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Multiple ribonucleases are widely found in living organisms, but the function and regulation of individual ribonucleases are still not clear. In the present study, we found that one oocytic ribonuclease, RC-RNase, is developmentally expressed in the liver and stored in the oocyte of the bullfrog, while another ribonuclease, RC-RNase L1, is constitutively expressed and retained in the liver at all stages. In females, the expression of RC-RNase increased with the degree of maturity and the concentration of plasma estradiol during oogenesis. In males, the RC-RNase gene was activated in the liver and the newly synthesized protein was secreted into plasma if estradiol was administered. To investigate the mechanism of estrogen-mediated activation of ribonuclease expression, we cloned the RC-RNase promoter and analyzed the putative transcription factor binding sites, e.g. TATA box, ERE, AP1 and CAAT box. Using luciferase as a reporter gene, we found that an estrogen response element in the promoter of RC-RNase was essential for both basic transcription and estradiol-mediated gene activation in estrogen receptor-positive MCF7 cells. These results support the hypothesis that RC-RNase is synthesized in the liver upon stimulation by estradiol during oogenesis, then secreted into the bloodstream and stored in oocytes for embryonic development.

The aims of this study were, firstly, to analyze the biochemical compositions of serum and follicular fluid (FF) from prepubertal gilts after PMSG (1,000 IU) treatment. The concentrations of total proteins, lipids, cholesterol, glucose and sex hormones (progesterone, P 4 ; estradiol-17β, E 2 ; testosterone, T) were measured. Secondary, the effects of porcine FF (pFF) addition (40% and 100%) in IVM media and different culture conditions [Exp. 1: mBMOC-2+20% porcine serum (PS), fresh IVM medium, filtered IVM-conditioned medium, or rabbit oviducts; Exp. 2: mBMOC-2+20%PS or stepwise medium replacement procedures (SMRP) cocultured with or without cumulus cells] on the in vitro development (IVD) of porcine oocytes were also examined. Results showed that no significant differences were found in total protein levels between serum and pFF from different sizes (large, >7 mm; medium, -5-7 mm; small, 0.05). Total lipid concentrations remained constant in serum (395-472 mg/dl), and reduced significantly in the pFF from large follicles (287 mg/dl) at 132 h after PMSG treatment when compared to those at other time points (441-480 mg/dl). Basal cholesterol levels in serum and pFF at 12 h were similar (153-161 mg/dl), but increased at 36 h (186-197 mg/dl). Basal P 4 and E 2 levels in serum (0.1 ng/ml and 5.5 pg/ml) were low, but increased from 0.34 ng/ml and 12.13 pg/ml at 24 h to 0.81 ng/ml and 61.70 pg/ml at 98 h, respectively, after PMSG treatment (p

Nanosilicate platelets (NSP), the form of natural silicate clay that was exfoliated from montmorillonite (MMT), is widely used as a feed additive for its high non-specific binding capacity with mycotoxins such as fumonisin B1 (FB1), and has been evaluated its safety for biomedical use including cytotoxicity, genotoxicity, and lethal dosage (LD). In the study, we further examined its toxicity on the development of CD1 mouse embryos and its capacity to prevent teratogenesis-induced by FB1. In vitro cultures, NSP did not disturb the development and the quality of intact pre-implantation mouse embryos. Further, newborn mice from females consumed with NSP showed no abnormalities. NSP had an unexpected high adsorption capacity in vitro. In contrast to female mice consumed with FB1 only, a very low residual level of FB1 in the circulation, reduced incidence of neutral tube defects and significantly increased fetal weight were observed in the females consumed with FB1 and NSP, suggesting a high alleviation effect of NSP on FB1 in vivo. Furthermore, FB1 treatment disturbed the gene expression of sphingolipid metabolism enzymes (longevity assurance homolog 5, LASS 5; sphingosine kinase 1, Sphk1; sphingosine kinase 2, Sphk2; sphingosine 1- phosphate lyase, Sgpl1; sphingosine 1-phosphate phosphatase, Sgpp1) in the maternal liver, uterus, fetus, and placenta, but NSP administration reversed the perturbations. Based on these findings, we conclude that NSP is a feasible and effective agent for supplementary use in reducing the toxicity of FB1 to animals.

Studies with intact preimplantation mouse embryos and some types of chimaeric aggregates have shown that the most advanced cells are preferentially allocated to the inner cell mass (ICM) rather than the trophectoderm. Thus, differences between 4-cell and 8-cell stage embryos could contribute to the tendency for tetraploid cells to colonise the trophectoderm more readily than the ICM in 4-cell tetraploid[harr ]8 cell diploid chimaeras. The aim of the present study was to test whether 4-cell stage embryos in 4-cell diploid[harr ]8-cell diploid aggregates contributed equally to all lineages present in the E12.5 conceptus. These chimaeras were compared with those produced from standard aggregates of two whole 8-cell embryos and aggregates of half an 8-cell embryo with a whole 8-cell embryo. As expected, the overall contribution of 4-cell embryos was lower than that of 8-cell embryos and similar to that of half 8-cell stage embryos. In the 4-cell[harr ]8-cell chimaeras the 4-cell stage embryos did not contribute more to the trophectoderm than the ICM derivatives. Thus, differences between 4-cell and 8-cell embryos cannot explain the restricted tissue distribution of tetraploid cells previously reported for 4-cell tetraploid[harr ]8-cell diploid chimaeras. It is suggested that cells from the more advanced embryo are more likely to contribute to the ICM but, for technical reasons, are prevented from doing so in simple aggregates of equal numbers of whole 4-cell and whole 8-cell stage embryos.

Heat stress causes a decrease of fertility in roosters. Yet, the way acute heat stress affects protein expression remains poorly understood. This study investigated differential protein expression in testes of the L2 strain of Taiwan country chickens following acute heat stress. Twelve 45-week-old roosters were allocated into four groups, including control roosters kept at 25 °C, roosters subjected to 38 °C acute heat stress for 4 hours without recovery, with 2 hours of recovery, and with 6 hours of recovery. Testis samples were collected for morphologic assay and protein analysis. Some of the differentially expressed proteins were validated by Western blot and immunohistochemistry. Abnormal and apoptotic spermatogenic cells were observed at 2 hours of recovery after acute heat stress, especially among the spermatocytes. Two-dimensional difference gel electrophoresis revealed that 119 protein spots were differentially expressed in chicken testes following heat stress, and peptide mass fingerprinting revealed that these spots contained 92 distinct proteins. In the heat-stressed samples, the heat shock proteins, chaperonin containing t-complex, and proteasome subunits were downregulated, and glutathione S-transferase, transgelin, and DJ-1 were upregulated. Our results demonstrate that acute heat stress impairs the processes of translation, protein folding, and protein degradation, and thus results in apoptosis and interferes with spermatogenesis. On the other hand, the increased expression of antioxidant enzymes, including glutathione S-transferase and DJ-1, may attenuate heat-induced damage. These findings may have implications for breeding chickens that can tolerate more extreme conditions.

Acute heat stress severely impacts poultry production. The hypothalamus acts as a crucial center to regulate body temperature, detect temperature changes, and modulate the autonomic nervous system and endocrine loop for heat retention and dissipation. The purpose of this study was to investigate global gene expression in the hypothalamus of broiler-type B strain Taiwan country chickens after acute heat stress. Twelve 30-week-old hens were allocated to four groups. Three heat-stressed groups were subjected to acute heat stress at 38 °C for 2 hours without recovery (H2R0), with 2 hours of recovery (H2R2), and with 6 hours of recovery (H2R6). The control hens were maintained at 25 °C. At the end, hypothalamus samples were collected for gene expression analysis. The results showed that 24, 11, and 25 genes were upregulated and 41, 15, and 42 genes were downregulated in H2R0, H2R2, and H2R6 treatments, respectively. The expressions of gonadotropin-releasing hormone 1 (GNRH1), heat shock 27-kDa protein 1 (HSPB1), neuropeptide Y (NPY), and heat shock protein 25 (HSP25) were upregulated at all recovery times after heat exposure. Conversely, the expression of TPH2 was downregulated at all recovery times. A gene ontology analysis showed that most of the differentially expressed genes were involved in biological processes including cellular processes, metabolic processes, localization, multicellular organismal processes, developmental processes, and biological regulation. A functional annotation analysis showed that the differentially expressed genes were related to the gene networks of responses to stress and reproductive functions. These differentially expressed genes might be essential and unique key factors in the heat stress response of the hypothalamus in chickens.

Basigin (Bsg), also called extracellular matrix metalloproteinase inducer (EMMPRIN), is highly expressed on the surface of tumor cells and stimulates adjacent fibroblasts or tumor cells to produce matrix metalloproteinases (mmps). It has been shown that Bsg plays an important role in growth, development, cell differentiation, and tumor progression. MicroRNAs (miRNAs) are a class of short endogenous non-protein coding RNAs of 20-25 nucleotides (nt) that function as post-transcriptional regulators of gene expression by base-pairing to their target mRNAs and thereby mediate cleavage of target mRNAs or translational repression. In this study, let-7b, one of the let-7 family members, was investigated for its effect on the growth and invasiveness of the mouse melanoma cell line B16-F10. We have shown that let-7b can suppress the expression of Bsg in B16-F10 cells and also provided evidence that this suppression could result in the indirect suppression of mmp-9. The ability of B16-F10 cells transfected with let-7b to invade or migrate was significantly reduced. In addition, let-7b transfected B16-F10 cells displayed an inhibition of both cellular proliferation and colony formation. Furthermore, it was shown that the overexpression of let-7b in B16-F10 cells could reduce lung metastasis. Taken together, the present study identifies let-7b as a tumor suppressor that represses cancer cell proliferation and migration as well as tumor metastasis in mouse melanoma cells.

Undecylprodigiosin (UP) is a bacterial bioactive metabolite produced by Streptomyces and Serratia. In this study, we explored the anticancer effect of UP. Human breast carcinoma cell lines BT-20, MCF-7, MDA-MB-231 and T47D and one nonmalignant human breast epithelial cell line, MCF-10A, were tested in this study. We found that UP exerted a potent cytotoxicity against all breast carcinoma cell lines in a dose- and time-dependent manner. In contrast, UP showed limited toxicity to MCF-10A cells, indicating UP's cytotoxic effect is selective for malignant cells. UP's cytotoxic effect was due to apoptosis, as confirmed by positive TUNEL signals, annexin V-binding, caspase 9 activation and PARP cleavage. Notably, UP-induced apoptosis was blocked by the pan-caspase inhibitor z-VAD.fmk, further indicating the involvement of caspase activity. Moreover, UP caused a marked decrease of the levels of antiapoptotic BCL-X(L), Survivin and XIAP while enhancing the levels of proapoptotic BIK, BIM, MCL-1S and NOXA, consequently favoring induction of apoptosis. Additionally, we found that cells with functional p53 (MCF-7, T47D) or mutant p53 (BT-20, MDA-MB-231) were both susceptible to UP's cytotoxicity. Importantly, UP was able to induce apoptosis in MCF-7 cells with p53 knockdown by RNA interference, confirming the dispensability of p53 in UP-induced apoptosis. Overall, our results establish that UP induces p53-independent apoptosis in breast carcinoma cells with no marked toxicity to nonmalignant cells, raising the possibility of its use as a new chemotherapeutic drug for breast cancer irrespective of p53 status.

The objective was to characterize the potential laying traits and underlying transcripts expressed in the hypothalamus and pituitary gland that were associated with egg production variability in five genetic stocks of chickens: two commercial lines, Red- (n = 12) and Black-feather (n = 14) Taiwan country chickens (TCCs); two selected lines of TCCs, B (high body weight/comb size; n = 17) and L2 (high-egg production; n = 14); and a commercial single comb White Leghorn (WL; n = 17). Six laying traits, age at first egg, clutch length, pause length, oviposition lag within clutch, follicle rapid growth period, and rate of yolk accumulation were measured. The significance of differential values among five chicken stocks and correlation coefficients between laying traits and number of eggs to 50 weeks of age or laying rate after first egg, and the expression level of 33 transcripts were determined. Longer clutch length and shorter oviposition lag within clutch contributed to a higher number of eggs to 50 weeks of age or laying rate after first egg in L2 (P < 0.05) and WL strains (P < 0.05). However, their rate of yolk accumulation (P < 0.05) and follicle rapid growth period (P < 0.05) were different, indicating the accumulation of different alleles after long-term, independent selection. Across all five strains, numbers of eggs to 50 weeks of age were positive correlated with average clutch length (P < 0.05) as well as the rate of yolk accumulation (P < 0.05). Expressions of PLAG1, STMN2, PGDS, PARK7, ANP32A, PCDHA@, SCG2, BDH and SAR1A transcripts contributed to number of eggs to 50 weeks of age (P < 0.05) or laying rate after first egg (P < 0.05). Analysis of correlation coefficients indicated that PLAG1 additionally played roles in decreasing average pause length. Two transcripts, PRL and GARNL1, specifically contributed to number of eggs to 50 weeks of age or laying rate after first egg by reducing oviposition lag within clutch (P < 0.05) and/or increasing average clutch length (P < 0.05), respectively. Expression level of NCAM1, contributed to laying rate after first egg by association with a shorter oviposition lag within clutch (P < 0.05). The current study attributed egg production phenotype in five strains into several laying traits; correlations between these traits and expression levels of underlying transcripts expressed in the hypothalamus and pituitary gland were also established.

The expression of testicular genes following acute heat stress has been reported in layer-type roosters, but few similar studies have been conducted on broilers. This study investigated the effect of acute heat stress on the gene expression in the testes of a broiler-type strain of Taiwan country chickens. Roosters were subjected to acute heat stress (38°C) for 4 h, and then exposed to 25°C, with testes collected 0, 2, and 6 h after the cessation of heat stress, using non-heat-stressed roosters as controls (n = 3 roosters per group). The body temperature and respiratory rate increased significantly (p

In the present study, we investigated the effects of specific cdc2 kinase inhibitor, butyrolactone I (BL I) on the prevention of germinal vesicle breakdown, changes of microtubular structures, and development of porcine oocytes after removal of the drug. In Experiment 1, cumulus-oocyte complexes (COCs) were cultured (44 h) in NCSU-23 medium containing different concentrations of BL I. The percentages of oocytes remaining at GV stage were 0, 0, 32, 80, and 84% (P

To identify transcripts related to high egg production expressed specifically in the hypothalamus and pituitary gland of the chicken, two subtracted cDNA libraries were constructed. Two divergently selected strains of Taiwan Country Chickens (TCCs), B (sire line) and L2 (dam line) were used; they had originated from a single population and were further subjected (since 1982) to selection for egg production to 40 wk of age and body weight/comb size, respectively. A total of 324 and 370 clones were identified from the L2-B (L2-subtract-B) and the B-L2 subtracted cDNA libraries, respectively. After sequencing and annotation, 175 and 136 transcripts that represented 53 known and 65 unknown non-redundant sequences were characterized in the L2-B subtracted cDNA library. Quantitative reverse-transcription (RT)-PCR was used to screen the mRNA expression levels of 32 randomly selected transcripts in another 78 laying hens from five different strains. These strains included the two original strains (B and L2) used to construct the subtracted cDNA libraries and an additional three commercial strains, i.e., Black- and Red-feather TCCs and Single-Comb White Leghorn (WL) layer. The mRNA expression levels of 16 transcripts were significantly higher in the L2 than in the B strain, whereas the mRNA expression levels of nine transcripts, BDH, NCAM1, PCDHA@, PGDS, PLAG1, PRL, SAR1A, SCG2 and STMN2, were significantly higher in two high egg production strains, L2 and Single-Comb WL; this indicated their usefulness as molecular markers of high egg production.

Two slow-growth local chicken strains, derived from a common base population, were bi-directionally selected over twenty generations for carcass traits (B strain) and egg production (L2 strain). The objective of the present study was to identify hypothalamic proteins associated with high egg production (by taking advantage of the similar genetic background of these two strains). Prior to and during egg laying, hypothalamic proteins of B and L2 hens were analyzed with two-dimensional gel electrophoresis (2-DE) and liquid chromatography–tandem mass spectrometry (LC–MS/MS). Approximately 430 well-resolved spots, ranging from 10 to 40kDa, pH 5–9, were quantified by image processing. Eight protein spots differed in quantity between B and L2 strains at either stage. Using LC–MS/MS, we identified six of eight protein spots, including proteins known for regulating gene expression, signal transduction and lipid metabolism. The mRNA expression levels of these six proteins were then evaluated by quantitative RT-PCR in five strains of hens, including B, L2 and another three commercial strains; heterogeneous nuclear ribonucleoprotein H3 ( HNRPH3 ) was higher in L2 than in the B strain (consistent with the findings in 2-DE). Increased levels of HNRPH3 mRNA were also present in the hypothalamus of high-egg-yield White Leghorn layers, but were absent in other domestic commercial strains with low egg production rates. In conclusion, the expression level of HNRPH3 may be a new molecular marker to screen for high egg production in slow-growth local chickens.

Ribonucleases with antitumor activity are mainly found in the oocytes and embryos of frogs, but the role of these ribonucleases in frog development is not clear. Moreover, most frog ribonuclease genes have not been cloned and characterized. In the present study, a group of ribonucleases were isolated from Rana catesbeiana (bullfrog). These ribonucleases in mature oocytes, namely RC-RNase, RC-RNase 2, RC-RNase 3, RC-RNase 4, RC-RNase 5 and RC-RNase 6, as well as liver-specific ribonuclease RC-RNase L1, were purified by column chromatographs and detected by zymogram assay and western blotting. Characterization of these purified ribonucleases revealed that they were highly conserved in amino acid sequence and had a pyroglutamate residue at their N-termini, but possessed different specific activities, base specificities and optimal pH values for their activities. These ribonucleases were cytotoxic to cervical carcinoma HeLa cells, but their cytotoxicities were not closely correlated to their enzymatic specific activities. Some other amino acid residues in addition to their catalytic residues were implicated to be involved in the cytotoxicity of the frog ribonucleases to tumor cells. Because the coding regions lack introns, the ribonuclease genes were cloned by PCR using genomic DNA as template. Their DNA sequences and amino acid sequences are homologous to those of mammalian ribonuclease superfamily, approximately 50 and approximately 25%, respectively.

In a previous study of mouse tetraploid[harr ]diploid chimaeric blastocysts, tetraploid cells were found to be more abundant in the trophectoderm than the inner cell mass (ICM) and more abundant in the mural trophectoderm than the polar trophectoderm. This non-random allocation of tetraploid cells to different regions of the chimaeric blastocyst may contribute to the restricted tissue distribution seen in postimplantation stage tetraploid[harr ]diploid chimaeras. However, the tetraploid and diploid embryos that were aggregated together differed in several respects: the tetraploid embryos had fewer cells and these cells were bigger and differed in ploidy. Each of these factors might underlie a non-random allocation of tetraploid cells to the chimaeric blastocyst. A combination of micromanipulation and electrofusion was used to produce two series of chimaeras that distinguished between the effects of cell size and ploidy on the allocation of cells to different tissues in chimaeric blastocysts. When aggregated cells differed in cell size but not ploidy, the derivatives of the larger cell contributed significantly more to the mural trophectoderm and polar trophectoderm than the ICM. When aggregated cells differed in ploidy but not cell size, the tetraploid cells contributed significantly more to the mural trophectoderm than the ICM. In both experiments the contributions to the polar trophectoderm tended to be intermediate between those of the mural trophectoderm and ICM. These experiments show that both the larger size and increased ploidy of tetraploid cells could have contributed to the non-random cell distribution that was observed in a previous study of tetraploid[harr ]diploid chimaeric blastocysts.

Embryo implantation is a complicated process involving interactions between the blastocyst and the luminal epithelium of the receptive uterus. Mucin 1 (MUC1) is an integral membrane glycoprotein expressed apically by secretory epithelial cells and the glandular epithelium in different organs, including the uterus. It is believed that loss of MUC1 on the surface of uterine epithelial cells is necessary for embryo implantation. The endogenous non-protein coding microRNAs (miRNAs) of 21–24 nucleotides are found in diverse organisms. It has been shown that miRNAs participate in a range of cellular processes by regulating gene expression at the post-transcriptional level. In the present study, the regulatory role of miRNA-199a on the expression of MUC1 in mouse uterus during implantation was investigated for its effect on embryo implantation. Western blotting and immunohistochemistry results showed high MUC1 expression on Day 0.5 and low expression by Day 4.5 of pregnancy. In contrast with MUC1 expression, increased miRNA-199a expression was evident at Day 4.5 of pregnancy, as measured by real-time reverse transcription–polymerase chain reaction. In addition, we demonstrated direct binding of miRNA-199a to the 3′-untranslated region of MUC1. Transfection of miRNA-199a into mouse uterine epithelial cells isolated from Day 0.5 of pregnancy also downregulated expression of MUC1. Therefore, the present study provides evidence that MUC1 is a direct target of miRNA-199a and suggests that development of novel strategies to facilitate a successful pregnancy and repair implantation failure humans may include miRNA.

Acute heat stress affects genes involved in spermatogenesis in mammals. However, there is apparently no elaborate research on the effects of acute heat stress on gene expression in avian testes. The purpose of this study was to investigate global gene expression in testes of the L2 strain of Taiwan country chicken after acute heat stress. Twelve roosters, 45 weeks old, were allocated into four groups, including control roosters kept at 25 °C, roosters subjected to 38 °C acute heat stress for 4 hours without recovery, with 2-hour recovery, and with 6-hour recovery, respectively. Testis samples were collected for RNA isolation and microarray analysis. Based on gene expression profiles, 169 genes were upregulated and 140 genes were downregulated after heat stress using a cutoff value of twofold or greater change. Based on gene ontology analysis, differentially expressed genes were mainly related to response to stress, transport, signal transduction, and metabolism. A functional network analysis displayed that heat shock protein genes and related chaperones were the major upregulated groups in chicken testes after acute heat stress. A quantitative real-time polymerase chain reaction analysis of mRNA expressions of HSP70, HSP90AA1, BAG3, SERPINB2, HSP25, DNAJA4, CYP3A80, CIRBP, and TAGLN confirmed the results of the microarray analysis. Because the HSP genes (HSP25, HSP70, and HSP90AA1) and the antiapoptotic BAG3 gene were dramatically altered in heat-stressed chicken testes, we concluded that these genes were important factors in the avian testes under acute heat stress. Whether these genes could be candidate genes for thermotolerance in roosters requires further investigation.

It has been reported that Xenopus egg extracts contain molecules that are capable of reprogramming mammalian somatic cells. The reprogrammed somatic cells, which are called extract treated cells (ETC), possess the potential for clinical therapy as embryonic stem (ES) cells do. Therefore, in addition to establishment of an efficient method to reprogram mouse NIH/3T3 cells by Xenopus egg extracts, the aim of this study was to select the ETC cells by the expression of Oct4. In Experiment 1, two methods, electroporation or permeabilization, were conducted to treat mouse NIH/3T3 cells with Xenopus egg extracts. 2 × 105 cells in 200 μL reprogramming mixture containing Xenopus egg extracts were stimulated by a direct current (DC) pulse (80 V mm–1 for 3 msec) three times followed by a pause of incubation at 37°C for 5 min and a single DC pulse (170 V mm–1, for 0.4 msec) subsequently. The electroporated cells were then incubated at 22°C for 1 h. In the other treatment group, NIH/3T3 cells (5 × 105) were permeabilized by streptolysin O (SLO, 500 ng mL–1 in PBS) for 50 min at 37°C before mixed with Xenopus egg extracts at 22°C for 2 h. Cells were cultured in DMEM supplemented with 10% FBS for the first 4 days and then changed to ES medium (DMEM supplemented with 15% FBS, 0.1 mm β-mercaptoethanol, 1000 unit mL–1 mLIF, 0.5% nonessential amino acids, 2 mm L-glutamine) for the last 6 days after Xenopus egg extract treatment. Cell colonies were found in both treatment groups at the end of culture. Examination by immunocytochemical staining, results showed that the extract-treated cell colonies expressed pluripotent marker proteins, such as alkaline phosphatase, Oct4, Nanog and Sox2. In Experiment 2, an enhanced green fluorescent protein (EGFP) expression vector was constructed and EGFP was driven by Oct4 enhancer and promoter (Oct4-EGFP). Mouse NIH/3T3 cells were then transfected with Oct4-EGFP plasmids and selected for stable clone by G418 screening. After 6 passages, the NIH/3T3-Oct4-EGFP cells were treated with egg extracts to induce reprogramming as Experiment 1, and monitored pluripotency based on the expression of EGFP. Results showed that some of the cells or cell colonies expressed green fluorescence driven by Oct4 regulatory element at the 8th day of culture after extract treatment. Our results demonstrated that both methods of electroporation and reversible permeabilization could introduce reprogramming molecules in Xenopus egg extract to the mammalian somatic cells and generate ETCs cells in vitro. Also, with the establishment of NIH/3T3-Oct4-EGFP cell line, the potentially reprogrammed colonies could be easily selected by EGFP expression. The changes of epigenetic modifications in the ETC cells would be investigated in the short future.