CRISPR-Cas9 Mediated Gene Deletion in Human Pluripotent Stem Cells Cultured Under Feeder-Free Conditions.

The CRISPR-Cas9 system for genome editing has revolutionized gene function studies in mammalian cells, including stem cells. However, the practical application of this technique, particularly in pluripotent stem cells, presents certain challenges, such as being time- and labor-intensive and having low editing efficiency. Here, we describe the generation of a CRISPR-mediated gene knockout in a human embryonic stem cell (hESC) line stably expressing sgRNAs for the L2HGDH gene, using a highly efficient and stable lentiviral-mediated gene delivery system.

O-GlcNAc transferase promotes glioblastoma by modulating genes responsible for cell survival, invasion, and inflammation.

Metabolic reprogramming has emerged as one of the key hallmarks of cancer cells. Various metabolic pathways are dysregulated in cancers, including the hexosamine biosynthesis pathway. Protein O-GlcNAcylation is catalyzed by the enzyme O-GlcNAc transferase (OGT), an effector of hexosamine biosynthesis pathway that is found to be upregulated in most cancers.

Developmental modeling of hepatogenesis using obese iPSCs-hepatocyte differentiation uncovers pathological features.

Obesity is a multigene disorder. However, in addition to genetic factors, environmental determinants also participate in developing obesity and related pathologies. Thus, obesity could be best described as a combination of genetic and environmental perturbations often having its origin during the early developmental period.

Lentiviral Mediated Delivery of shRNAs to hESCs and NPCs Using Low-cost Cationic Polymer Polyethylenimine (PEI).

The current protocol describes the use of lentiviral particles for the delivery of short hairpin RNAs (shRNAs) to both human embryonic stem cells (hESCs) as well as neural progenitor cells (NPCs) derived from hESCs at high efficiency. Lentiviral particles were generated by co-transfecting HEK293T cells using entry vectors (carrying shRNAs) along with packaging plasmids (pAX and pMD2.G) using the low-cost cationic polymer polyethylenimine (PEI).

RA-Induced Transcriptional Silencing of Checkpoint Kinase-2 through Promoter Methylation by Dnmt3b Is Required for Neuronal Differentiation of P19 Cells.

In a previous study, we identified several novel targets of Dnmt3b using a chromatin library from retinoic acid (RA)-treated P19 cells. The present study describes the regulation of expression and function of checkpoint kinase (Chk2), which was one of the target genes of Dnmt3b. Chromatin immunoprecipitation followed by quantitative PCR analysis showed that recruitment of Dnmt3b on Chk2 promoter is induced following RA treatment of P19 cells.

Polylysine-modified polyethylenimine (PEI-PLL) mediated VEGF gene delivery protects dopaminergic neurons in cell culture and in rat models of Parkinson's Disease (PD).

Unlabelled: Parkinson's Disease (PD) is a chronic neurodegenerative disorder characterized by motor deficits which result from the progressive loss of dopaminergic neurons. Gene therapy using growth factors such as VEGF seems to be a viable approach for potential therapeutic treatment of PD. In this study, we utilized a novel non-viral gene carrier designated as PEI-PLL synthesized by our laboratory to deliver VEGF gene to study its effect by using both cell culture as well as animal models of PD.

Epigenetic regulation of Dpp6 expression by Dnmt3b and its novel role in the inhibition of RA induced neuronal differentiation of P19 cells.

DNA methylation is an important mechanism of gene silencing in mammals catalyzed by a group of DNA methyltransferases including Dnmt1, Dnmt3a, and Dnmt3b which are required for the establishment of genomic methylation patterns during development and differentiation. In this report, we studied the role of DNA methyltransferases during retinoic acid induced neuronal differentiation of P19 cells. We observed an increase in the mRNA and protein level of Dnmt3b, whereas the expression of Dnmt1 and Dnmt3a was decreased after RA treatment of P19 cells which indicated that Dnmt3b is more important during neuronal differentiation of P19 cells.

Doxycycline can stimulate cytoprotection in neural stem cells with oxygen-glucose deprivation-reoxygenation injury: a potential approach to enhance effectiveness of cell transplantation therapy.

A substantial loss of transplanted neural stem cells is a major limitation to cell transplantation therapy of stroke. In this study, we provided in vitro evidence that doxycycline preconditioning of neural stem cells have resulted in decreased cell death and increased cell viability after oxygen-glucose deprivation-reoxygenation conditions that best mimics cerebral ischemia-reperfusion injury. Resistance to oxidative stress is one of the mechanisms of doxycycline-induced cytoprotection in neural stem cells as it significantly reduced the superoxide anion production.

PtdIns (3,4,5) P3 recruitment of Myo10 is essential for axon development.

Myosin X (Myo10) with pleckstrin homology (PH) domains is a motor protein acting in filopodium initiation and extension. However, its potential role has not been fully understood, especially in neuronal development. In the present study the preferential accumulation of Myo10 in axon tips has been revealed in primary culture of hippocampal neurons with the aid of immunofluorescence from anti-Myo10 antibody in combination with anti-Tuj1 antibody as specific marker.

Production of surfactin from Bacillus subtilis MZ-7 grown on pharmamedia commercial medium.

Background: Commercial medium (Pharmamedia) was investigated for the production of surfactin by Bacillus subtilis MZ-7. Different media (defined, semi-defined, and complex media) were compared for the production of surfactin after fixing the least influential variables in standardized fermentation conditions. Carbohydrate and nitrogen supplements were also tried to improve production in Pharmamedia.