| PGC cells are precursors of highly specified EG cell lineage which possess the ability of unlimited proliferation and multi-differentiation. EG cells share some common features with ES cells, including morphology, biochemical features and the capacity for in vitro differentiation. The establishment of EG cell lines of augulate animal such as pigs has far-reaching influence on the modern bio-medical research, the establishment of animal physiology or pathology models, and animal breeding. But so far, porcine EG cell linages with stable and long generations have not been established, and so, the pluripotency of porcine EG cell still have been identified. In addition, vast modification of genetic imprint happened during the evolution of PGCs to germ cells, and EG cells may inherit the imprinted memory from those PGCs with erroneous imprint code, which finally result in the aberrant in vivo development of cloned embryos if used as donor cells. This is the major difference in function between ES cell and EG cell. So this research was aimed to analyze the effect of KSR and cytokines on porcine EG cell culture. Besides that, imprint status of porcine PGCs derived from day 27 and day 35 fetuses were detected,which aimed to to provide with the foundation for the applications of porcine EG cell lineage in future.First, the effect of Knock-out serum replacement (KSR) on porcine EG cell colony was tested in this study. The isolated PGCs from porcine gonads was seeded with the same density (0.4 embryo/ well) on mouse embryonic fibroblasts(MEF)pretreated with mitomycin C. KSR, specified FBS and ordinary FBS was added in the medium according to previous design. During the culture in vitro of porcine PGCs, we could observe small colonies were observed on day 3-4 of primary culture in the groups of KSR and defined FBS. With the further development, various colony types can be observed, such as nest, flat and roots. The common features of ES cell were displayed in these colonies:indistinct cell edge inside EG colony, and obvious demarcated margin between colony and feeder and good refraction. Average number of EG colony in the primary culture between defined KSR and FBS is 24-28 and 24-28 per well, and the efficiency in the passage 3rd culture were 11/24 and 14/24, with no statistical difference in them. After continued culture, these EG colony still growed well and retained the good proliferation ability. EG colony grew larger and formed new colony with good refractivity and clear edge. Hardly differentiated EG cell appeared in the part or the whole of porcine EG colony.But, porcine isolated PGCs in the group of ordinary FBS cultured in the same EG culture medium still displayed the different features: various colony types being observed during early culture growed comparably slower and flatter. 7-8 days, the edge between feeder and EG colony appreared unclear with the further development in vitro. Differentiated cells were observed in a portion or a whole of porcine EG colony, even worse after subculture. More differentiated EG cell, neuron or fibroblast cell can be observed companying with fewer undifferentiated cell colonies.The efficiency of porcine EG colony formation in primary culture and 3rd passage was used to analyze the difference between KSR and special FBS group, and the results showed that no difference existed in these groups, whereas significantly higher than that in ordinary FBS, which demonstrated ordinary FBS was not appropriately used as the long culture of porcine EG.To investigate the potential use of BIO in maintaining porcine EG cell colony in culture was investigated either by using itself or in combination with LIF or a mixture of 3 cytokines, LIF, SCF and FGF, porcine PGCs were isolated from day 24 and day 28 fetuses, and cultured in stem cell culture medium supplemented with LIF, SCF, FGF and BIO. BrdU, one chemical component similar to thymine, was added in on the 2nd day of primary culture. BrdU immunostaining was subjected to lalbel the actively mitotic PGCs 20 hours later and and positive BrdU labeling PGCs per the total PGCs was counted as mitosis index for further statistical analysis among groups. The results showed the mitosis index (26.8%) in BIO combined with 3F treatment group was significant higher than those in other groups (18.9%, 7.0%,9.1%). In addition, the mitosis index in the treatment of 3F only (18.9%) was observed significantly higher than those in BIO treatment, either itself (7.0%) or combined with LIF(9.1%), whereas no obvious difference existed in the treatment groups of BIO only and BIO combined with LIF.The effect of BIO by single use, combination with LIF, and combination with LIF, SCF and FGF on the efficiency of porcine EG cell colony was further observed. Porcine PGCs derived from day 24 and day 28 fetuses was subjected to in vitro culture of EG cell colony. All the colonies in the primary cultures were passaged onto new MEF feeder cells in the ratio of 1:4 after 7-8 days of primary culture on the 24-well plates, and then subjected to the third passage culture in the splitting ratios of 1:2-1:6, depending on cell proliferation status. The efficacy of EG cell colony in passage 3rd ,which was counted as the wells containing EG cell colonies per the total wells at the third passage culture were used as a index statistics to evaluate the the effect of BIO combination on porcine EG cell by one way–ANOVA statistics. The results showed the combined treatment of porcine day 24 PGCs with BIO and 3F resulted in a significantly higher rate of EG colony formation (17/24) than those of other groups(12/24, 1/8, 0/8), whereas rare colonies (0/8-1/8) formed in single BIO treatment or combination treatment of LIF and BIO, the efficiency in which was even obviously lower than that in 3F group(12/24), indicating single use or combined used with LIF, SCF, and FGF of BIO at current concentration could not replace the role of LIF, SCF, and FGF in porcine EG culture. However, lower efficiencies of EG cell colony derived from day 28 PGCs were displayed (0/8-1/8)and no difference in groups. In sum, total efficient of EG cell colony formation derived from day 28 PGCs is obvious lower than that derived from day 24 PGCs, indicating the combined use of BIO and 3F can not result in the increasing efficiency of porcine EG cell colony.In addition, the effect of the combinated cytokines on the differentiated EG cell was presented in the next assay: porcine differentiated EG cells were derived from embryoid body and re-seeded on newly MEF feeder and cultured in the same medium besides cytokines. 6-7days later, the differentiated EG cell displayed again the characterization of EG cell colony as well as the expression of pluripotency factors such as NANOG and OCT4,demonstrating the combined use of BIO and 3F had the the apparent anti-differentiated performance by reversingthe differentiated EG cell to the undifferentiated status. By contrast, the combined treatment of BIO and LIF did not produce the same effect like this. In sum, our results demonstrate the combined treatment of BIO with SCF, LIF, and FGF could significantly contribute to the establishment of porcine EG cell colony and maintain the undifferentiated status, whereas the single use or combined use of BIO could not replace the role of 3F.To investigate the imprinted status of porcine PGCs from day 27 and day 35 embryos, a SNP site from PEG10 gene, one of maternally imprinted gene, were select in this study. And then, the SNP pattern of PEG10 gene in Yorkshire pigs from local farm was examined by restriction fragment length polymorphism (RFLP) and sequencing and Yorkshire pigs showing different homozygous SNP sites of PEG10 gene were mated to produce the offspring containing heterozygous SNP site. Finally, the sows were sacrificed on day 27 and day 35 of pregnancy, respectively, and porcine embryos containing hyterzygous sites were obtained. Next, porcine PGCs were isolated from embryos and further used for construction of PGC cloned embryo and culture of porcine EG cells in vitro. The cloned embryos were cultured in PZM3 medium for six days till the formation of blastocysts. Besides that, porcine PGCs were cultured on mouse embryonic fibroblasts (MEF) for the derivation of porcine EG cell colony. which were characterized by AP staining, immunostaining,molecular identification of pluripotent factors and EB formation. Finally, RNA was extracted from PGCs, cloned embryos and porcine EG cell colonies, then subjected for imprinted analysis by detecting the SNP site of PEG10 gene. The cloned embryos constructed with porcine fetal fibroblasts and MEF feeder cell were used as a control. Our results showed that monoallelic expression occurred on porcine fibroblast cloned embryo and MEF feeder control, validating the reliability of PCR results. The imprinted analysis of PGC cloned embryo and EG cell colony derived from day 27 PGCs showed biallelic expression of the SNP of PEG10 gene; However, the colony and constructed blastocysts derived from day 35 PGCs displayed monoallelic expression. Also, the same biallelic expression was observed from almost all the PGCs, no matter they were derived from male or female ones, indicating that the parental genomic imprint is erased in male and female germlines derived from day 27 PGCs, and reestablished in those from day 35 PGCs. This work is a basic study attempting to explore the epigenetics of primordial germ cells, for a better application of embryonic germ stem cells in transgenic animals, chimeras'animals etc.
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