Gerelchimeg Bou

Transgene Expression Is Associated with Copy Number and Cytomegalovirus Promoter Methylation in Transgenic Pigs

Transgenic animals have been used for years to study gene function, produce important proteins, and generate models for the study of human diseases. However, inheritance and expression instability of the transgene in transgenic animals is a major limitation. Copy number and promoter methylation are known to regulate gene expression, but no report has systematically examined their effect on transgene expression. In the study, we generated two transgenic pigs by somatic cell nuclear transfer (SCNT) that express green fluorescent protein (GFP) driven by cytomegalovirus (CMV). Absolute quantitative real-time PCR and bisulfite sequencing were performed to determine transgene copy number and promoter methylation level. The correlation of transgene expression with copy number and promoter methylation was analyzed in individual development, fibroblast cells, various tissues, and offspring of the transgenic pigs. Our results demonstrate that transgene expression is associated with copy number and CMV promoter methylation in transgenic pigs.

Unfaithful Maintenance of Methylation Imprints Due to Loss of Maternal Nuclear Dnmt1 during Somatic Cell Nuclear Transfer

The low success rate of somatic cell nuclear transfer (SCNT) in mammalian cloning is largely due to imprinting problems. However, little is known about the mechanisms of reprogramming imprinted genes during SCNT. Parental origin-specific DNA methylation regulates the monoallelic expression of imprinted genes. In natural fertilization, methylation imprints are established in the parental germline and maintained throughout embryonic development. However, it is unclear whether methylation imprints are protected from global changes of DNA methylation in cloned preimplantation embryos. Here, we demonstrate that cloned porcine preimplantation embryos exhibit demethylation at differentially methylated regions (DMRs) of imprinted genes; in particular, demethylation occurs during the first two cell cycles. By RNAi-mediated knockdown, we found that Dnmt1 is required for the maintenance of methylation imprints in porcine preimplantation embryos. However, no clear signals were detected in the nuclei of oocytes and preimplantation embryos by immunofluorescence. Thus, Dnmt1 is present at very low levels in the nuclei of porcine oocytes and preimplantation embryos and maintains methylation imprints. We further showed that methylation imprints were rescued in nonenucleated metaphase II (MII) oocytes. Our results indicate that loss of Dnmt1 in the maternal nucleus during SCNT significantly contributes to the unfaithful maintenance of methylation imprints in cloned embryos.

Sox2 is the faithful marker for pluripotency in pig: Evidence from embryonic studies

Background: Mammalian first lineage segregation generates trophectoderm (TE) and pluripotent inner cell mass (ICM), which provides an ideal model for studying the mechanisms of maintenance and loss of pluripotency. In mouse, the transcription factor OCT4 restricts to ICM and plays a key role in TE/ICM specification and pluripotent regulatory networks. However, in pig, OCT4 does not restrict to ICM cells, suggesting a different molecular basis in TE/ICM specification and pluripotent regulatory networks. Results: To explore molecular basis of porcine TE/ICM specification and pluripotent regulatory networks, we examined expression pattern of pluripotency factors, including SOX2, REX1, SALL4, ESG1, NANOG, TBX3, LIN28, KLF2, and KLF5, in porcine blastocysts. We found that SOX2 is a faithful pluripotent marker that anchored to the pluripotent cells including embryonic part cells, ICM cells and newly EPI cells along with developmental progress, whereas OCT4 expressed in almost all the cells at the same time. Consistently, analysis of spatiotemporal distribution of SOX2 and the TE marker CDX2 revealed an exclusive expression pattern in D6 blastocysts, whereas no correlation was observed between OCT4 and CDX2 at the same stage. Conclusions: Our results provide a molecular basis in porcine embryonic patterning and a clue for further studying porcine pluripotent regulatory networks. Developmental Dynamics 244:619–627, 2015.

A novel long intergenic noncoding RNA indispensable for the cleavage of mouse two‐cell embryos

Endogenous retroviruses (ERVs) are transcriptionally active in cleavage stage embryos, yet their functions are unknown. ERV sequences are present in the majority of long intergenic noncoding RNAs (lincRNAs) in mouse and humans, playing key roles in many cellular processes and diseases. Here, we identify LincGET as a nuclear lincRNA that is GLN‐, MERVL‐, and ERVK‐associated and essential for mouse embryonic development beyond the two‐cell stage. LincGET is expressed in late two‐ to four‐cell mouse embryos. Its depletion leads to developmental arrest at the late G2 phase of the two‐cell stage and to MAPK signaling pathway inhibition. LincGET forms an RNA–protein complex with hnRNP U, FUBP1, and ILF2, promoting the cis‐regulatory activity of long terminal repeats (LTRs) in GLN, MERVL, and ERVK (GLKLTRs), and inhibiting RNA alternative splicing, partially by downregulating hnRNP U, FUBP1, and ILF2 protein levels. Hnrnpu or Ilf2 mRNA injection at the pronuclear stage also decreases the preimplantation developmental rate, and Fubp1 mRNA injection at the pronuclear stage causes a block at the two‐cell stage. Thus, as the first functional ERV‐associated lincRNA, LincGET provides clues for ERV functions in cleavage stage embryonic development.

rRNA genes are not fully activated in mouse somatic cell nuclear transfer embryos

Background: The nucleolus always shows delayed development and malfunction in somatic cell nuclear transfer (NT) embryos. Results: NT embryos showed low rDNA activity and developmental competence when donor cells with low rDNA activity were used. Conclusion: rDNA reprogramming efficiency in NT embryos was determined by the rDNA activity in donor cells from which they derived. Significance: Developmental potential of NT embryos with rDNA activity in the donor cells was correlated. The well known and most important function of nucleoli is ribosome biogenesis. However, the nucleolus showed delayed development and malfunction in somatic cell nuclear transfer (NT) embryos. Previous studies indicated that nearly half rRNA genes (rDNA) in somatic cells were inactive and not transcribed. We compared the rDNA methylation level, active nucleolar organizer region (NORs) numbers, nucleolar proteins (upstream binding factor (UBF), nucleophosmin (B23)) distribution, and nucleolar-related gene expression in three different donor cells and NT embryos. The results showed embryonic stem cells (ESCs) had the most active NORs and lowest rDNA methylation level (7.66 and 6.76%), whereas mouse embryonic fibroblasts (MEFs) were the opposite (4.70 and 22.57%). After the donor cells were injected into enucleated MII oocytes, cumulus cells and MEFs nuclei lost B23 and UBF signals in 20 min, whereas in ESC-NT embryos, B23 and UBF signals could still be detected at 60 min post-NT. The embryos derived from ESCs, cumulus cells, and MEFs showed the same trend in active NORs numbers (7.19 versus 6.68 versus 5.77, p < 0.05) and rDNA methylation levels (6.36 versus 9.67% versus 15.52%) at the 4-cell stage as that in donor cells. However, the MEF-NT embryos displayed low rRNA synthesis/processing potential at morula stage and had an obvious decrease in blastocyst developmental rate. The results presented clear evidences that the rDNA reprogramming efficiency in NT embryos was determined by the rDNA activity in donor cells from which they derived.

CDX2 is essential for cell proliferation and polarity in porcine blastocysts.

The role of CDX2 in trophectoderm (TE) cells has been extensively studied, yet the results are contradictory and species specific. Here, CDX2 expression and function were explored in early porcine embryos. Notably, siRNA-mediated gene knockdown and lentivirus-mediated TE-specific gene regulation demonstrated that CDX2 is essential for the maintenance of blastocyst integrity by regulating the BMP4-mediated blastocyst niche and classic protein kinase C (PKC)-mediated TE polarity in mammalian embryos. Mechanistically, CDX2-depleted porcine embryos stalled at the blastocyst stage and exhibited apoptosis and inactive cell proliferation, possibly resulting from BMP4 downregulation. Moreover, TE cells in CDX2-depleted blastocysts displayed defective F-actin apical organization associated with downregulation of PKCα (PRKCA). Collectively, these results provide further insight into the functional diversity of CDX2 in early mammalian embryos. Summary: The transcription factor CDX2 functions in the trophectoderm of pig embryos to maintain polarity and integrity of the blastocyst, revealing further diversity in CDX2 activities across mammals.

Overexpression Nanog Activates Pluripotent Genes in Porcine Fetal Fibroblasts and Nuclear Transfer Embryos

Nanog as an important transcription factor plays a pivotal role in maintaining pluripotency and in reprogramming the epigenome of somatic cells. Its ability to function on committed somatic cells and embryos has been well defined in mouse and human, but rarely in pig. To better understand Nanogs function on reprogramming in porcine fetal fibroblast (PFF) and nuclear transfer (NT) embryo, we cloned porcine Nanog CDS and constructed pcDNA3.1 (+)/Nanog and pEGFP‐C1/Nanog overexpression vectors and transfected them into PFFs. We studied the cell biological changes and the expression of Nanog, Oct4, Sox2, Klf4, C‐myc, and Sall4 in transfected PFFs. We also detected the development potential of the cloned embryos harboring Nanog stably overexpressed fibroblasts and the expression of Oct4, Sox2, and both endogenous and exogenous Nanog in these embryos. The results showed that transient overexpression Nanog in PFF could activate the expression of Oct4 (5‐fold), C‐myc (2‐fold), and Sall4 (5‐fold) in somatic cells, but they could not be maintained during G418 selection. In NT embryos, although Nanog overexpression did not have a significant effect on blastocyst development rate and blastocyst cell number, it could significantly activate the expression of endogenous Nanog, Oct4, Sox2 to 160‐fold, 93‐fold, and 182‐fold, respectively (P < 0.05). Our results demonstrate that Nanog could interact with and activate other pluripotent genes both in PFFs and embryos. Anat Rec, 2011.

Porcine Pluripotent Stem Cells Derived from IVF Embryos Contribute to Chimeric Development In Vivo

Although the pig is considered an important model of human disease and an ideal animal for the preclinical testing of cell transplantation, the utility of this model has been hampered by a lack of genuine porcine embryonic stem cells. Here, we derived a porcine pluripotent stem cell (pPSC) line from day 5.5 blastocysts in a newly developed culture system based on MXV medium and a 5% oxygen atmosphere. The pPSCs had been passaged more than 75 times over two years, and the morphology of the colony was similar to that of human embryonic stem cells. Characterization and assessment showed that the pPSCs were alkaline phosphatase (AKP) positive, possessed normal karyotypes and expressed classic pluripotent markers, including OCT4, SOX2 and NANOG. In vitro differentiation through embryonic body formation and in vivo differentiation via teratoma formation in nude mice demonstrated that the pPSCs could differentiate into cells of the three germ layers. The pPSCs transfected with fuw-DsRed (pPSC-FDs) could be passaged with a stable expression of both DsRed and pluripotent markers. Notably, when pPSC-FDs were used as donor cells for somatic nuclear transfer, 11.52% of the reconstructed embryos developed into blastocysts, which was not significantly different from that of the reconstructed embryos derived from porcine embryonic fibroblasts. When pPSC-FDs were injected into day 4.5 blastocysts, they became involved in the in vitro embryonic development and contributed to the viscera of foetuses at day 50 of pregnancy as well as the developed placenta after the chimeric blastocysts were transferred into recipients. These findings indicated that the pPSCs were porcine pluripotent cells; that this would be a useful cell line for porcine genetic engineering and a valuable cell line for clarifying the molecular mechanism of pluripotency regulation in pigs.

Cdx2 represses Oct4 function via inducing its proteasome-dependent degradation in early porcine embryos.

Reciprocal repression of inner cell mass specific factor OCT4 and trophectoderm specific factor CDX2 promotes mouse first lineage segregation. Studies in mouse embryonic stem (ES) cells revealed that they bind to each others regulatory regions to reciprocally suppress transcription, additionally they form protein complex for mutual antagonism. However, so far the molecular interaction of Oct4 and Cdx2 in other mammals early embryo is not yet investigated. Here, over-expression of Cdx2 in early porcine embryo showed CDX2 represses Oct4 through neither the transcriptional repression nor forming repressive complex, but promoting OCT4 nuclear export and proteasomal degradation. The results showed novel molecular regulation of CDX2 on Oct4, and provided important clues for clarifying the mechanism of interaction between CDX2 and Oct4 in embryo of mammals other than mouse.

Tbx3 and Nr5α2 improve the viability of porcine induced pluripotent stem cells after dissociation into single cells by inhibiting RHO-ROCK-MLC signaling

Porcine induced pluripotent stem cells (piPSCs) had been reported during the past 5years, but there were few reports on how the cell signaling works in piPSCs. In order to clarify the signaling work that dominated the characteristic difference of two types of piPSCs which were derived from Oct4, Sox2, Klf4 and c-Myc (termed 4F piPSCs) and Oct4, Sox2, Klf4, c-Myc, Tbx3 and Nr5α2 (termed 6F piPSCs) respectively, we performed this study. 4F piPSCs and 6F piPSCs were cultured in medium with or without the ROCK inhibitor Y27632 after dissociating into single cells, the efficiency of a single cell colony and the number of AP positive colonies were assessed. The total RhoA and GTP-bind RhoA were detected in 4F piPSCs and 6F piPSCs before and after digestion into single cells. To explore the relationship between RHO-ROCK-MLC signaling pathway and the two factors Tbx3 and Nr5α2, the 4F piPSCs were infected with lenti-virus Tbx3 and Nr5α2 (termed 4F+TND). Results showed that the viability of cells could be enhanced by Y27632 and the RHO-ROCK-MLC signaling pathway was activated after dissociation into single cells in 4F piPSCs but not in 6F piPSCs. And, the 4F+TND piPSCs could be passaged and keep in high viability after dissociation into single cells, though the morphology of colonies did not change. These results indicated that the Tbx3 and Nr5α2 can improve the viability of piPSCs after dissociation into single cells by inhibiting the RHO-ROCK-MLC signaling pathway. And this provides useful information for establishing porcine pluripotent cells in future study.