Diabetes mellitus, the most common metabolic disorder, is divided into two types: type 1 and type 2. The essential treatment of type 1 diabetes, caused by immune-mediated destruction of β-cells, is transplantation of the pancreas; however, this treatment is limited by issues such as the lack of donors for islet transplantation and immune rejection. As an alternative approach, stem cell therapy has been used as a new tool. The present study revealed that bone marrowderived mesenchymal stromal cells (BM-MSCs) could be transdifferentiated into pancreatic cells by the insertion of a key gene for embryonic development of the pancreas, the pancreatic and duodenal homeobox factor 1 (PDX1). To avoid immune rejection associated with xenotransplantation and to develop a new cell-based treatment, BM-MSCs from α-1,3-galactosyltransferase knockout (GalT KO) pigs were used as the source of the cells. Transfection of the EGFP-hPDX1 gene into GalT KO pig-derived BM-MSCs was performed by electroporation. Cells were evaluated for hPDX1 expression by immunofluorescence and RT-PCR. Transdifferentiation into pancreatic cells was confirmed by morphological transformation, immunofluorescence, and endogenous pPDX1 gene expression. At 3∼4 weeks after transduction, cell morphology changed from spindle-like shape to round shape, similar to that observed in cuboidal epithelium expressing EGFP. Results of RT-PCR confirmed the expression of both exogenous hPDX1 and endogenous pPDX1. Therefore, GalT KO pig-derived BM-MSCs transdifferentiated into pancreatic cells by transfection of hPDX1. The present results are indicative of the therapeutic potential of PDX1-expressing GalT KO pig-derived BM-MSCs in β-cell replacement. This potential needs to be explored further by using in vivo studies to confirm these findings.

A major barrier to progress in pig to primate organ transplantation or cell therapy is the presence of terminal α -1,3-galactosyl epitopes on the surface of pig cells. Therefore, the purpose of this experiment was to establish and cha- racterize mesenchymal stromal/stem cells (MSCs) derived from α-1,3-galactosyltransferase (GalT) knock out (GalT KO) pig to confirm their potential for cell therapy. Bone marrow (BM)-MSCs from GalT KO pig of 1 month old were isolated by Ficoll-Paque PLUS gradient and cultured with A-DMEM + 10% FBS on plastic dishes in 5% CO2 incubator at 38.5. GalT KO BM-MSCs were analyzed for the expression of CD markers (CD45－, 29+, 90+ and 105+) and in vitro differentiation ability (adiopogenesis and osteogenesis). Further, cell proliferation capacity and cell aging of GalT KO BM-MSCs were compared to Wild BM-MSCs by BrdU incorporation assay (Roche, Germany) using ELISA at intervals of two days for 7 days. Finally, the cell size was also evaluated in GalT KO and Wild BM-MSCs. Statistical analysis was performed by T-test (P<0.05). GalT KO BM-MSCs showed fibroblast-like cell morphology on plastic culture dish at passage 1 and exhibited CD45－, 29+, 90+ and 105+ expression profile. Follow in ginduction in StemPro adipogenesis and osteogenesis media for 3 weeks, GalT KO BM-MSCs were differentiated into adipocytes, as demonstrated by Oilred Ostaining of lipid vacuoles and osteocytes, as confirmed by Alizarinred Sstaining of mineral dispositions, respectively. BrdU incorporation assay showed a significant decrease in cell proliferation capacity of GalT KO BM-MSCs compared to Wild BM-MSCs from 3 day, when they were seeded at 1×103 cells/well in 96-well plate. Passage 3 GalT KO and Wild BM-MSCs at 80% confluence in culture dish were allowed to form single cells to calculate cell size. The results showed that GalT KO BM-MSCs (15.0 ± 0.4 μm) had a little larger cell size than Wild BM-MSCs (13.5 ± 0.3 μm). From the above findings, it is summarized that GalT KO BM-MSCs possessed similar biological properties with Wild BM-MSCs, but exhibited a weak cell proliferation ability and resistance to cell aging. Therefore, GalT KO BM-MSCs might form a good source for cell therapy after due consideration to low proliferation potency in vitro.

This study was conducted to analyze the transgenic efficiency and sex ratio in -1,3-galactosyltransferase (GalT) knock-out (KO) transgenic pigs according to generation. GalT KO piglets were produced by artificial insemination or natural mating. The transgenic confirmation of GalT KO was evaluated by PCR amplification using specific primers. After electrophoresis, three types of bands were detected such as 2.3 kb single band (Wild), 2.3 and 3.6kb double bands (GalT KO -/+; heterozygote), and 3.6kb single band (GalT KO -/-; homozygote). Transgenic efficiency in F1 generation was 64.5% (23/35) of GalT KO (-/+). In F2 generation, GalT KO transgenic efficiency was 36.4% (21/57, Wild), 47.5% (28/57, GalT KO -/+), and 16.1% (8/57, GalT KO -/-), respectively. Interestingly, no homozygote piglets were born in 6 deliveries among total 11 deliveries, although they were pregnant between male (M) and female (F) heterozygote. In the 5 litters including at least one GalT KO -/- piglet, the transgenic efficiency was 13.3% (2/24, Wild), 51.3% (14/24, GalT KO -/+), and 35.3% (8/24, GalT KO -/-), respectively. The sex ratio of M and F was 40:60 in and 49:51 in generation, respectively. Based on these results, GalT KO transgenic pigs have had a reproductive ability with a normal range of transgenic efficiency and sex ratio.

The purpose of this study was undertaken to evaluate of cryopreservation efficiency in α 1,3-galactosyltransferase knock-out(GalT KO) cloned miniature pig sperm. To compare ability of frozen-thawed sperm characteristics, three different pig strains (GalT KO) cloned miniature pig, PWG miniature pig and Duroc were used. The ejaculated semen from the three pig species was diluted with same volume extender and added to LEY solution for freezing. The diluted semen was placed in 0.5 ml straws, and freezing was initiated by exposing the straws to liquid nitrogen (LN2) vapours for 10 min before placing them into LN2 for cryopreservation. A fter thawing, the sperm ability were assessed for viability (SYBR-14/PI staining), abnormality (Rose Bengal staining), and acrosome status (intactness, intensity and capacitation) (chlorotetracycline, CTC staining). The viability of frozen-thawed GalT KO pig sperm had no significant difference as compared with Duroc and PWG miniature pig sperm. However, The CTC pattern of frozen-thawed GalT KO cloned miniature pig spermatozoa showed significantly lower rates in F pattern and AR pattern (p<0.05) and significantly higher rates in B pattern than Duroc and PWG miniature pig (p<0.05). The abnormality of GalT KO cloned miniature pig sperm was significantly lower as compared to Duroc and PWG miniature pig sperm (p<0.05). In conclusion, GalT KO cloned miniature pig semen can be cryopreserved successfully and used for artificial insemination reasonably.

본 연구의 목적은 집단논리적사고력검사의 이용 목적에 따라 일반화가능도이론을 적용하여 문항과 피험자만을 고려한 단일국면의 오차원, 그리고 문항과 피험자, 그리고 영역을 고려한 다국면의 오차원을 분석하는 데 있다. 연구는 지방 소재 초 중 고등학생 총 1016명을 대상으로 이루어졌으며, 21문항의 GALT 완본을 40분 동안 실시하고, 이 중 축소본에 해당하는 12문항을 별도로 추출하여 일반화가능도이론을 이용한 신뢰도 분석에 이용하였다. 자료의 분석을 위해 일반화가능도이론을 적용하여 p×i설계와 p×(i:h)설계로 나누어 G 연구와 D 연구를 실시하였다. 분석결과는 다음과 같다. 첫째, 완본과 축소본을 p×I설계로 D 연구를 수행한 결과 완본의 경우 21문항을 평가했을 때 0.87로 적정 수준의 일반화가능도 계수인 0.80을 상회하였으며, 13문항에서도 적정 수준의 일반화가능도 계수에 도달하였다. 축소본의 경우 12문항을 평가했을 때 0.77로 적정 수준의 일반화가능도 계수에 미치지 못하였으며, 최소 15문항 이상에서 신뢰도가 적정 수준에 도달하였다. 둘째, 축소본을 p×(I:H)설계로 D 연구를 수행한 결과 6영역에 대해 영역별로 2문항씩 구성될 경우 0.71로 적정 수준의 일반화가능도계수인 0.80 보다 낮게 측정되었으며, 최소 영역별 5문항 이상에서 신뢰도가 적정 수준에 도달하였다.