Expression of Recombinant Human Octamer-Binding Transcription Factor 4 in Rice Suspension Cells.

The rice cell suspension culture system is a good way to produce recombinant human proteins, owing to its high biosafety and low production cost. Human Octamer-binding Transcription Factor 4 (Oct4) is a fundamental transcription factor responsible for maintaining human pluripotent embryonic stem cells. Recombinant Oct4 protein has been used to induce pluripotent stem cells. In this study, recombinant Oct4 proteins are produced via a sugar starvation-inducible αAmy3/RAmy3D promoter-signal peptide-based rice recombinant protein expression system. Oct4 mRNAs accumulate in the transgenic rice suspension cells under sugar starvation. The Oct4 recombinant protein is detected in the transgenic rice suspension cells, and its highest yield is approximately 0.41% of total cellular soluble proteins after one day of sugar starvation. The rice cell-synthesized recombinant human Oct4 protein show DNA-binding activity in vitro, which implies that the protein structure is correct for enabling specific binding to the target DNA motif.

An electrochemical sensor for Oct4 detection in human tissue based on target-induced steric hindrance effect on a tetrahedral DNA nanostructure.

Here, we report an electrochemical sensor for rapid and sensitive detection of octamer-binding transcription factor 4 (Oct4) in human tissue samples by utilizing a designed tetrahedral DNA nanostructure (TDN). In the design, the TDN is also extended with two additional strands from two vertices. When Oct4 is absent, the strands are linked together by complementary pairing bases. Owing to the rigid structure of TDN, contact of the redox labels on the signal strand and electrode surface is greatly prohibited, resulting in a lower electrochemical signal. However, the specific binding of Oct4 to the edge of the tetrahedron will liberate the signal strand and increase the redox current dramatically. Experimental results reveal that the proposed sensor shows a linear range of 0.5-1000 ng/mL with a detection limit of 60 pg/mL. Moreover, it can be directly applied to clinical sample detection. This sensor can also achieve one-step detection of Oct4 in less than 30 min. Furthermore, through replacing the binding site, this sensor can be easily extended to a wide application range of DNA binding proteins.

Isolation and characterization of mesenchymal progenitors derived from the bone marrow of goats native from northeastern Brazil.

PURPOSE: To characterize bone marrow progenitors cells grown in vitro, using native goats from northeastern Brazil as animal model. METHODS: Ten northeastern Brazil native goats of both genders were used from the Piauí Federal University Agricultural Science Center's (UFPI) - Goat Farming Sector. Bone marrow aspirates where taken from the tibial ridge and seeded on culture plates for isolation, expansion and Flow Cytometry (expression markers - Oct-3/4, PCNA, Ck-Pan, Vimentina, Nanog). RESULTS: Progenitor cells showed colonies characterized by the presence of cell pellets with fibroblastoid morphology. Cell confluence was taken after 14 days culture and the non-adherent mononuclear cell progressive reduction. After the first passage, 94.36% cell viability was observed, starting from 4.6 x 106 cell/mL initially seeded. Cells that went through flow cytometry showed positive expression for Oct-3/4, PCNA, Ck-Pan, Vimentina, and Nanog. CONCLUSIONS: Bone marrow progenitor isolated of native goats from northeastern Brazil showed expression markers also seen in embryonic stem cells (Oct-3/4, Nanog), markers of cell proliferation (PCNA) and markers for mesenchymal cells (Vimentina and Ck-pan), which associated to morphological and culture growth features, suggest the existence of a mesenchymal stem cell (MSC) population in the goat bone marrow stromal cells studied.

Oct4 links multiple epigenetic pathways to the pluripotency network.

Oct4 is a well-known transcription factor that plays fundamental roles in stem cell self-renewal, pluripotency, and somatic cell reprogramming. However, limited information is available on Oct4-associated protein complexes and their intrinsic protein-protein interactions that dictate Oct4's critical regulatory activities. Here we employed an improved affinity purification approach combined with mass spectrometry to purify Oct4 protein complexes in mouse embryonic stem cells (mESCs), and discovered many novel Oct4 partners important for self-renewal and pluripotency of mESCs. Notably, we found that Oct4 is associated with multiple chromatin-modifying complexes with documented as well as newly proved functional significance in stem cell maintenance and somatic cell reprogramming. Our study establishes a solid biochemical basis for genetic and epigenetic regulation of stem cell pluripotency and provides a framework for exploring alternative factor-based reprogramming strategies.

In silico tandem affinity purification refines an Oct4 interaction list.

INTRODUCTION: Octamer-binding transcription factor 4 (Oct4) is a master regulator of early mammalian development. Its expression begins from the oocyte stage, becomes restricted to the inner cell mass of the blastocyst and eventually remains only in primordial germ cells. Unearthing the interactions of Oct4 would provide insight into how this transcription factor is central to cell fate and stem cell pluripotency. METHODS: In the present study, affinity-tagged endogenous Oct4 cell lines were established via homologous recombination gene targeting in embryonic stem (ES) cells to express tagged Oct4. This allows tagged Oct4 to be expressed without altering the total Oct4 levels from their physiological levels. RESULTS: Modified ES cells remained pluripotent. However, when modified ES cells were tested for their functionality, cells with a large tag failed to produce viable homozygous mice. Use of a smaller tag resulted in mice with normal development, viability and fertility. This indicated that the choice of tags can affect the performance of Oct4. Also, different tags produce a different repertoire of Oct4 interactors. CONCLUSIONS: Using a total of four different tags, we found 33 potential Oct4 interactors, of which 30 are novel. In addition to transcriptional regulation, the molecular function associated with these Oct4-associated proteins includes various other catalytic activities, suggesting that, aside from chromosome remodeling and transcriptional regulation, Oct4 function extends more widely to other essential cellular mechanisms. Our findings show that multiple purification approaches are needed to uncover a comprehensive Oct4 protein interaction network.

Successful co-immunoprecipitation of Oct4 and Nanog using cross-linking.

The transcription factors Oct4 and Nanog are essential for the maintenance of an undifferentiated and pluripotent state in early embryonic cells, embryonic stem cells and embryonal carcinoma cells in humans and mice. These factors are co-localized to promoters of more than 300 genes, and synergistically regulate their activities. Currently, the molecular interaction between these two factors has not been well-characterized. During attempts to co-immunoprecipitate Oct4 and Nanog we found that cross-linking with dithiobis[succinimidylpropionate] was necessary to maintain their interaction. This result was supported by gel filtration analysis. Surprisingly, formaldehyde, a cross-linker commonly used during chromatin immunoprecipitation of Oct4 and Nanog, did not preserve the complex. Our findings demonstrate the effectiveness of using DSP to mitigate the instability of the interaction between these two particular proteins. Additionally, this solution may potentially allow us to identify novel members of the Oct4-Nanog complex, leading to better understanding of the regulatory mechanisms behind pluripotency.

Stem cell regulatory function mediated by expression of a novel mouse Oct4 pseudogene.

Multiple pseudogenes have been proposed for embryonic stem (ES) cell-specific genes, and their abundance suggests that some of these potential pseudogenes may be functional. ES cell-specific expression of Oct4 regulates stem cell pluripotency and self-renewing state. Although Oct4 expression has been reported in adult tissues during gene reprogramming, the detected Oct4 signal might be contributed by Oct4 pseudogenes. Among the multiple Oct4 transcripts characterized here is a approximately 1 kb clone derived from P19 embryonal carcinoma stem cells, which shares a approximately 87% sequence homology with the parent Oct4 gene, and has the potential of encoding an 80-amino acid product (designated as Oct4P1). Adenoviral expression of Oct4P1 in mesenchymal stem cells promotes their proliferation and inhibits their osteochondral differentiation. These dual effects of Oct4P1 are reminiscent of the stem cell regulatory function of the parent Oct4, and suggest that Oct4P1 may be a functional pseudogene or a novel Oct4-related gene with a unique function in stem cells.