Bovine NANOG Research Articles

NANOG is required to form the epiblast and maintain pluripotency in the bovine embryo.

During preimplantation development, the embryo undergoes two consecutive lineages specifications. The first cell fate decision determines which cells give rise to the trophectoderm (TE) and the inner cell mass (ICM). Subsequently, the ICM differentiates into hypoblast and epiblast, the latter giving rise to the embryo proper. The transcription factors that govern these cell fate decisions have been extensively studied in the mouse, but are still poorly understood in other mammalian species. In the present study, the role of NANOG in the formation of the epiblast and maintenance of pluripotency in the bovine embryo was investigated. Using a CRISPR-Cas9 approach, guide RNAs were designed to target exon 2, resulting in a functional deletion of bovine NANOG at the zygote stage. Disruption of NANOG resulted in the embryos that form a blastocoel and an ICM composed of hypoblast cells. Furthermore, NANOG-null embryos showed lower expression of epiblast cell markers SOX2 and HA2AFZ, and hypoblast marker GATA6; without affecting the expression of TE markers CDX2 and KRT8. Results indicate that NANOG, has no apparent role in segregation or maintenance of the TE, but it is required to derive and maintain the pluripotent epiblast and during the second lineage commitment in the bovine embryo.

Cloning and Characterization of a Nanog Pseudogene in Sika Deer (Cervus nippon).

Nanog plays a crucial role in the maintenance of stem cell pluripotency. Annual full regeneration of deer antlers has been shown to be a stem cell-based process, and antler stem cells (ASCs) reportedly express Nanog. In the present study, we found that Nanog RNA expressed by ASCs was a pseudogene (Nanog-ps). The coding sequence of Nanog-ps was 93.1% homologous to that of bovine Nanog, but with two missing nucleotides after position 391. Deletion of the two nucleotides in Nanog-ps resulted in a frame-shift mutation, suggesting that Nanog-ps would not encode a normal Nanog protein. Overexpression of Nanog-ps failed to affect downstream genes of Nanog or to enhance cell proliferation in the ASCs. However, this pseudogene was transcribed in the ASCs and encoded a nuclear protein; the expression levels of Nanog-ps were also related to the degree of stemness in antler cells. Here, we reported this pseudogene, because it could serve as a useful marker for identifying ASCs and evaluating the degree of their stemness.

Characterization of the Nanog 5'-flanking Region in Bovine.

Bovine embryonic stem cells have potential for use in research, such as transgenic cattle generation and the study of developmental gene regulation. The Nanog may play a critical role in maintenance of the undifferentiated state of embryonic stem cells in the bovine, as in murine and human. Nevertheless, efforts to study the bovine Nanog for pluripotency-maintaining factors have been insufficient. In this study, in order to understand the mechanisms of transcriptional regulation of the bovine Nanog, the 5′-flanking region of the Nanog was isolated from ear cells of Hanwoo. Results of transient transfection using a luciferase reporter gene under the control of serially deleted 5′-flanking sequences revealed that the −134 to −19 region contained the positive regulatory sequences for the transcription of the bovine Nanog. Results from mutagenesis studies demonstrated that the Sp1-binding site that is located in the proximal promoter region plays an important role in transcriptional activity of the bovine Nanog promoter. The electrophoretic mobility shift assay with the Sp1 specific antibody confirmed the specific binding of Sp1 transcription factor to this site. In addition, significant inhibition of Nanog promoter activity by the Sp1 mutant was observed in murine embryonic stem cells. Furthermore, chromatin-immunoprecipitation assay with the Sp1 specific antibody confirmed the specific binding of Sp1 transcription factor to this site. These results suggest that Sp1 is an essential regulatory factor for bovine Nanog transcriptional activity.

Characterization of Bovine <i>NANOG</i>5’-flanking Region during Differentiation of Mouse Embryonic Stem Cells

Embryonic stem cells (ESCs) have been used as a powerful tool for research including gene manipulated animal models and the study of developmental gene regulation. Among the critical regulatory factors that maintain the pluripotency and self-renewal of undifferentiated ESCs, NANOG plays a very important role. Nevertheless, because pluripotency maintaining factors and specific markers for livestock ESCs have not yet been probed, few studies of the NANOG gene from domestic animals including bovine have been reported. Therefore, we chose mouse ESCs in order to understand and compare NANOG expression between bovine, human, and mouse during ESCs differentiation. We cloned a 600 bp (−420/+181) bovine NANOG 5′-flanking region, and tagged it with humanized recombinant green fluorescent protein (hrGFP) as a tracing reporter. Very high GFP expression for bovine NANOG promoter was observed in the mouse ESC line. GFP expression was monitored upon ESC differentiation and was gradually reduced along with differentiation toward neurons and adipocyte cells. Activity of bovine NANOG (−420/+181) promoter was compared with already known mouse and human NANOG promoters in mouse ESC and they were likely to show a similar pattern of regulation. In conclusion, bovine NANOG 5-flanking region functions in mouse ES cells and has characteristics similar to those of mouse and human. These results suggest that bovine gene function studied in mouse ES cells should be evaluated and extrapolated for application to characterization of bovine ES cells.

CRISPR/Cas9 nuclease-mediated gene knock-in in bovine-induced pluripotent cells.

Efficient and precise genetic engineering in livestock such as cattle holds great promise in agriculture and biomedicine. However, techniques that generate pluripotent stem cells, as well as reliable tools for gene targeting in livestock, are still inefficient, and thus not routinely used. Here, we report highly efficient gene targeting in the bovine genome using bovine pluripotent cells and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 nuclease. First, we generate induced pluripotent stem cells (iPSCs) from bovine somatic fibroblasts by the ectopic expression of yamanaka factors and GSK3β and MEK inhibitor (2i) treatment. We observed that these bovine iPSCs are highly similar to naïve pluripotent stem cells with regard to gene expression and developmental potential in teratomas. Moreover, CRISPR/Cas9 nuclease, which was specific for the bovine NANOG locus, showed highly efficient editing of the bovine genome in bovine iPSCs and embryos. To conclude, CRISPR/Cas9 nuclease-mediated homologous recombination targeting in bovine pluripotent cells is an efficient gene editing method that can be used to generate transgenic livestock in the future.

Monitoring bovine fetal fibroblast reprogramming utilizing a bovine NANOG promoter‐driven EGFP reporter system

NANOG is an essential transcription factor involved in the proliferation and maintenance of embryonic stem cells (ESC) and reprogramming of somatic cells to a pluripotent state. Oct4 and Nanog promoter-driven enhanced green fluorescent protein (EGFP) reporters have been employed for establishing lines of induced pluripotent stem cells (iPSC) from mouse, human, and pig. In ruminants, including cattle, in which no fully validated ESC lines have been established, iPSC generated by reprogramming somatic cells to an ESC-like state may prove useful in the production of genetically modified livestock. In this study, utility of the bovine NANOG reporter was tested for use with cattle. Seven proximal bovine NANOG promoter fragments of different size were fused to the LUC gene, and were tested in mouse ESC lines using a dual-luciferase assay. Three of the bovine NANOG promoters, 315 bp (-134/+181), 446 bp (-265/+181), and 1,100 bp (-919/+181), were fused to a nuclear localized signal EGFP reporter gene. The fidelity of these constructs was analyzed by transfection into mouse ESC and bovine fetal fibroblasts (bFFs), and subsequent reprogramming of the bFF. Fusion of the transgenic bFF with human teratocarcinoma (NTERA2) cells induced nuclear expression of the EGFP reporter. Similarly, bFF-derived somatic cell nuclear transfer (SCNT) embryos expressed EGFP in a stage- and location-appropriate manner. Following reprogramming of transgenic bFFs for 10 days with an Oct4-Sox2-Klf4-cMyc vector, iPSC expressed EGFP and alkaline phosphatase. These results indicate that NANOG reporters can be used to monitor nuclear reprogramming of bFFs and to distinguish cell allocation in SCNT-derived embryos.

Deciphering Induction of Pluripotency in a Bovine Model.

Strategies utilized in deriving embryonic stem (ES) cells from mouse and primate embryos have not been sufficient for maintaining self renewal and inhibiting spontaneous differentiation in ungulate embryo explant cultures, implying that ungulate ESC may require a unique signature of signal transduction pathways, transcription factor hierarchies, and chromatin modifications. To determine how key transcription factors OCT4 (POU5F1) and SOX2 induce changes in developmental potential within differentiated bovine cells, we constructed inducible bOCT4 and bSOX2 expression constructs and a live cell, pluripotency-specific transcriptional marker using the bovine NANOG promoter. The open reading frames of transcription factors bOCT4 and bSOX2 were obtained via RT-PCR from bovine CT-1 and bovine fetal fibroblast (BFF) cell lines, respectively. The bovine NANOG promoter was obtained by PCR from genomic DNA. The bOCT4 ORF was joined to a hemagglutinin (HA) epitope tag sequence and cloned into the tetracycline-inducible pTHE vector, which also contains a bicistronic tetracycline repressor (TetR)-IRES-neomycin resistance cassette. The bSOX2 ORF was joined to a MYC epitope tag sequence and cloned into a modified pTHE vector containing a bicistronic TetR-IRES-nuclear localized dsRED2 cassette. Each construct was transiently transfected into HEK293 cells and then doxycycline induced for initial protein expression analysis. Recombinant protein levels were analyzed by western blot. Both constructs were shown to be responsive to doxycycline in a dose dependent manner (0 ng/ml - 10 ng/ml). Immunocytochemistry of induced, transfected HEK293 cells demonstrated that these recombinant transcription factors localized to nuclei, indicating that the HA and MYC epitopes did not obstruct proper protein transport and localization. The transcriptional marker construct, bNANOG promoter linked to nuclear localized green fluorescent protein (bNANOGp-nGFP) was also created using a portion of the bNANOG promoter 440 base pairs upstream of the translational start site. Stable expression of nGFP driven by the bNANOG promoter was obtained in both NANOG expressing R1 mouse ESC and human NTERA cell lines. Expression of nGFP was down-regulated in differentiating R1 mES cells and was not observed in BFF. These studies demonstrated that the bNANOG promoter functioned as expected and that when linked to a marker gene can be used to study induction, maintenance, and loss of pluripotency. These results validate the constructs for appropriate use in studies exploring induction and loss of pluripotency, thus allowing us to follow changes in the transcriptome of selected BFF cells throughout the initial stage of reprogramming and uncover signature markers indicating increased developmental potential. This research was supported by CSREES USDA NRI grant 200405447 and Maryland Agricultural Experimental Station grants. (poster)

246 EXPRESSION PATTERN OF NANOG AND Par3 GENES IN IN VITRO-DERIVED BOVINE EMBRYOS

Homeobox genes have been demonstrated to be important in patterning and lineage specification during early embryogenesis. Nanog belongs to the family of DNA-binding transcription factors and has been shown to maintain pluripotency of embryonic stem cells, both in murine and human. Par3 plays an essential role in determining cell fate of the early mouse embryo, leading to the generation of the inner cell mass and the trophectoderm. No information is available on these genes in the bovine; therefore, the aim of the present study was to identify and characterize Nanog and Par3 expression in bovine embryos. Oocytes recovered from slaughterhouse ovaries were matured for 22 h, fertilized in vitro and then cultured in mSOFaa medium. RNA was extracted from pools of five oocytes and embryos at different stages of development (2-, 4-, 8-, 16-cell, morula and blastocyst). It was then reverse transcribed, and PCR runs were carried out with primers specifically designed for Nanog and Par3, based on the sequence data bank available. The amplified products were separated on a 2% TAE agarose gel, purified, sequenced and aligned using Clustal W. Comparison of the bovine Nanog cDNA sequence (EMBL AM039957) with databases revealed a 84% degree of homology with the human, 97% with the mouse, and 82% with the goat genes. IVF bovine embryos express Nanog only upon genome activation, becoming detectable from the 8-cell stage onward indicating that Nanog is zygotically expressed in the bovine similar to what happens in mouse, pig and goat. Bovine Par3 cDNA sequence (EMBL AM039956) shows a high degree of homology with human (83%), mouse (81%), and rat (79%). Also Par3 is expressed only upon the maternal to embryonic transition (MET) at the 8-cell stage. As opposed to the expression patterns of other early embryo genes, like Oct-4 and Zar-1, Nanog and Par3 expression patterns in bovine embryos closely resemble those described in the mouse. Since both are absent in the ooplasm and before MET, they represent useful markers for genome activation. This work was supported by FIRB RBNE01HPMX, FIRST 2004 and ESF-EuroStells.