have been successful for several years; however, the lack of an oocyte in vitro maturation system has limited their application. The aim of this study was to evaluate the effect of canine oviduct epithelial cells (cOECs) on canine oocyte maturation in vitro. Specifically, the method used for isolation of cOECs did not affect the expression of epithelial markers, E-cadherin and cytokeratin, on fresh, cultured and cryopreserved cells. Moreover, BrdU analysis showed that cOECs cultured in Medium 171 supplemented with mammary epithelial growth supplement were more proliferative than counterparts in advanced Dulbecco's modified Eagle medium or Medium 199. Maturation rate of canine oocytes collected from bitches at diestrus was significantly increased when oocytes were co-cultured with either fresh, cultured or frozen/thawed cOECs (13.23 ± 1.15%, 10.38 ± 4.89%, or 10.54 ± 2.96%, respectively) than that of control oocytes cultured without cOECs (2.48 ± 2.16%, p < 0.05). Additionally, the number of oocytes collected from bitches at estrus the reached metaphase II was increased ∼4 fold in co-culture with fresh, cultured, or frozen/thawed cOECs (47.2 ± 3.82%, 45.4 ± 7.34%, and 46.9 ± 1.51%, respectively) as compared with oocytes cultured without cOECs (11.9 ± 3.18%, p < 0.05). Nuclear maturation was further confirmed by assessing the formation of normal metaphase-II spindles, whereas cytoplasmic maturation was confirmed by inducing parthenogenetic oocyte activation. Embryonic development to the 8-cell stage was similar between in vivo and in vitro matured oocytes. These results suggested that co-culturing immature canine oocytes with cOECs facilitated canine oocyte maturation and early stages of embryonic development.

Scriptaid improves the reprogramming of donor cells and enhances canine-porcine interspecies embryo development.

Histone methylation, histone acetylation, and DNA methylation are the important factors for somatic cell nuclear transfer (SCNT). Histone deacetylase inhibitors (HDACi) and DNA methyltransferase inhibitors (DNMTi) have been used to improve cloning efficiency. In particular, scriptaid, an HDACi, has been shown to improve SCNT efficiency. However, no studies have been performed on canines. Here, we evaluated the effects of scriptaid on histone modification in canine ear fibroblasts (cEFs) and cloned canine embryos derived from cEFs. The early development of cloned canine-porcine interspecies SCNT (iSCNT) embryos was also examined. cEFs were treated with scriptaid (0, 100, 250, 500, 750, and 1000nM) in a medium for 24h. Scriptaid treatment (all concentrations) did not significantly affect cell apoptosis. Treatment with 500nM scriptaid caused a significant increase in the acetylation of H3K9, H3K14, and H4K5. cEFs treated with 500nM scriptaid showed significantly decreased Gcn5, Hat1, Hdac6, and Bcl2 and increased Oct4 and Sox2 expression levels. After SCNT with canine oocytes, H3K14 acetylation was significantly increased in the one- and two-cell cloned embryos from scriptaid-treated cEFs. In iSCNT, the percentage of embryos in the 16-cell stage was significantly higher in the scriptaid-treated group (21.6±2.44%) than in the control (7.5±2.09%). The expression levels of Oct4, Sox2, and Bcl2 were significantly increased in 16-cell iSCNT embryos, whereas that of Hdac6 was decreased. These results demonstrated that scriptaid affected the reprogramming of canine donor and cloned embryos, as well as early embryo development in canine-porcine iSCNT, by regulating reprogramming and apoptotic genes.

Dog cloning with in vivo matured oocytes obtaining using serum estradiol levels for predicting time of ovulation.

Dog cloning using in vivo-matured oocytes has been carried out for a decade. To obtain mature oocytes, serum progesterone (P4) levels are used to evaluate ovulation. However, the accuracy of these methods is not sufficient. Thus, the aim of the present study was to verify the feasibility of serum estradiol (E2) on canine ovulation determination as assessed by the percentage of dogs yielding mature oocytes. In vivo-matured oocytes were utilized for canine somatic cell nuclear transfer (SCNT), and serum P4 and E2 levels were assessed to determine ovulation and oocyte maturation. Canine serum P4 and E2 concentrations during both pro-estrus and estrus were analyzed by electrochemiluminescence immunoassay. The percentage of dogs yielding mature oocytes using each of the two ovulation prediction methods were compared, and correlations between the percentage of each method and temperature were analyzed. Following evaluation, oocytes were collected surgically, and a significantly higher percentage (P < 0.05) of dogs yielding mature oocytes was observed using E2 (56.43%) for ovulation detection as compared with that using P4 (39.60%). The percentage of dogs yielding mature oocytes using P4 significantly lower (P < 0.05) than E2 in autumn (P4, 37.50% vs. E2, 52.00%) and winter (P4, 29.17% vs. E2, 59.09%). Using E2, the percentage was maintained at about 52.00-66.67% regardless of the season and temperature. Correlation analysis showed that the dynamic of percentage of dogs yielding mature oocyte using P4 was highly correlated with environmental temperature (RP4 = 0.862), whereas E2 was not affected by temperature (RE2 = 0.199). To determine whether serum E2 could be used for ovulation prediction for canine cloning, ovulation of 25 and 19 dogs (P < 0.05) were predicted using P4 or E2 methods, respectively and two puppies, one from each ovulation prediction method, were obtained after SCNT and embryo transfer. Thus, compared with the P4 method, E2 was an accurate and reliable method for canine cloning.

Dog cloning with in vivo matured oocytes obtained using electric chemiluminescence immunoassay-predicted ovulation method.

Radioactive immunoassay (RIA) is a traditional serum hormone assay method, but the application of the method in reproductive studies is limited by the associated radioactivity. The aim of present study was to evaluate the reliability of RIA and to compare its canine serum progesterone concentration determination accuracy to that of the electric chemiluminescence immunoassay (ECLI). In vivo matured oocytes were utilized for canine somatic cell nuclear transfer (SCNT), and serum progesterone levels were assessed to accurately determine ovulation and oocyte maturation. Canine serum progesterone concentrations during both proestrus and estrus were analyzed by RIA and ECLI to determine the ovulation day. Although both methods detected similar progesterone levels before ovulation, the mean progesterone concentration determined using ECLI was significantly higher than of RIA three days before ovulation. Following ovulation, oocytes were collected by surgery, and a lower percentage of mature oocytes were observed using ECLI (39%) as compared to RIA (67%) if 4-8ng/ml of progesterone were used for determination of ovulation. A high percentage of mature oocytes was observed using ECLI when 6-15 ng/mL of progesterone was used for ovulation determination. To determine whether ECLI could be used for canine cloning, six canines were selected as oocyte donors, and two puppies were obtained after SCNT and embryo transfer. In conclusion, compared to the traditional RIA method, the ECLI method is a safe and reliable method for canine cloning.

Ascorbic acid increases demethylation in somatic cell nuclear transfer embryos of the pig (Sus scrofa).

OBJECTIVE: Investigated the effect and mechanism of ascorbic acid on the development of porcine embryos produced by somatic cell nuclear transfer (SCNT). METHODS: Porcine embryos were produced by SCNT and cultured in the presence or absence of ascorbic acid. Ten-eleven translocation 3 (TET3) in oocytes was knocked down by siRNA injection. After ascorbic acid treatment, reprogramming genes were analyzed by realtime reverse transcription-polymerase chain reaction (RT-PCR). Furthermore, relative 5-methylcytosine and 5-hydroxymethylcytosine content in pronucleus were detected by realtime PCR. RESULTS: Ascorbic acid significantly increased the development of porcine embryos produced by SCNT. After SCNT, transcript levels of reprogramming genes, Pou5f1, Sox2, and Klf were significantly increased in blastocysts. Furthermore, ascorbic acid reduced 5-methylcytosine content in pronuclear embryos compared with the control group. Knock down of TET3 in porcine oocytes significantly prevents the demethylation of somatic cell nucleus after SCNT, even if in the presence of ascorbic acid. CONCLUSION: Ascorbic acid enhanced the development of porcine SCNT embryos via the increased TET3 mediated demethylation of somatic nucleus.

Cell-free extract from porcine induced pluripotent stem cells can affect porcine somatic cell nuclear reprogramming.

Pretreatment of somatic cells with undifferentiated cell extracts, such as embryonic stem cells and mammalian oocytes, is an attractive alternative method for reprogramming control. The properties of induced pluripotent stem cells (iPSCs) are similar to those of embryonic stem cells; however, no studies have reported somatic cell nuclear reprogramming using iPSC extracts. Therefore, this study aimed to evaluate the effects of porcine iPSC extracts treatment on porcine ear fibroblasts and early development of porcine cloned embryos produced from porcine ear skin fibroblasts pretreated with the porcine iPSC extracts. The Chariot(TM) reagent system was used to deliver the iPSC extracts into cultured porcine ear skin fibroblasts. The iPSC extracts-treated cells (iPSC-treated cells) were cultured for 3 days and used for analyzing histone modification and somatic cell nuclear transfer. Compared to the results for nontreated cells, the trimethylation status of histone H3 lysine residue 9 (H3K9) in the iPSC-treated cells significantly decreased. The expression of Jmjd2b, the H3K9 trimethylation-specific demethylase gene, significantly increased in the iPSC-treated cells; conversely, the expression of the proapoptotic genes, Bax and p53, significantly decreased. When the iPSC-treated cells were transferred into enucleated porcine oocytes, no differences were observed in blastocyst development and total cell number in blastocysts compared with the results for control cells. However, H3K9 trimethylation of pronuclear-stage-cloned embryos significantly decreased in the iPSC-treated cells. Additionally, Bax and p53 gene expression in the blastocysts was significantly lower in iPSC-treated cells than in control cells. To our knowledge, this study is the first to show that an extracts of porcine iPSCs can affect histone modification and gene expression in porcine ear skin fibroblasts and cloned embryos.

In vitro development of canine somatic cell nuclear transfer embryos in different culture media.

The objective of the present study was to investigate the effects of three different culture media on the development of canine somatic cell nuclear transfer (SCNT) embryos. Canine cloned embryos were cultured in modified synthetic oviductal fluid (mSOF), porcine zygote medium-3 (PZM-3), or G1/G2 sequential media. Our results showed that the G1/G2 media yielded significantly higher morula and blastocyst development in canine SCNT embryos (26.1% and 7.8%, respectively) compared to PZM-3 (8.5% and 0%or mSOF (2.3% and 0%) media. In conclusion, this study suggests that blastocysts can be produced more efficiently using G1/G2 media to culture canine SCNT embryos.