Rats are an important laboratory animal for biomedical research. Though rats have some physiology and genetic similarities to human, several technical issues such as delicate in vitro culture system and low survival rate after pronuclear microinjection have hindered the development of transgenic rat generation. Accordingly, in this study, to produce transgenic rat, we established transposon-mediated insertional mutagenesis by cytoplasmic microinjection. The sleeping beauty transposon (SB) and SB-transposase recognize the precise genome integration into a TA nucleotide by ‘cut-and-paste’ mechanism. It mediates stable integration and reliable long-term expression. DNA, 0.4ng/ul SB vector (IR/DR-EF1a-eGFP-2A-IL2-pA-IR/DR) and mRNA, 5ng/ul SB-transposase were injected to 1-cell stage embryo and one transgenic rat was generated after full-term gestation. To confirm the genome insertion, GFP was detected by PCR. Further, this method was applied to generate transgenic rats producing Cas9 protein. DNA, 0.4ng/ul SB vector (IR/DR-CAG-Cas9-2A-eGFP-pA-IR/DR) and mRNA, 5ng/ul SB-transposase were injected to 1-cell stage embryo. Some of the in vitro cultured embryos showed GFP positive at blastocyst stage and Cas9 sequence was detected by PCR. One stillbirth pup was born to date and genome PCR on Cas9 was positive. In summary, the SB transposon system could be a highly effective method that contribute to the production of transgenic rats. If the protocols will be optimized, we successfully generated efficiently transgenic rats for human models by SB system. 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.

Interferon tau (IFNT), has known as a key signal molecule for a period of pregnancy in ruminants owing to the need on maternal recognition of pregnancy. It is generated in trophectoderm cells of the elongation bovine conceptus at day 13-21 and a peak output is at day 15-17 of pregnancy period. Moreover, other studies indicated that it can be effective in the embryonic development and quality. In previous study, there were 8 bovine IFNT, but only 2 forms of IFNTs, IFNT2 and IFN-tau-c1, were expressed by the conceptuses during the peri-implantation. In this study, we target the one between the two, IFN-tau-c1 and then the effect of IFNT knockout in donor cells to bovine cloned embryonic development by somatic cell nuclear transfer (SCNT) was investigated. In order to proceed this study, the immature oocytes from the ovaries at local slaughterhouse have been matured in vitro for 22 hours. For preparing the donor cell that have a mutation on IFNT gene, somatic cells were transiently transfected with Cas9 protein and single guide RNA targeting IFNT, and various single derived colonies with high proliferation were isolated and confirm the mutation by PCR. Finally, one colony had mono-allelic mutation (4bps deletion) was picked out and applied as the donor cell to SCNT. A donor cell was injected into an oocyte that nucleus was removed. Reconstructed oocytes with the donor cell were fused by electrical shock, activated by chemical stimulation and cultured for 7 days in chemically defined medium. In this study, control (n=199) and IFNT knockout-group (n=219) were compared with four replications. As results, there was no significant difference between control-and IFNT-knockout group not only in cleavage rate, but also blastocyst formation rate (Control: 12.3% ± 9.2, IFNT knockout-group: 20.1 ± 11%). In addition, the number of blastocyst cell was not different between control (91.7 ± 26.2) and IFNT knockout group (83.5 ± 21.3). 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 indicated that the interruption of IFNT did not influence the embryonic development. In future study, we will transfer these mutated embryos toto test the effect of IFNT for pregnancy period. 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 purpose of this study is to improve practical skill evaluation method of mushroom - trained certified technician's practical skill evaluation which is one of national qualification tests based on national incompetency standards. One of the current National Competency Standards (NCS), mushroom - trained certified technician uses NCS based practical assessment method. In order to improve the current practical evaluation method, we try to improve practical evaluation method based on field customized problem solving ability and improve the practical evaluation method, various evaluation methods should be constructed. On the purpose of identify the diversity and problems of the evaluation method, the experts of the group consultation, the mushroom-related research institute and the related industry collaborated to identify the problems of the actual mushroom - trained certified technician practicum test, This study on the evaluation improvement method was carried out. In this study, the contents of practical test of the current mushroom traits were analyzed and the trends of the latest mushroom industry were widely reflected.

Recently, we published a microinjection method for generating transgenic cattle using the DNA transposon system and their analysis by next-generation sequencing (Yum et al. Sci Rep. 2016 Jun 21;6:27185). In that study, we generated transgenic cattle using two different types of DNA transposon system, sleeping beauty (SB) and piggybac (PB), carrying Yellow fluorescent protein with SB (SB-YFP, female) and green fluorescent protein with PB (PB-GFP, male) under the control of the ubiquitous CAG promoter, respectively. The female and male founder cattle have been grown up to date (the female age: 40 months old, the male age: 33 months old) without any health issues. In genomic instability and blood analysis, there was no significant differences between wild type and founder cattle. In the present study, we confirmed germ-line transmission of the transposon-mediated transgene integrations and ubiquitous and persistent expression of transgene in second generation of offspring (F1). The F1 was born without any assistance and expressed GFP in the eyes without UV light. The ubiquitous expression of GFP was detected in skin fibroblast from the ear tissue and confirmed by genomic DNA PCR, which suggest that the transgene from the PB-GFP was successfully transmitted. Unfortunately, no transgene from SB-YFP were identified. To confirm the transgene integration site, the genomic DNA from blood was extracted and performed next-generation sequencing (NGS). The GFP gene was integrated in chromosome 4 (two copies), and 6. As results, a total of two copies of paternal transgene transmitted into the F1. All the integrated position was not related with coding region and there was no significant difference in genomic variants between transgenic and non-transgenic cattle. To our knowledge, this is the first report of germ-line transmission through non-viral transgenic founder cattle. Those transgenic cattle will be valuable resource to many fields of biomedical research and agricultural science.

CRISPR/Cas9-induced knock-out/-in can be occurred at specific locus in the genome by non-homologous end joining (NHEJ) or homology directed repair (HDR). Here, we demonstrate the targeted insertion into the specific loci of embryo fertilized by semen from transgenic cattle via CRISPR/Cas9 system. Recently, we published on the efficient generation of transgenic cattle using the DNA transposon system (Yum et al. Sci Rep. 2016 Jun 21;6:27185). In the study, eight transgenic cattle were born following transposon-mediated gene delivery system (Sleeping Beauty and Piggybac transposon system) via microinjection. In the analysis of their genome stability using next-generation sequencing, there was no significant difference in the number of genetic variants between transgenic and non-transgenic cattle. All the transgenic cattle have grown up to date (the oldest age: 33 months old, the youngest age: 15 months old) without any health issue. One of transgenic male cattle expressing GFP reached puberty and semen was collected. Over 200 frozen semen straws were produced and some were used for in vitro fertilization (IVF). On seven days after IVF, expression of GFP was observed at blastocyst stage and was seen in 80% of the embryos. Another application is to edit the GFP locus of the transgenic cattle because long-term and ubiquitous expression of transgene didn’t affect their health. In one cell stage embryos produced using GFP frozen-thawed semen, microinjection of sgRNA for GFP, Cas9, together with donor DNA that included RFP and homology arms to link the double-strand break of sgRNA target site into fertilized eggs resulted in expression of RFP. This indicated that the GFP locus of transgenic cattle shows potential candidates for stable insertion of the functional transgene. Knock-out/-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 P-112 that transgenic cattle via transposon system are healthy to date and germ-line competence was confirmed. The GFP locus will be used as the potential target site 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 biomedical research and medicine. This work was supported by BK21 PLUS Program for Creative Veterinary Science and Seoul Milk Coop (SNU 550-20160004).

The CRISPR/Cas9 system is proved to be a powerful tool for knock-out and knock-in in various species. By introducing genetic materials of two components (Cas9 and small guide (sg) RNA) into cells or pronuclear of the fertilized embryo, gene editing occurs. Some studies reported that efficiency of gene editing would be increased as Cas9 was integrated into cells or animals since Cas9 is indispensable in the CRISPR/Cas9 system. Accordingly, the production of Cas9 expressing cattle may provide the broadly used gene editing platform in cattle. For this study, Cas9 and RFP genes were cloned into PiggyBac (PB) transposon system. PB-Cas9-RFP and transposase were microinjected into 1436 in vitro fertilized embryos and 241 blastocysts were formed. Blastocysts with RFP expression accounting for 14.1% of total formed blastocysts were selected and transferred into 5 recipient cow. After gestation periods, four transgenic cattle were delivered without any veterinary assistance. From a transgenic cattle, ear skin tissue was collected for primary culture. On those primary cells, sgRNAs in DNA form for various genes such as PRNP, RB1 and BLG were transfected as 2ug of sgRNA per 5x105 cells using Nucleo factor system (Neon®, invitrogen, program#16). As expected, every group of each sgRNA delivered was confirmed to be mutated by T7E1assay. Those data demonstrated that for the first time, transgenic cattle with Cas9 expression were born, grown up to date and will be avaluable resource for genome-editing in cattle. This work was supported by BK21PLUS Program for Creative Veterinary Science and Seoul Milk Coop (SNU550-20160004).

Mesenchymal stem cell (MSC) based cell therapy has emerged as a promising therapeutic approach for treatment of several degenerative, infectious and non-infectious diseases. Numerous studies have demonstrated the remarkable immunosuppressive and antibacterial effects of MSCs both in vitro and in vivo, in animal models and in humans. However, the antibacterial effects of MSCs rely heavily on their paracrine factors rather than direct cell-to-cell contact and the effect is specific to disease and site of infection or injury. Furthermore, recent studies have demonstrated the double-edged sword effect of MSCs in bacterial infectious diseases. Despite their inherent potential for repair of damaged tissues, immunosuppression, and alleviation of various autoimmune as well as infectious diseases, MSCs also play a critical role in promoting persistent bacterial infection and disease progression. Therapeutic administration of MSCs successfully inhibited the bacterial growth and enhances survival by improved clearance of pathogenic bacteria in sepsis and pneumonic conditions. However, due to their abnormal transformation, they assist in long lasting survival and persistent infection of Mycobacterium tuberculosis (M. tuberculosis) and may also be responsible for progression of gastric cancer. This review focuses on recent advances that have broadened our understanding of MSC based therapy for bacterial diseases and provides new insight into the possible therapeutic targets of fatal bacterial diseases.

Semen can be divided into two parts. One is cellular part which contains sperms the other is liquid part which is called by seminal plasma. The seminal plasma is a nutritive and protective medium for the sperms. Fructose, which is major energy source, is supplied to sperms swim to female oocyte. Alkalic property protects sperms from hostile environment of female reproductive organ. Also, seminal plasma induces tolerance to preexisted immune cells, and changes intra‐uterine environment to better conditions for fertilized embryos to implant. However, the effects of seminal plasma in in vitro culture of fertilized embryos are unclear. Second fraction of fresh semen was obtained from a normal farm pig. The semen was centrifuged to remove sperms, and then supernatant was filtrated. The filtered seminal plasma was stored in — 30℃. In this study, electrically activated and chemically activated porcine embryos were employed to investigate the developmental rate after 2 hours treatment of none, 0.1%, 0.5%, and 1% seminal plasma in culture media by two days of activation. Both electrically and chemically activated embryos, cleavage rate and cell numbers of blastocysts were not significant difference within four groups. Blastocyst formation rate of electrically activated embryos also did not show significant difference within any groups. However 0.1% seminal plasma treatment group showed significantly increase of blastocyst formation rate in chemically activated group (None; 24.8%, 0.1%; 31.7%, 0.5%; 19.4, and 1%; 16.5%, respectively. p<0.05).

Minipigs are regarded as one of the most important laboratory animal in that anatomical and physiological properties are similar to human and their reproduction efficiency is relatively higher compared to other large animal species. Particularly, several diseases that cannot be mimicked in rodent models are successfully occurred or induced in pig models therefore it has been interested in a valuable model for human diseases. Pigs are also ‘standard’ species in xenotransplantation research. To maximize experimental outcome using minipigs, establishment and management of proper animal facility, right animal husbandry and control of pathogens are very important. In this review, we summarized several international guidelines related with minipigs published by several companies or governments and discuss optimal conditions for providing informative ideas to the researchers who want to use minipigs in their future studies.

The necessity of conditional gene expression in pigs for transgenic models is raised. Thus, in this study, Cre-loxP conditional expression in porcine fetal fibroblasts was investigated and the transformed fibroblasts were reprogrammed in enucleated oocytes for further early embryonic development. Fetal fibroblasts from miniature pigs were used for transfection with pCALNL-DsRed including floxed neomycin resistant gene and selected with 750 ug/mL neomycin for two weeks. The transfected cells did not express DsRed under fluorescence microscope. After transient transfection of plasmid DNA expressing Cre, the fibroblasts began to express DsRed. The cells expressing Ds- Red were employed into somatic cell nuclear transfer (SCNT). A total of 121 oocytes were used for SCNT and 76 cloned embryos (62.8%)were cleaved. Six blastocysts were grown up after SCNT and expressed DsRed. Deletion of floxed neomycin resistant gene was confirmed by RT-PCR in cloned blastocysts. Taken together, this study demonstrated that Cre-loxP recombination in miniature pig fibroblasts were successfully worked and those sequential transformed cells were developed into pre-implantation stage via SCNT.

The present study investigated the effects of follicle stimulating hormone (FSH) and human chorionic gonadotrophin (hCG) on the nuclear maturation of canine oocytes. Oocytes were recovered from mongrel female ovaries in various reproductive states; follicular, luteal or anestrous stage. Oocytes were cultured in serum-free tissue culture medium (TCM)-199 supplemented with various concentrations of FSH (Exp. 1: 0, 0.5, 1.0 or 10 IU) or hCG (Exp. 2: 0, 0.5, 1.0 or 10 IU) or both (Exp. 3: 1 IU FSH + 1 IU hCG) for 72 hr to determine the effective concentration of these hormones, and to examine their combined effect. After maturation culture, oocytes were denuded in PBS containing 0.1% (w/v) hyaluronidase by gentle pipetting. The denuded oocytes were stained with 1.9 μM. Hoechst 33342 in glycerol and the nuclear state of oocytes was evaluated under UV light. More (p<0.05) oocytes matured to MII stage when follicular stage oocytes were supplemented with 1 IU FSH (6.2%) compared with the control, 0.1 or 10.0 IU FSH (0 to 1.2%). Significantly higher (p<0.05) maturation rate to MII stage was observed in follicular stage oocytes supplemented with 1.0 IU hCG (7.2%) compared with the control or other hCG supplemented groups (0 to 1.5%). However, the combination of FSH and hCG did not improve the nuclear maturation rate of canine oocyte (2.4 %) compared with FSH (6.2%) and hCG alone (7.2%). In conclusion, FSH or hCG alone significantly increased the maturation of canine oocytes to MII stage.