Variation occurred in treated groups compared to control groups, but higher levels were still found in cloned embryos compared with their in vivo counterparts. Similar results were observed for the XIST gene in only one treated group, whereas variation in XIST gene expression was notably higher in the other treated groups than in the control groups. These results indicate that not all X-linked gene expression in cloned embryos responded to Sc treatment in the same manner; moreover, the effectiveness of this treatment during the SCNT process may be limited to a few genes. Similar findings were reported by Whitworth et al. [43], with increased XIST expression in porcine IVF embryos and up- or downregulated expression in male cloned embryos from ex vivo collections compared with in vivo controls. Unfortunately, because this study was performed using pooled embryo cDNA samples of mixed sex, making a direct comparison among embryos for this gene is difficult. Nevertheless, this finding appears to indicate that aberrant regulation of X-linked gene expressions can occur within a short exposure time to in vitro environments, even before the fertilization process, or throughout the SCNT process. Additionally, note that porcine IVM systems can lead to epigenetic defects in oocytes that mature in vitro, which may vary GSK2606414 site greatly among individuals [44]. Considering the possibility of misinterpretation due to individual variation in Xlinked gene expression in in vitro-produced IVF and cloned embryos, transcriptional analyses of individual embryos may benecessary to gain a more robust measure of gene expression in preimplantation embryos. This will not only advance our understanding of the reprogramming process, but will also permit more accurate predictions of the factors affecting the in vitro environments of developing embryos. Moreover, a more refined analysis, such as RNA FISH for XIST, is GSK2256098 web needed to determine XCI patterns during embryonic development and to clarify how XCI occurs in cloned embryos.Materials and Methods Ethics StatementThe pig experiments were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Veterinary and Quarantine Service and were supervised by Gyeonggido Livestock and Veterinary Service. Each study was approved by the animal ethics committee of Sooam biotech research foundation (license number AEC-20081021-0001). Porcine ovaries were provided by the regional slaughterhouse (Hyup-Shin, Anyang, Korea).In vitro Maturation (IVM)Ovaries were collected from prepubescent gilts at a local slaughterhouse and transported to the laboratory in 0.9 (w/v) NaCl supplemented with 100 mg/ml streptomycin sulfate (Amresco, Solon, OH) within 1 h at 37uC. Cumulus ocyte complexes (COCs) were obtained from follicles that were 3? mm in diameter using 18-gauge microneedles. Oocytes possessing an evenly granulated cytoplasm and a compact surrounding cumulus mass were collected and washed twice with TL EPES VA medium (Tyrode’s lactate EPES medium supplemented with 0.01 polyvinyl alcohol). After washing, 40?0 COCs were transferred to 500 ml of IVM medium (TCM-199; Invitrogen, Carlsbad, CA) supplemented with 10 ng/ml epidermal growth factor (EGF), 1 mg/ml insulin (Sigma-Aldrich, St. Louis, MO), 4 IU/ml eCG (Intervet, Boxmeer, The Netherlands), hCG (Intervet), and 10 (v/v) porcine follicular fluid (pFF). After 22 h of culture, the COCs were transferred to an IVM medium witho.Variation occurred in treated groups compared to control groups, but higher levels were still found in cloned embryos compared with their in vivo counterparts. Similar results were observed for the XIST gene in only one treated group, whereas variation in XIST gene expression was notably higher in the other treated groups than in the control groups. These results indicate that not all X-linked gene expression in cloned embryos responded to Sc treatment in the same manner; moreover, the effectiveness of this treatment during the SCNT process may be limited to a few genes. Similar findings were reported by Whitworth et al. [43], with increased XIST expression in porcine IVF embryos and up- or downregulated expression in male cloned embryos from ex vivo collections compared with in vivo controls. Unfortunately, because this study was performed using pooled embryo cDNA samples of mixed sex, making a direct comparison among embryos for this gene is difficult. Nevertheless, this finding appears to indicate that aberrant regulation of X-linked gene expressions can occur within a short exposure time to in vitro environments, even before the fertilization process, or throughout the SCNT process. Additionally, note that porcine IVM systems can lead to epigenetic defects in oocytes that mature in vitro, which may vary greatly among individuals [44]. Considering the possibility of misinterpretation due to individual variation in Xlinked gene expression in in vitro-produced IVF and cloned embryos, transcriptional analyses of individual embryos may benecessary to gain a more robust measure of gene expression in preimplantation embryos. This will not only advance our understanding of the reprogramming process, but will also permit more accurate predictions of the factors affecting the in vitro environments of developing embryos. Moreover, a more refined analysis, such as RNA FISH for XIST, is needed to determine XCI patterns during embryonic development and to clarify how XCI occurs in cloned embryos.Materials and Methods Ethics StatementThe pig experiments were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Veterinary and Quarantine Service and were supervised by Gyeonggido Livestock and Veterinary Service. Each study was approved by the animal ethics committee of Sooam biotech research foundation (license number AEC-20081021-0001). Porcine ovaries were provided by the regional slaughterhouse (Hyup-Shin, Anyang, Korea).In vitro Maturation (IVM)Ovaries were collected from prepubescent gilts at a local slaughterhouse and transported to the laboratory in 0.9 (w/v) NaCl supplemented with 100 mg/ml streptomycin sulfate (Amresco, Solon, OH) within 1 h at 37uC. Cumulus ocyte complexes (COCs) were obtained from follicles that were 3? mm in diameter using 18-gauge microneedles. Oocytes possessing an evenly granulated cytoplasm and a compact surrounding cumulus mass were collected and washed twice with TL EPES VA medium (Tyrode’s lactate EPES medium supplemented with 0.01 polyvinyl alcohol). After washing, 40?0 COCs were transferred to 500 ml of IVM medium (TCM-199; Invitrogen, Carlsbad, CA) supplemented with 10 ng/ml epidermal growth factor (EGF), 1 mg/ml insulin (Sigma-Aldrich, St. Louis, MO), 4 IU/ml eCG (Intervet, Boxmeer, The Netherlands), hCG (Intervet), and 10 (v/v) porcine follicular fluid (pFF). After 22 h of culture, the COCs were transferred to an IVM medium witho.
