Endoplasmic reticulum (ER) stress is well known as a suppressor in osteoblast differentiation and activating transcription factor 3 (ATF3) could be induced by a various extracellular signals including cytokines, hormones, DNA damage. Up to date, although the role of ATF3 have been studied, the function of ATF3 in osteoblast differentiation is still not clear yet. Our study showed that expression level of ATF3 could be incresed by tunicamycin which is ER stress inducer in preosteoblasts. BMPs, which are secreted by osteoblasts, can be important regulators in osteogenic differentiation. The stress-responsive transcription factor ATF3 is a negative regulator of osteoblast differentiation in MC3T3-E1 cells. In this study, we verified that BMP2-stimulated osteoblast differentiation could be inhibited by over-expressed ATF3 through regulating alkaline phosphatase (ALP) expression and activation.

The α-Gal epitope (Galα1,3Galα1,4GlcNAc-R) is responsible for hyperacute rejection (HAR) during transgenic pig-to-non-human primate xenotransplantation. To overcome HAR after xenografts, it is essential for the inactivation of α1,3Galactosyltransferase (GT) gene by the homozygotic knocked out of GT-/- and the isoglobotrihexosylceramide synthase (iGb3s-/-). This study was performed to investigate the generation and characterization of the α1,3GT-MCP/-MCP+iGb3-/- transgenic cells. Ear fibroblast cells from the GT-MCP/-MCP pig were cultured and used to positive control. For iGb3s knock out, the Cas9-GFP-iGb3s vector was transfected into the GT-MCP/-MCP cells. The Cas9-GFP-iGb3s transfected cells were sorted and sequenced for the selection of GT-MCP/-MCP+ iGb3s-/- cells. Among the three sorted cell lines, one transgenic cell line was homozygously deleted 3 bases and 10 bases in each chromosome, respectively. To characterize an expression of α-Gal epitope, a wild type and the transgenic cells were measured by FACS Aria using BS-IB4 lectin antibody. The expression of α-Gal epitope in GT-MCP/-MCP cells (<0.01 %) were significantly down-regulated to the range of wild type (99.4 %) fibroblast cells (p<0.05). To analyze the function of iGb3s, α -Gal epitope expressions were measured for the GT-MCP/-MCP, GT-MCP/-MCP+iGb3s-/+, and GT-MCP/-MCP+iGb3s-/-. The range was 95.8%, 94.2%, and 75.8%, respectively. Interestingly, there was a negative range (16.2%) of α-Gal epitope -/- section in GT-MCP/-MCP+iGb3s-/-, compared to 2.74% of GT-MCP/-MCP+iGb3s-/+ and 1.4% of WT, respectively. Our results demonstrated that iGb3s-/-combined with GT-/- had a function to inhibit α-Gal epitope expression in pig cells. Further studies are needed to evaluate the functions of double gene knock out to minimize a HAR response after xenotransplantation.

The α-Gal epitope (Galα1,3Galα1,4GlcNAc-R) is responsible for hyperacute rejection (HAR) during transgenic pig-to-non-human primate xenotransplantation. There are genes related to the expression of α-Gal epitope such as α1,3Galactosyltransferase gene (GT-/-) and the isoglobotrihexosylceramide synthase (iGb3s-/-). This study was performed to investigate the expression of α-Gal epitope in the skin derived from GT-/- transgenic pig. The skin (7/1000 inches) was obtained by dermatome (Zimmer® Electric Dermatome) from one month old of wildtype (WT) and GT-/- piglets, respectively. The skins were fixed, dehydrated, cleaned, and embedded. To analyze the expression of α-Gal epitope, the paraffin section of WT and GT-/- were stained with BS-IB4 lectin and isoglobotrihexosylceramide synthase antibody. There was a strong BS-IB4 lectin signal in the skin of WT, but not detected in GT-/-. However, the iGb3s positive signals were stained in the skin of both WT and GT-/-. Taken together, it can be postulated that the knocked out of GT gene may not enough to inhibit the expression of α-Gal epitope. Further studies are needed to evaluate the functions of the double knock out of GT and iGb3s on the expression of α-Gal epitope.

Ribosomal protein L21 (RPL21) plays an important role in ribosome assembly. It is considered to be a major cause for the occurrence of the hypotrichosis simplex (HTS), a type of sustained hair loss from early childhood to adulthood. In this study, the full-length sequence of pig RPL21 gene (GenBank accession number: KU891824) was cloned and identified for the first time. We found it contains a 483-bp open reading frame (ORF) encoding 160 amino acids. It is located in the plus strand of chromosome 11, which spans 2,167 bp from 4,199,792 to 4,201,958. We found RPL21 expression level is closely related to cell proliferation and cell cycle arrest. In the knockdown group, the cell proliferation activity was significantly decreased (P<0.01) and an obvious accumulation of cells at the G2/M phase with a simultaneous up-regulation of p53 and p21 was observed. This likely due to knockdown of RPL21 triggered ribosomal stress, which affected the normal ribosome assembly and caused defective ribosome biogenesis. The unassembled RPs were released consequently from the nucleolus to the nucleoplasm where they can activate p53-dependent cell-cycle responsive factors and led to a G2/M arrest. We expect these results may provide valid information for further study on the pig RPL21 gene and the cause of hypo trichosis simplex.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas9) system can be applied to produce transgenic pigs. Therefore, we applied CRISPR/Cas9 system to generate FoxN1-targeted pig parthenogenetic embryos. Using single guided RNA targeted to pig FoxN1 genes was injected into cytoplasm of in vitro matured oocyte before electrical activation. In results, regardless of the concentrations of vector, the cleavage rate were significantly (p<0.05) decreased (4 ng/μl, 51.24%; 8 ng/μl, 40.88%; and 16 ng/μl; 45.22%) compared to no injection group (70.44%). The blastocyst formation rates were also decreased in vector injected 3 groups (4 ng/μl, 7.96%; 8 ng/μl, 6.4%; and 16 ng/μl; 9.04%) compared to no injection group (29.07%). In addition, the blastocyst formation rates between sham injected group (13.51%) and no injection group (29.07%) also showed significant difference (p<0.05). The mutation rates were comparable between groups (4 ng/μl, 18.4%; 8 ng/μl, 12.5%; and 16 ng/μl; 20.0%). The sequencing analysis showed that blastocysts derived from each group were successfully mutated in FoxN1 loci regardless of the vector concentrations. However, the deletion patterns were higher than the patterns of point mutation and insertion regardless of the vector concentrations. In conclusion, we described that cytoplasmic microinjection of FoxN1-targeted CRISPR/Cas9 vector could efficiently generate transgenic pig parthenogenetic embryos in one-step.

The shortage of human organs for transplantation has induced the research on the possibility of using animal as porcine. However, pig to human transplantation as known as xeno-transplantation has major problem as immunorejection. Recently, the solutions of pig to human xenotransplantation are commonly mentioned as having a genetically modification which include alpha 1, 3 galatosyl transferase knockout (GTKO) and immune-suppressing gene transgenic model. Unfortunately, the expression level of transgenic gene is very low activity. Therefore, development of gene overexpression system is the most urgent issue. Also, the tissue specific overexpression system is very important. Because most blood vessels are endothelial cells, establishment of the endothelial-specific promoter is attractive candidates for the introduction of suppressing immunorejection. In this study, we focus the ICAM2 promoter which has endothelial-specific regulatory region. To detect the regulatory region of ICAM2 promoter, we cloned 3.7 kb size mini-pig ICAM2 promoter. We conduct serial deletion of 5' flanking region of mini-pig ICAM2 promoter then selected promoter size as 1 kb, 1.5 kb, 2 kb, 2.5 kb, and 3 kb. To analyze promoter activity, luciferase assay system was conducted among these vectors and compare endothelial activity with epithelial cells. The reporter gene assay revealed that ICAM2 promoter has critical activity in endothelial cells (CPAE) and 1 kb size of ICAM2 promoter activity was significantly increased. Taken together, our studies suggest that mini-pig ICMA2 promoter is endothelial cell specific overexpression promoter and among above all size of promoters, 1 kb size promoter is optimal candidate to overcome the vascular immunorejection in pig to human xenotransplantation.

Here we report the productions of genetically modified cloned Massachusetts General Hospital miniature pig (MGH minipig) using freshly thawed donor cells equilibrated with roscovitine. Fibroblasts were isolated from the ear skin of a 10-day-old male MGH minipig. The donor cells were divided into two groups: cultured for 3 days (culture) and freshly thawed with 500 nM roscovitine. The viability of the donor cells was significantly higher at 0 h (94.6±3.5) compared with 1 h (81.7±5.7) after thawing (p=0.028). After 1 hr of equilibration time, the proportion of G0/G1 stage in roscovitine group was not different from 0 hr group, but not in culture medium group (p<0.01), respectively. Although the developmental characteristics were not different in both methods, the pregnancy and delivery rate in freshly thawed group were significantly higher than that of culture group (p<0.01), respectively. In total, 12 TG cloned MGH minipigs were delivered and the individual cloning efficiency was from 0 to 2.54%. Taken together, the use of freshly thawed donor cells equilibrated with roscovitine may be helpful method to increase the productivity of the genetically modified cloned MGH minipigs.

This study was performed to comprehend the developmental characteristics of cloned embryos knocked out (KO) of α-1,3-galactosyltransferase (GalT) gene. Immature oocytes were collected and cultured for 40 hrs (1-step) or 20hrs (with hormone) + 20hrs (without hormone) (2-step). The embryos transferred with miniature pig ear fibroblast cell were used as control. The reconstructed embryos were cultured in PZM-3 with 5% CO2 in air at 38.5℃ for 6 days. To determine the quality of the blstocysts, TUNEL and quantitative realtime RT-PCR were performed. The embryos were transferred to a surrogate (Landrace) at an earlier stage of the estrus cycle. The maturation rate was significantly higher in 2-step method than that of 1-step (p<0.05). The blastocyst development of GalT KO embryos was significantly lower than that of normal cloned embryos (p<0.05). The total and apoptotic cell number of GalT KO blastocysts was not different statistically from control. The relative abundance of Bax-α/Bcl-xl ratio was significantly higher in both cloned blastocysts than that of in vivo blastocysts (p<0.05). Taken together, it can be postulated that the lower developmental potential and higher expression of apoptosis related genes in GalT KO SCNT embryos might be a cause of a low efficiency of GalT KO cloned miniature pig production.

The Korean native pig (KNP) have been considered as animal models for animal biotechnology research because of their relatively small body size and their presumably highly inbred status due to the closed breeding program. However, little is reported about the use of KNP for animal biotechnology researches. This study was performed to establish the somatic cell nuclear transfer (SCNT) protocol for the production of swine leukocyte antigens (SLA) homotype-defined SCNT KNP. The ear fibroblast cells originated from KNP were cultured and used as donor cell. After thawing, the donor cells were cultured for 1 hour with 15 μM roscovitine prior to the nuclear transfer. The numbers of reconstructed and parthenogenetic embryos transferred were 98 ± 35.2 and 145 ± 11.2, respectively. The pregnancy and delivery rate were 3/5 (60%) and 2/5 (40%). One healthy SLA homotype-defined SCNT KNP was successfully generated. The recipient-based individual cloning efficiency ranged from 0.65 to 1.08%. Taken together, it can be postulated that the methodological establishment of the production of SLA homotype-defined cloned KNP can be applied to the generation of transgenic cloned KNP as model animals for human disease and xenotransplantation researches.