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Gene integration at site-specific loci is a critical approach for understanding the function of a gene in cells or animals. The AAVS1 locus is a well-known safe harbor for human and mouse studies. In this study, we found an AAVS1-like sequence (pAAVS1) in the porcine genome using the Genome Browser and designed TALEN and CRISPR/Cas9 to target the pAAVS1. The efficiency of CRISPR/Cas9 in porcine cells was superior to that of TALEN. We added a loxP-lox2272 sequences to the pAAVS1 targeting donor vector containing GFP for further exchange of various transgenes via recombinase-mediated cassette exchange (RMCE). The donor vector and CRISPR/Cas9 components were transfected into porcine fibroblasts. Targeted cells of CRISPR/Cas9-mediated homologous recombination were identified by antibiotic selection. Gene knock-in was confirmed by PCR. To induce RMCE, another donor vector containing the loxP-lox2272 and inducible Cre recombinase was cloned. The Cre-donor vector was transfected into the pAAVS1 targeted cell line, and RMCE was induced by adding doxycycline to the culture medium. RMCE in porcine fibroblasts was confirmed using PCR. In conclusion, gene targeting at the pAAVS1 and RMCE in porcine fibroblasts was successful. This technology will be useful for future porcine transgenesis studies and the generation of stable transgenic pigs.

Although the production of several founder animals (F0) for gene editing in livestock has been reported in cattle, very few studies have assessed germline transmission to the next generation due to the long sexual maturation and gestation periods. The present study aimed to assess the germline transmission of MSTN mutations (-12bps deletion) in MSTN mutant F0 male and female cattle. For this purpose, oocytes and semen were collected after the sexual maturation of MSTN cattle, and embryos produced by in vitro fertilization were analyzed. In addition, the embryos were subjected to additional gene (PRNP) editing using electroporation. Embryos produced by in vitro fertilization with MSTN male and female cattle were transferred to a surrogate, and one calf was successfully born. MSTN heterozygous mutation was shown by sequencing of the F1 calf, which had no health issues. As a further experiment, using electroporation, additional gene-edited embryos fertilized with the MSTN male sperm showed a high mutation rate of PRNP (86.2 ± 3.4%). These data demonstrate that the cattle produced through gene editing matured without health issues and had transmitted MSTN mutation from the germ cells. Also, additional mutation of embryos fertilized with the MSTN male sperm could enable further mutagenesis using electroporation.

Simple Summary CRISPR/Cas9 driven multiplex genome editing may induce genotoxicity and chromosomal rearrangements due to DNA double-strand breaks at multiple loci simultaneously. To overcome this problem in porcine cells we utilized Target-AID, a base editing system, to edit multiple loci in the porcine genome. We showed that the Target-AID system works well in porcine fibroblasts with up to 63.15% efficiency. This is the first report demonstrating that the Target-AID system works well in porcine cells and can be used to generate genome-edited pigs. Abstract Multiplex genome editing may induce genotoxicity and chromosomal rearrangements due to double-strand DNA breaks at multiple loci simultaneously induced by programmable nucleases, including CRISPR/Cas9. However, recently developed base-editing systems can directly substitute target sequences without double-strand breaks. Thus, the base-editing system is expected to be a safer method for multiplex genome-editing platforms for livestock. Target-AID is a base editing system composed of PmCDA1, a cytidine deaminase from sea lampreys, fused to Cas9 nickase. It can be used to substitute cytosine for thymine in 3–5 base editing windows 18 bases upstream of the protospacer-adjacent motif site. In the current study, we demonstrated Target-AID-mediated base editing in porcine cells for the first time. We targeted multiple loci in the porcine genome using the Target-AID system and successfully induced target-specific base substitutions with up to 63.15% efficiency. This system can be used for the further production of various genome-engineered pigs.

Gene integration at site-specific loci, such as safe harbor regions for stable expression via transgenesis, is a critical approach for understanding the function of a gene in cells or animals. The AAVS1 locus is a well-known safe harbor site for human and mouse studies. In the present study, we found an AAVS1-like sequence in the porcine genome using the UCSC Genome Browser and designed TALEN and CRISPR/Cas9 to target AAVS1. The efficiency of CRISPR/Cas9 for targeting the AAVS1 locus in porcine cells was superior to that of TALEN. An AAVS1-targeting donor vector containing GFP was designed and cloned. We added a loxP-lox2272 cassette sequence to the donor vector for further exchange of various transgenes in the AAVS1-targeted cell line. The donor vector and CRISPR/Cas9 components targeting AAVS1 were transfected into a porcine fibroblast cell line. Targeted cells of CRISPR/Cas9-mediated homologous recombination were identified by antibiotic selection. Gene knock-in at the AAVS1 locus was confirmed by PCR analysis. To induce recombinase-mediated cassette exchange (RMCE), another donor vector containing the loxP-lox2272 cassette and inducible (Tet-on) Cre recombinase was cloned. The Cre-donor vector was transfected into the AAVS1-targeted cell line, and RMCE was induced by adding doxycycline to the culture medium. RMCE in porcine fibroblasts was confirmed using PCR analysis. In conclusion, gene targeting at the AAVS1 locus and RMCE in porcine fibroblasts was successful. This technology will be useful for future porcine transgenesis studies and the generation of stable transgenic pigs.

The potential of induced pluripotent stem (iPS) cells, which have self-renewal ability and can differentiate into three germ layers, led us to hypothesize that iPS cells in pigs can be useful and suitable source for producing transgenic pigs. In this study, we generated iPS-like cells using doxycycline-inducible piggyBac (PB) expression vectors encoding porcine 4 transcription factors. After transfection, transfected cells were cultured until the formation of outgrowing colonies taking least of 7-10 days. The iPS-like cells demonstrated pluripotent characteristics such as self-renewal, high proliferation, expression of pluripotent markers, and aggregation ability. The embryo development through somatic cell nuclear transfer (SCNT), cleavage rate, and blastocyst formation rate did not show any significant differences. However, the total cell number of blastocysts was significantly increased with the established cell line. In conclusion, the iPS-like cell line, generated from porcine transcriptional factors using the PB transposon system, demonstrated pluripotency with the capacity for unlimited self-renewal, and could be used as donor cells to produce cloned embryos by SCNT. These cells will be suitable for gene modification and would contribute to the stability or safety of pig models in biomedical research.

Hanwoo (Bos taurus coreanae) are well adapted to the environment of Korea and exhibit unique genetic traits; however, the perinatal mortality rate of Hanwoo is 2–3%, which imposes an economic burden. The timing of parturition is often predicted subjectively due to insufficient data on hormonal changes around parturition, and few studies have examined hormones in Hanwoo. Therefore, we measured the changes in various hormones around parturition in Hanwoo to seek an objective predictor of parturition time. We measured progesterone, prolactin, and cortisol concentrations daily in jugular vein blood samples beginning 6 days before parturition until 7 days after parturition in 14 female Hanwoo. Five were induced to conceive with artificial insemination and nine were induced to conceive via embryo transfer. Progesterone concentration decreased significantly during parturition in the embryo transfer (n = 9) and total (n = 14) groups, but did not change significantly in the artificial insemination (n = 5) ​​group. Prolactin concentration increased on the day of parturition, but did not differ significantly among the groups. Cortisol remained constant throughout. We concluded that the parturition time can be predicted in Hanwoo using progesterone concentration. This knowledge can reduce perinatal mortality, which will help to improve farm income and animal welfare.

International audience; We prepared tetrahedral DNAzymes (TDzs) to overcome potential limitations such as insufficient serum stability and poor cellular uptake of single-stranded DNAzymes (ssDzs). TDzs showed enhanced serum stability and higher cellular uptake efficiency compared to those of ssDzs, providing significantly improved intracellular gene-silencing activity to down-regulate the target mRNA level.

Best estimate plus uncertainty (BEPU) is a promising approach to the safety analysis of nuclear reactors, and the uncertainty calculation is a very important concern about it. BEPU ensures realistic safety margins and secures higher reactor effectiveness by adopting best-estimate codes and realistic input data with uncertainties, whereas the previous conservative analysis generates excessive conservatism by considering each input parameter separately. A loss of flow accident (LOFA) of a 5 MW open-pool type research reactor was selected as a sample problem for a BEPU uncertainty assessment. We selected the failures of all primary cooling system (PCS) pumps, which would cause the abrupt reduction of flow and the reversal of core flow. The significant contributors to the reactor safety were identified and then input sets were sampled. For the uncertainty evaluation, 124 calculations were performed. This is the number of code runs required for a 95%/95% level with the 3rd order Wilk’s formula. The MOSAIQUE software developed by Korean Atomic Energy Research Institute (KAERI) was used for automated sampling of the uncertainty parameters, a global uncertainty calculation, and post processing of the results. The critical heat flux ratio (CHFR) and the fuel centerline temperature (FCT) were calculated at the 95%/95% level and were compared with those from conservative analyses. In addition, the impact of each design variables on the safety parameters was estimated by sensitivity analysis.

Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) exhibit potency for the regeneration of infarcted hearts. Vascular endothelial growth factor (VEGF) is capable of inducing angiogenesis and can boost stem cell-based therapeutic effects. However, high levels of VEGF can cause abnormal blood vessel growth and hemangiomas. Thus, a controllable system to induce therapeutic levels of VEGF is required for cell therapy. We generated an inducible VEGF-secreting stem cell (VEGF/hUCB-MSC) that controls the expression of VEGF and tested the therapeutic efficacy in rat myocardial infarction (MI) model to apply functional stem cells to MI. To introduce the inducible VEGF gene cassette into a safe harbor site of the hUCB-MSC chromosome, the transcription activator-like effector nucleases system was used. After confirming the integration of the cassette into the locus, VEGF secretion in physiological concentration from VEGF/hUCB-MSCs after doxycycline (Dox) induction was proved in conditioned media. VEGF secretion was detected in mice implanted with VEGF/hUCB-MSCs grown via a cell sheet system. Vessel formation was induced in mice transplanted with Matrigel containing VEGF/hUCB-MSCs treated with Dox. Moreover, seeding of the VEGF/hUCB-MSCs onto the cardiac patch significantly improved the left ventricle ejection fraction and fractional shortening in a rat MI model upon VEGF induction. Induced VEGF/hUCB-MSC patches significantly decreased the MI size and fibrosis and increased muscle thickness, suggesting improved survival of cardiomyocytes and protection from MI damage. These results suggest that our inducible VEGF-secreting stem cell system is an effective therapeutic approach for the treatment of MI.

Animal models, particularly pigs, have come to play an important role in translational biomedical research. There have been many pig models with genetically modifications via somatic cell nuclear transfer (SCNT). However, because most transgenic pigs have been produced by random integration to date, the necessity for more exact gene-mutated models using recombinase based conditional gene expression like mice has been raised. Currently, advanced genome-editing technologies enable us to generate specific gene-deleted and -inserted pig models. In the future, the development of pig models with gene editing technologies could be a valuable resource for biomedical research.

Mesenchymal stem cells (MSCs) promote therapeutic angiogenesis to cure serious vascular disorders. However, their survival period and cytokine-secretory capacity are limited. Although hepatocyte growth factor (HGF) can accelerate the rate of angiogenesis, recombinant HGF is limited because of its very short half-life (

In animal reproduction technologies, the in vitro embryo culture system has advanced over the past few decades. However, in vitro cultured embryos still have reduced functional and physiological abilities compared with those from in vivo conditions, and many factors of oviduct and uterine environments have not yet been revealed. Here, we demonstrated the in vitro culture of domestic goat (Capra hircus) embryos using two types of culture media, modified synthetic oviductal fluid (mSOF) and a two-step chemically defined medium (DI/II). To obtain parthenogenetic goat embryos, oocytes were matured in vitro in tissue culture media-199 supplemented with 10% fetal bovine serum for 22 to 24 hours, and activated with 5 μM, Ca(2+) ionomycin for 4 minutes, followed by 1.9 mM, 6-dimethylaminopurine treatment for 4 hours. After 2 days of embryo culture in different culture media, there were no significant differences in cleavage rates (96.6% vs. 95.4% in mSOF vs. DI/II, respectively). However, the DI/II group showed improved development competence to blastocysts (64.6% vs. 82.3% in mSOF vs. DI/II, respectively) and the total cell number of blastocysts (144.3 ± 9.2 vs. 264.4 ± 15.2 in mSOF vs. DI/II, respectively) at Day 7. After the cryopreservation of early-stage blastocysts at Day 6 via the conventional slow-freezing procedure, the surviving embryos were analyzed. The re-expansion rate after freezing and thawing was significantly higher in DI/II (39.66% vs. 67.69% in mSOF vs. DI/II, respectively), but there were no statistical differences in total cell numbers (142.3 ± 12.1 vs. 172.1 ± 11.6 in mSOF vs. DI/II, respectively), apoptotic index (4.9 ± 0.8% vs. 3.8 ± 0.7 in mSOF vs. DI/II, respectively), and the gene expression levels (BAX, GLUT1, MnSOD, and OCT4) among the re-expanded blastocysts. Overall, our data reported that the defined in vitro culture media for goat embryos were established with high efficiency, which will be very useful for goat embryo production.

Determining cell lineage and function is critical to understanding human physiology and pathology. Although advances in lineage tracing methods provide new insight into cell fate, defining cellular diversity at the mammalian level remains a challenge. Here, we develop a genome editing strategy using a cytidine deaminase fused with nickase Cas9 (nCas9) to specifically target endogenous interspersed repeat regions in mammalian cells. The resulting mutation patterns serve as a genetic barcode, which is induced by targeted mutagenesis with single-guide RNA (sgRNA), leveraging substitution events, and subsequent read out by a single primer pair. By analyzing interspersed mutation signatures, we show the accurate reconstruction of cell lineage using both bulk cell and single-cell data. We envision that our genetic barcode system will enable fine-resolution mapping of organismal development in healthy and diseased mammalian states., Lineage tracing has provided new insights into cell fate but defining cellular diversity remains a challenge. Here the authors target endogenous repeat regions in mammalian cells with cytidine deaminase fused to nCas9 to create genetic barcodes for fine-resolution mapping.

Background Transposon-mediated, non-viral gene delivery is a powerful tool for generating stable cell lines and transgenic animals. However, as multi-copy insertion is the preferred integration pattern, there is the potential for uncontrolled changes in endogenous gene expression and detrimental effects in cells or animals. Our group has previously reported on the generation of several transgenic cattle by using microinjection of the Sleeping Beauty (SB) and PiggyBac (PB) transposons and seeks to explore the long-term effects of this technology on cattle. Results Transgenic cattle, one female (SNU-SB-1) and one male (SNU-PB-1), reached over 36 months of age with no significant health issues and normal blood parameters. The detection of transgene integration and fluorescent signal in oocytes and sperm suggested the capacity for germline transmission in both of the founder animals. After natural breeding, the founder transgenic cow delivered a male calf and secreted milk containing fluorescent transgenic proteins. The calf expressed green fluorescent protein in primary cells from ear skin, with no significant change in overall genomic stability and blood parameters. Three sites of transgene integration were identified by next-generation sequencing of the calf’s genome. Conclusions Overall, these data demonstrate that transposon-mediated transgenesis can be applied to cattle without being detrimental to their long-term genomic stability or general health. We further suggest that this technology may be usefully applied in other fields, such as the generation of transgenic animal models. Electronic supplementary material The online version of this article (10.1186/s12864-018-4760-4) contains supplementary material, which is available to authorized users.

Even though prion (encoded by the PRNP gene) diseases like bovine spongiform encephalopathy (BSE) are fatal neurodegenerative diseases in cattle, their study via gene deletion has been limited due to the absence of cell lines or mutant models. In this study, we aim to develop an immortalized fibroblast cell line in which genome-engineering technology can be readily applied to create gene-modified clones for studies. To this end, this study is designed to 1) investigate the induction of primary fibroblasts to immortalization by introducing Bmi-1 and hTert genes; 2) investigate the disruption of the PRNP in those cells; and 3) evaluate the gene expression and embryonic development using knockout (KO) cell lines. Primary cells from a male neonate were immortalized with Bmi-1and hTert. Immortalized cells were cultured for more than 180 days without any changes in their doubling time and morphology. Furthermore, to knockout the PRNP gene, plasmids that encode transcription activator-like effector nuclease (TALEN) pairs were transfected into the cells, and transfected single cells were propagated. Mutated clonal cell lines were confirmed by T7 endonuclease I assay and sequencing. Four knockout cell lines were used for somatic cell nuclear transfer (SCNT), and the resulting embryos were developed to the blastocyst stage. The genes (CSNK2A1, FAM64A, MPG and PRND) were affected after PRNP disruption in immortalized cells. In conclusion, we established immortalized cattle fibroblasts using Bmi-1 and hTert genes, and used TALENs to knockout the PRNP gene in these immortalized cells. The efficient PRNP KO is expected to be a useful technology to develop our understanding of in vitro prion protein functions in cattle.

In recent years, CRISPR/Cas9 has become known as a powerful tool for generating genetically modified cells or animal models with its site-specific gene editing capability. Because myostatin (MSTN) is associated with muscle differentiation and growth, ablation of MSTN would be ideal for studying phenotypes associated with muscle growth in beef cattle. The aim of this study is to produce Korean beef cattle with MSTN mutation through microinjection of CRISPR/Cas9. For this study, ovaries of Korean beef cattle were obtained from slaughterhouses; 122 immature oocytes were aspirated from 8 Korean beef cattle. The oocytes were matured in tissue culture medium 199-based medium for 22h and inseminated with frozen-thawed semen to produce zygotes. All 122 zygotes were directly injected with 200ng of Cas9 mRNA and 100ng of sgRNA for MSTN after IVF and were cultured for 7 days in two-step chemically defined medium. Each embryo from the individual cattle was cultured separately to distinguish its lineage. After 7 d, 29 (23.8±8.9) blastocysts were recovered, and all blastocysts were assessed with T7 endonuclease (T7E1) mutation assay to check for mutations at the MSTN locus. Based on T7E1, 26 out of 29 blastocysts (89.7±10.5%) had heterozygous mutation in the MSTN gene. After analysis, additional blastocysts were produced and transferred into 14 surrogates. All surrogates were diagnosed for pregnancy via rectal palpation and ultrasonography at Day 55, at which time normal fetuses and embryonic sacs were confirmed in 10 surrogates. In conclusion, these data demonstrate that CRISPR/Cas9 could be employed as an efficient method to genetically modify the MSTN gene in Korean beef cattle.

By considering the requirements for a DEMO-relevant blanket concept, Korea (KO) has proposed a He cooled molten lithium (HCML) test blanket module (TBM) for testing in ITER. A performance analysis for the thermal–hydraulics and a safety analysis for the KO TBM have been carried out using a commercial CFD code, ANSYS-CFX, and a system code, GAMMA (GAs multicomponent mixture analysis), which was developed by the gas cooled reactor in Korea. To verify the codes, a preliminary study was performed by Lee using a single TBM first wall (FW) mock-up made from the same material as the KO TBM, ferritic martensitic steel, using a 6 MPa nitrogen gas loop. The test was performed at pressures of 1.1, 1.9 and 2.9 MPa, and under various ranges of flow rate from 0.0105 to 0.0407 kg/s with a constant wall temperature condition. In the present study, a thermal–hydraulic test was performed with the newly constructed helium supplying system, in which the design pressure and temperature were 9 MPa and 500 °C, respectively. In the experiment, the same mock-up was used, and the test was performed under the conditions of 3 MPa pressure, 30 °C inlet temperature and 70 m/s helium velocity, which are almost same conditions of the KO TBM FW. One side of the mock-up was heated with a constant heat flux of 0.3–0.5 MW/m 2 using a graphite heating system, KoHLT-2 (Korea heat load test facility-2). Because the comparison result between CFX 11 and GAMMA showed a difference tendency, the modification of heat transfer correlation included in GAMMA was performed. And the modified GAMMA showed the strong parity with CFX 11 calculation results.

The design scheme and system codes for fusion application have been developed for the ITER Test Blanket Module (TBM) program in Korea in parallel with the breeding blanket development, which were based on the developed system codes in Gen. IV reactor development projects such as MARS (Multi-dimensional Analysis of Reactor Safety) and GAMMA (GAs Multi-component Mixture Analysis). Considering the unique and common features with both the Fusion and Gen. IV reactors, four approaches have been carried out: (1) modifying the heat transfer model and suggesting a 3D analysis for considering the one-sided heating with extreme temperature differences, (2) implementing a tritium permeation model for a simulation of its behavior and amount simulation in a fusion coolant system, (3) developing a physical properties generation model for PbLi and Li considering the liquid metal breeders in these codes, and (4) implementing the magnetohydrodynamics (MHD) model by Miyazaki et.al. To integrate these separate codes ...

Here, we efficiently generated transgenic cattle using two transposon systems (Sleeping Beauty and Piggybac) and their genomes were analyzed by next-generation sequencing (NGS). Blastocysts derived from microinjection of DNA transposons were selected and transferred into recipient cows. Nine transgenic cattle have been generated and grown-up to date without any health issues except two. Some of them expressed strong fluorescence and the transgene in the oocytes from a superovulating one were detected by PCR and sequencing. To investigate genomic variants by the transgene transposition, whole genomic DNA were analyzed by NGS. We found that preferred transposable integration (TA or TTAA) was identified in their genome. Even though multi-copies (i.e. fifteen) were confirmed, there was no significant difference in genome instabilities. In conclusion, we demonstrated that transgenic cattle using the DNA transposon system could be efficiently generated, and all those animals could be a valuable resource for agriculture and veterinary science.

The forces acting on a bubble were analyzed to determine the criteria for bubble departure and lift-off from a heated wall. Mechanistic models for the bubble departure and lift-off diameters were developed from the analysis results to predict the evaporation heat flux under low heat flux and low flow velocity conditions. The developed model for the departure diameter depends strongly on the contact angle, but the model for the lift-off diameter was found to be a function of the lift-off number, which indicates the ratio between the shear lift force and the growth force. Wall boiling experiments were performed to measure bubble departure and lift-off diameters using digital image processing. From the experimental data, the developed models were validated, and the results showed good agreement between the experimental data and the predicted diameters.

Interferon tau (IFNT), a type I interferon, is known as a key signal molecule during pregnancy in ruminants because of the necessity in maternal recognition of pregnancy. It is produced in trophectoderm cells of the elongation bovine conceptus at Day 13-21, and peak production is at Day 15 to 17 of pregnancy. In addition, other studies show that it can affect embryonic development and quality. In this study, the effect of IFNT knockout in donor cells to bovine cloned embryonic development by somatic cell nuclear transfer (SCNT) was investigated. To proceed with this experiment, immature oocytes from ovaries at a local slaughterhouse were matured in vitro for 22h. To prepare the donor cell with IFNT knockout, somatic cells were transfected with Cas9 and single guide RNA targeting IFNT, and several single derived colonies with high proliferation were isolated for mutation assay. Finally, one colony that had mono-allelic mutation (4bp deletion) was selected and used as the donor cell for SCNT. A donor cell was injected into an enucleated oocyte. Reconstructed oocytes with the donor cell were fused by electrical shock, activated by chemical stimulation, and cultured for 7 days in chemically defined medium. For this study, control (n=94) and IFNT knockout groups (n=140) were compared with 4 replications. The results showed no significant difference between control and IFNT knockout groups not only in cleavage rate, but also in blastocyst formation rate (control: 15.7±8.3%, IFNT knockout group: 26.3±13.1%). In addition, the number of blastocyst cell was not different between control (88.2±27.0) and IFNT knockout group (88.0±21.1). Some IFNT mutated blastocysts from SCNT were randomly selected for confirmation of the deletion of IFNT, and all samples were positive for mutation. In conclusion, these data demonstrated that the disruption of IFNT did not affect embryonic development. In future study, we would transfer these embryos and check the effect of IFNT during pregnancy status. This work was supported by BK21 PLUS Program for Creative Veterinary Science, the National Research Foundation of Korea (2017R1A2B3004972), and the Technology Development Program (S2566872) by MSS.

The p53 gene is a tumour suppressor gene with many roles in cellular reprogramming, senescence, or cancers. Knockdown of p53 in somatic cells improves generation of induced pluripotent stem cells and developmental competence of cloned embryos. In this study, we hypothesised that disruption of p53 in bovine somatic cells using CRISPR-Cas9 may improve reprogramming and result in increased blastocyst formation. For the study, ovary-derived immature bovine cumulus-oocyte complexes were matured in vitro. To prepare the donor cells with disruption of p53, primary cells from the skin tissue were transfected with spCas9 and single guide RNA for bovine p53. Two days later, genomic DNA was extracted and the mutation was evaluated on the target region using the T7E1 enzyme. Several single cell colonies were isolated to investigate the mutation and sequencing for mutation positive colonies was performed. One colony with high proliferative activity and heterozygotic mutation (−7bp deletion) was selected and used as the donor cell for somatic cell nuclear transfer. The donor cell was microinjected into an enucleated oocyte. Reconstructed oocytes were fused by electrical pulse, activated by chemical stimulation, and cultured in chemically defined media for 7 days at 5% CO2 and 5% O2. Data were subjected to ANOVA and least significant different test to determine differences between experimental groups by using SAS software (SAS Institute Inc., Cary, NC, USA). Statistical significance was determined when the P-value was less than 0.05. Blastocyst formation rate (13.5±7.6%) from the mutated cell line was significantly lower than wild type cells (43.9±12.1%). Moreover, total cell number (115.4±13.3) of the blastocysts from knockout cells was significantly decreased compared with the control group (143.6±22.5). Some randomly selected blastocysts derived from mutated cells were subjected to mutation assay to confirm deletion of p53. As expected, all the samples were positive to mutation. These data did not support our hypothesis that p53 disruption could improve blastocyst formation competence of bovine cloned embryos. In the future, the reason for impaired embryonic development of clones of p53 deleted somatic cells will be further investigated. This work was supported by BK21 PLUS Program for Creative Veterinary Science, the National Research Foundation of Korea (2017R1A2B3004972), and the Technology Development Program (S2566872) by MSS.

This study was performed to get basic information about properties of high blaine slag cement(HSC) to use shotcrete(or sprayed concrete and mortar). Particle size distribution, setting time and compressive strength test, analysis like as SEM, DSC thermal analysis, XRD was carried out to investigate principle properties of HSC. Setting time of HSC was delayed slightly, but influence of accelerators was more bigger than ordinary portland cement(OPC). Compressive strength of HSC at 28 days was more higher than OPC regardless of using accelerators . Results of analysis showed early period hydration products of HSC is more small and located widely, because of the interface of between cement particle and water is increased as specific surface of cement increase. From the SEM observation and analysis of DSC and XRD results, aluminates accelerators bring on some hydration products like as calcium aluminium hydrates, alkali free accelerators increases ettringite and monosulfates. Aluminates accelerators has a advantage of setting time and early strength, alkali free accelerators increases strength after 7 days

Adipose tissue mesenchymal stem cells (ATMSCs) represent an attractive tool for the establishment of a successful stem cell-based therapy in the field of liver regeneration medicine. ATMSCs overexpressing Oct4 and Sox2 (Oct4/Sox2-ATMSCs) showed enhanced proliferation and multipotency. Hence, we hypothesized that Oct4 and Sox2 can increase "transdifferentiation" of ATMSCs into cells of the hepatic lineage. In this study, we generated Oct4- and Sox2-overexpressing human ATMSCs by liposomal transfection. We confirmed the expression of mesenchymal stem cell surface markers without morphological alterations in both red-fluorescent protein (RFP) (control)- and Oct4/Sox2-ATMSCs by flow cytometry. After induction of differentiation into hepatocyte-like cells, the morphology of ATMSCs changed and they began to appear as round or polygonal epithelioid cells. Hepatic markers were evaluated by reverse transcription-polymerase chain reaction and confirmed by immunofluorescence. The results showed that albumin was strongly expressed in hepatogenic differentiated Oct4/Sox2-ATMSCs, whereas the expression level of α-fetoprotein was lower than that of RFP-ATMSCs. The functionality of hepatocytes was evaluated by periodic acid-Schiff (PAS) staining and urea assays. The number of PAS-positive cells was significantly higher and urea production was significantly higher in Oct4/Sox2-ATMSCs compared to that in RFP-ATMSCs. Taken together, the hepatocyte-like cells derived from Oct4/Sox2-ATMSCs were mature hepatocytes, possibly functional hepatocytes with enhanced capacity to store glycogen and produce urea. In this study, we demonstrated the enhanced transdifferentiation of Oct4- and Sox2-overexpressing ATMSCs into hepatocyte-like cells that have enhanced hepatocyte-specific functions. Therefore, we expect that Oct4/Sox2-ATMSCs may become a very useful source for hepatocyte regeneration or liver cell transplantation.

A large number of oocytes and adequate recipient conditions are required to produce genetically modified rats by embryo transfer. In this study, we investigated the effect of a synthetic gonadotropin-releasing hormone (GnRH) agonist, buserelin, on (1) superovulatory response in donor rats, and (2) oestrous cycle synchrony in recipient female Sprague Dawley rats at 8 weeks of age. First, for superovulation, donors were divided into 2 groups: control and buserelin injection group (42 μg mL−1). Superovulation was performed in both groups by injection of pregnant mare’s serum gonadotropin (PMSG, 150 IU) at Day −3 and human chorionic gonadotropin (hCG, 150 IU) at Day −1 (Day 0 = oocyte collection). In addition, the buserelin group was given 42 μg mL−1 buserelin on Day −4. Oviduct and ampulla were removed surgically to collect the oocytes. As results, more ovulations were detected in the 42 μg mL−1 buserelin group (6.18 ± 1.10 oocyte/rat, n = 240) than in the control group (5.06 ± 0.82 oocyte/rat; n = 124; t-test P = 0.038). Second, 2 groups were set up to investigate the effect of administration of buserelin on synchronizing the oestrus cycle of recipients. Because the oestrous cycle of the rat was 4 to 5 days, we examined the difference of vaginal impedance between the 2 groups on Day 4 after injection and checked whether there was a significant difference in the synchronization of the oestrous cycle. An impedance value >3.0 kΩ was considered as an indication for proestrus stage; therefore, we analysed the data >3.0 kΩ in both groups. In results, the incidence of proestrus was significantly increased in buserelin injection group (n = 128) than in the control group (n = 70). In addition, there was a significant difference in mean impedance in the 2 groups (buserelin injection group: 4.25 ± 3.79 kΩ; control group: 1.43 ± 1.20 kΩ; P

The CRISPR/Cas9 system has proved to be a powerful tool for knockout and knock-in in various species. When 2 components—Cas9 and single guide (sg)RNA—are delivered into cells or embryos, the events of gene editing occur. Because Cas9 is essential for gene editing in the CRISPR/Cas9 system, some studies have reported the production of Cas9-expressing animals, such as mice, which could be used to increase gene editing efficiency in subsequent experiments. In previous reports, we successfully produced 4 Cas9-expressing cattle via microinjection (Hahn et al. 2016 Reprod. Fertil. Dev. 29, 211). Primary cells from these calves had Cas9 activity because transfection of only sgRNA resulted in gene deletion. The aim of this study was to analyse the blood of the transgenic cattle to investigate the effect of Cas9 expression on health. Two of 4 transgenic calves died; one had severe ruminant tympany, failed to respond to treatment, and died at 4 months of age, and the other died at 5 months of age due to accidental ingestion of a needle from a feed bunk. Blood samples were obtained from the surviving 2 transgenic cattle (1 male and 1 female) at 7 and 12 months for blood analysis. Five milliliters of whole blood samples was collected from the jugular vein. Portions were used for CBC (Hemavet 950, Drew Scientific, Miami Lakes, FL, USA) and for serum chemistry analysis (BS-400, Mindray, Shenzhen, China). Average values for white blood cells (9600 and 1057/mm3), neutrophils (4590 and 3870/mm3), lymphocytes (4020 and 5910/mm3), red blood cells (732,000 and 798,000/mm3), hemoglobin (9.5 and 10.2 g dL−1), packed cell volume (24.3 and 25.3%), platelet (439,000 and 327,500/mm3), AST (76 and 104 IU), ALP (140 and 133 IU), BUN (7.5 and 10.5 mg dL−1), and creatinine (1.3 and 1.0 mg dL−1) of male and female transgenic calves were within the reference range. Additionally, there was no difference in general health information, including body temperature and feeding. In conclusion, we demonstrated that continuous Cas9 expression in transgenic cattle did not affect health status of the surviving calves in terms of blood analysis. They have grown up without any health issues and are currently 14 (female) and 15 (male) months old. In the near future, we will evaluate their germline transmission by natural breeding or in vitro fertilization. This work was supported by BK21 PLUS Program for Creative Veterinary Science, NRF (NRF-2017R1A2B3004972), and Seoul Milk Coop (SNU 550–20160004).

SummaryGenome-editing technologies are considered to be an important tool for generating gene knockout cattle models. Here, we report highly efficient disruption of a chromosomally integrated eGFP gene in bovine somatic cells using RNA-guided endonucleases, a new class of programmable nucleases developed from a bacterial Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system. In the present study, we obtained homogenously eGFP-expressing primary fibroblasts from cloned bovine transgenic embryonic tissues and employed them for further analysis. CRISPR/Cas9 plasmids specifically targeting the eGFP gene were transfected into the eGFP fibroblasts by electroporation. After 10 days of culture, more than 40% of the cells had lost eGFP expression in fluorescence activated cell sorting (FACS) analysis. Targeted sequences of the transfected cells were analyzed, and various small indel mutations (6–203 bp deletions) in the target sequence were found. The fibroblasts mutated with the CRISPR/Cas9 system were applied for somatic cell nuclear transfer, and the reconstructed embryos were successfully developed into the blastocyst stage. In conclusion, the CRISPR/Cas9 system was successfully utilized in bovine cells and cloned embryos. This will be a useful technique to develop livestock transgenesis for agricultural science.

Cell penetrating peptides (CPPs) are peptides that can be translocated into cells and used as a carrier platform for the intracellular uptake of cargo molecules. Subject to the source of CPP sequences and their positively charged nature, the cytotoxicity and immunogenicity of conventional CPPs needs to be optimized to expand their utility for biomedical applications. In addition to these safety issues, the stability of CPPs needs to be addressed since their positively charged residues are prone to interact with the biological milieu. As an effort to overcome these limitations of the current CPP technology, we isolated CPP candidate sequences and synthesized peptides from twelve isoforms of annexin, a family of membrane-interacting human proteins. The candidate screen returned a CPP rich in hydrophobic residues that showed more efficient cellular uptake than TAT-CPP. We then investigated the uptake mechanism, subcellular localization and biophysical properties of the newly found CPP, verifying low cytotoxicity, long-term serum stability and non-immunogenicity. Finally, model proteins conjugated to this peptide were successfully delivered into mammalian cells both in vitro and in vivo, indicating a potential use of the peptide as a carrier for the delivery of macromolecular cargos.

Recently, we published on the efficient production of transgenic cattle using the DNA transposon system (Yum et al. 2016 Sci. Rep. 6, 27185). In that study, 8 transgenic cattle were born following transposon-mediated gene delivery system (Sleeping Beauty and Piggybac transposon) via microinjection of zygotes. In the analysis of their genomic stability using next-generation sequencing, there was no significant difference in the number of genomic variants between transgenic and nontransgenic cattle. In this study, we have described current status of those transgenic cattle in term of health, germ-line transmission, and application. All the transgenic cattle have grown up to date (the oldest being 30 months old, the youngest being 12 months old) without any health issue. In general blood analysis, there were not any significant changes between transgenic cattle and wild type. Because the transgene (green fluorescent protein; GFP) expression is constitutively active and has strong expression, it could be visualised without fluorescence equipment. One of transgenic male cattle reached puberty and semen was collected. Over 200 frozen semen straws were produced and some were used for IVF. In every IVF replication, around 80% blastocysts expressed the GFP. Over 36 GFP blastocysts were frozen for embryo transfer in the future, and we are planning to crossbreed for generating homozygotic transgenic cattle. Another application is to use the GFP locus to gene-edit the transgenic cattle, as long-term expression of transgene did not affect their health. In 1 cell stage, embryos produced using GFP frozen-thawed semen, single guide RNA for GFP, Cas9, together with donor DNA that included RFP and homology arms to link the double-strand break of single guide RNA target site, were co-injected and RFP was observed. Knockout/-in for editing GFP locus using CRISPR-Cas9 might be a valuable approach for the next generation of transgenic models by microinjection. In conclusion, we demonstrated that transgenic cattle via transposon are healthy to date and germ-line competence was confirmed. The GFP locus will be used as the target region for future gene engineering via genome-editing technology. Finally, all those animals could be a valuable agricultural and veterinary science resource for studying the effects of gene manipulation on disease resistance and food production. This work was supported by BK21 PLUS Program for Creative Veterinary Science and Seoul Milk Coop (SNU 550–20160004).

A genome-editing technology, CRISPR/Cas9 system is proved to be a powerful tool for knockout and knock-in in various species. When 2 components [Cas9 and single guide (sg) RNA] are delivered into cells or embryos, the events of gene editing occur. Because Cas9 is essential for every gene editing based on the CRISPR/Cas9 system, some studies reported that efficiency of gene editing would be increased as Cas9 was integrated into cells or animals. Accordingly, if the Cas9-expressing cattle is born, it would be broadly used for gene editing in cattle. For this study, the Cas9 and RFP genes were cloned into the PiggyBac transposon system. PiggyBac-Cas9-RFP and transposase were microinjected into 1436 IVF embryos and 241 blastocysts were formed. Blastocysts with RFP expression accounted for 14.1% of total formed blastocysts. Five blastocysts were selected and transferred into 5 recipient cow (1 embryo per recipient). After gestation periods, 4 transgenic cattle were delivered without any veterinary assistance. From transgenic cattle, ear skin tissue was collected for primary culture. On those primary cells, sgRNA in DNA form for various genes such as PRNP, RB1, and BLG were transfected with 2 μg of sgRNA per 5 × 105 cells using electroporation. As expected, every group of each sgRNA delivered was confirmed to be mutated by T7E1 assay. The data demonstrated that, for the first time, transgenic cattle with Cas9 expression were born, grown up to date (age = 5 months) and will be a valuable resource for genome editing in cattle. This work was supported by BK21 PLUS Program for Creative Veterinary Science and Seoul Milk Coop (SNU 550–20160004).

Site-specific recombinases (SSR), such as Cre and Flp recombinases, which enable DNA excision, insertion, and translocation, have been used for conditional target gene expression in mouse and other vertebrates. In this study, we evaluated another SSR, Dre-recombinase (Dre), which is functionally similar to Cre recombinase in porcine fibroblasts and embryos. For this study, 2 fragment DNA constructs (rox GFP-polyA and rox RFP-polyA) were combined with piggybac transposition expression vector (Kim et al. 2011 J. Vet. Med. Sci.) using a multisite gateway cloning system (MultiSite Gateway® Pro, Invitrogen, Carlsbad, CA, USA). The expression vector carrying rox-flanked green fluorescent protein (GFP) followed by red fluorescent protein (RFP) and transposase were transfected into kidney-derived porcine cells by nucleofection (Neon® Transfection System, Invitrogen). A GFP-expressing cell line, which was not expressing RFP, was established. And then rox-flanked GFP were removed by Dre transfection and RFP was expressed in the kidney cells. At the cellular level, this excision was confirmed by site-specific RT-PCR and sequencing. The rox-flanked GFP cells were reconstructed with enucleated oocytes and then the cloned embryos were cultured in porcine zygote medium-5. Dre was micro-injected into 1 of the 2-cell-stage blastomeres. After 6 days, RFP expression was observed on the part of embryos after microinjection. In conclusion, the data demonstrated that, like other SSR, Dre might be applied in conditional target gene expression for generating porcine biomedical models.