Accelerated generation of gene-engineered monoclonal CHO cell lines using FluidFM nanoinjection and CRISPR/Cas9.

Chinese hamster ovary (CHO) cells are the commonly used mammalian host system to manufacture recombinant proteins including monoclonal antibodies. However unfavorable non-human glycoprofile displayed on CHO-produced monoclonal antibodies have negative impacts on product quality, pharmacokinetics, and therapeutic efficiency. Glycoengineering such as genetic elimination of genes involved in glycosylation pathway in CHO cells is a viable solution but constrained due to longer timeline and laborious workflow.

Inhibition of FGF and TGF-β Pathways in hESCs Identify STOX2 as a Novel SMAD2/4 Cofactor.

The fibroblast growth factor (FGF) and the transforming growth factor-β (TGF-β) pathways are both involved in the maintenance of human embryonic stem cells (hESCs) and regulate the onset of their differentiation. Their converging functions have suggested that these pathways might share a wide range of overlapping targets. Published studies have focused on the long-term effects (24-48 h) of FGF and TGF-β inhibition in hESCs, identifying direct and indirect target genes.

Gaining Insights into the Function of Post-Translational Protein Modification Using Genome Engineering and Molecular Cell Biology.

Modifications by kinases are a fast and reversible mechanism to diversify the function of the targeted proteins. The OCT4 transcription factor is essential for preimplantation development and pluripotency of embryonic stem cells (ESC), and its activity is tightly regulated by post-transcriptional modifications. Several phosphorylation sites have been identified by systemic approaches and their functions proposed.

Comparative analysis of differential gene expression tools for RNA sequencing time course data.

RNA sequencing (RNA-seq) has become a standard procedure to investigate transcriptional changes between conditions and is routinely used in research and clinics. While standard differential expression (DE) analysis between two conditions has been extensively studied, and improved over the past decades, RNA-seq time course (TC) DE analysis algorithms are still in their early stages. In this study, we compare, for the first time, existing TC RNA-seq tools on an extensive simulation data set and validated the best performing tools on published data.

A Concise Protocol for siRNA-Mediated Gene Suppression in Human Embryonic Stem Cells.

Human embryonic stem cells hold great promise for future biomedical applications such as disease modeling and regenerative medicine. However, these cells are notoriously difficult to culture and are refractory to common means of genetic manipulation, thereby limiting their range of applications. In this protocol, we present an easy and robust method of gene repression in human embryonic stem cells using lipofection of small interfering RNA (siRNA).

Switch enhancers interpret TGF-β and Hippo signaling to control cell fate in human embryonic stem cells.

A small toolkit of morphogens is used repeatedly to direct development, raising the question of how context dictates interpretation of the same cue. One example is the transforming growth factor β (TGF-β) pathway that in human embryonic stem cells fulfills two opposite functions: pluripotency maintenance and mesendoderm (ME) specification. Using proteomics coupled to analysis of genome occupancy, we uncover a regulatory complex composed of transcriptional effectors of the Hippo pathway (TAZ/YAP/TEAD), the TGF-β pathway (SMAD2/3), and the pluripotency regulator OCT4 (TSO).

The TGFβ superfamily in stem cell biology and early mammalian embryonic development.

Background: Members of the Transforming Growth Factor-beta (TGFβ) superfamily of cytokines are essential for early embryonic development and play crucial roles in pluripotency and differentiation of embryonic stem cells in vitro.

Scope Of Review: In this review, we discuss how TGFβ family signals are read by cells and how they are modulated by the cellular context. Furthermore, we review recent advances in our understanding of TGFβ function in embryonic stem cells and point out hot topics at the intersection of TGFβ signaling and stem cell biology fields.

Functional genomics reveals a BMP-driven mesenchymal-to-epithelial transition in the initiation of somatic cell reprogramming.

Somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) by expression of defined embryonic factors. However, little is known of the molecular mechanisms underlying the reprogramming process. Here we explore somatic cell reprogramming by exploiting a secondary mouse embryonic fibroblast model that forms iPSCs with high efficiency upon inducible expression of Oct4, Klf4, c-Myc, and Sox2.

The Nrf2 transcription factor protects from toxin-induced liver injury and fibrosis.

The liver is frequently exposed to insults, including toxic chemicals and alcohol, viral infection or metabolic overload. Although it can fully regenerate after acute injury, chronic liver damage causes liver fibrosis and cirrhosis, which can result in complete liver failure. In this study, we demonstrate that the NF-E2-related factor 2 (Nrf2) transcription factor protects the liver from acute and chronic toxin-mediated damage.

The cytoprotective Nrf2 transcription factor controls insulin receptor signaling in the regenerating liver.

The Nrf2 transcription factor is a crucial regulator of the cellular redox homeostasis through its capacity to induce the expression of enzymes, which detoxify reactive oxygen species, and of other antioxidant proteins. Therefore, it plays an important role in the protection from carcinogenesis induced by various insults. In addition, recent results identified a novel role of Nrf2 in tissue repair.

Impaired liver regeneration in Nrf2 knockout mice: role of ROS-mediated insulin/IGF-1 resistance.

The liver is frequently challenged by surgery-induced metabolic overload, viruses or toxins, which induce the formation of reactive oxygen species. To determine the effect of oxidative stress on liver regeneration and to identify the underlying signaling pathways, we studied liver repair in mice lacking the Nrf2 transcription factor. In these animals, expression of several cytoprotective enzymes was reduced in hepatocytes, resulting in oxidative stress.

Electrophilic chemicals but not UV irradiation or reactive oxygen species activate Nrf2 in keratinocytes in vitro and in vivo.

The NF-E2-related factor 2 (Nrf2) transcription factor is a potent inducer of cytoprotective genes, which encode--among others--enzymes that detoxify reactive oxygen species (ROS). As we demonstrated a crucial role of Nrf2 in the prevention of skin carcinogenesis, it is of interest to identify Nrf2-activating factors in keratinocytes. For this purpose, keratinocytes from mice transgenic for an Nrf2-responsive reporter gene were analyzed.

Nrf transcription factors in keratinocytes are essential for skin tumor prevention but not for wound healing.

The Nrf2 transcription factor is a key player in the cellular stress response through its regulation of cytoprotective genes. In this study we determined the role of Nrf2-mediated gene expression in keratinocytes for skin development, wound repair, and skin carcinogenesis. To overcome compensation by the related Nrf1 and Nrf3 proteins, we expressed a dominant-negative Nrf2 mutant (dnNrf2) in the epidermis of transgenic mice.

Fibroblast growth factor receptor 1-IIIb is dispensable for skin morphogenesis and wound healing.

Alternative splicing in the extracellular domain is a characteristic feature of members of the fibroblast growth factor receptor (FGFR) family. This splicing event generates receptor variants, which differ in their ligand binding specificities. A poorly characterized splice variant is FGFR1-IIIb, recently found to be a functional FGF receptor predominantly expressed in the skin.

Fibroblast growth factor 22 and its potential role during skin development and repair.

We have isolated, using RT-PCR, a cDNA from mouse skin wounds that encodes fibroblast growth factor (FGF) 22, a recently discovered member of the FGF family, which is closely related to FGF-7 and FGF-10. Transient expression of tagged FGF-22 protein in COS-1 and MCF-7 cells revealed that the protein was present within the cell and at the cell surface but was not apparently released from the cell. Analysis of RNA expression revealed that FGF-22 transcripts were not detected in the developing mouse embryo until day E16.