Co-expression Of EGFP Research Articles

Generation of human induced pluripotent stem cells carrying albumin-sfGFP reporter

Current methodologies for hepatocyte induction from human induced pluripotent stem cells (hiPSCs) have limited efficacy due to lack of a functional hepatocyte reporter. To address this, we developed an endogenous albumin (ALB)-sfGFP reporter system in hiPSCs using homologous directed recombination (HDR)-mediated knock-in. The hiPSCs maintained the characteristic morphology, pluripotency, and normal karyotype while demonstrating successful differentiation into all three germ layers both in vitro and in vivo. Co-expression of EGFP and ALB was observed in the derived hepatocyte-like cells (HLCs). This reporter system holds promise for functional hepatocyte induction.

A Newly Designed EGFP-2A Peptide Monocistronic Baculoviral Vector for Concatenating the Expression of Recombinant Proteins in Insect Cells

Recombinant proteins produced by the baculovirus expression vector system (BVES) have been widely applied in the agricultural and medical fields. However, the procedure for protein expression is inefficient and needs to be improved. Herein, we propose a simple construct that incorporates a selectable marker (enhanced green fluorescent protein, EGFP) and a picorna viral-derived “self-cleaving” 2A-like peptide to separate the EGFP and target proteins in a monocistronic baculovirus vector to facilitate isolation of the recombinant baculovirus in the BVES. In this study, porcine adiponectin (ADN), a secreted, multimeric protein with insulin-sensitizing properties, was used to demonstrate its utility in our EGFP-2A-based expression system. EGFP and ADN were simultaneously expressed by a recombinant alphabaculovirus. Co-expression of EGFP facilitates the manipulation of the following processes, such as determining expression kinetics and harvesting ADN. The results showed that the 2A “self-cleaving” process does not interfere with EGFP activity or with signal peptide removal and the secretion of recombinant ADN. Posttranslational modifications, including glycosylation, of the recombinant ADN occurred in insect cells, and the formation of various multimers was further verified. Most importantly, the insect-produced ADN showed a similar bioactivity to that of mammalian cells. This concept provides a practical and economic approach that utilizes a new combination of alphabaculovirus/insect cell expression systems for future applications.

Adipose tissue-derived stem cells boost vascularization in grafted ovarian tissue by growth factor secretion and differentiation into endothelial cell lineages.

Adipose tissue-derived stem cells (ASCs) have multilineage differentiation potential, proangiogenic properties, and the ability to enhance vascularization in xenografted human ovarian tissue. The aim of the present study was to identify the mechanisms behind the proangiogenic effects of ASCs. For this purpose, severe combined immunodeficient (SCID) mice were grafted with frozen-thawed human ovarian tissue. ASCs were labeled by lentiviral transfection for expression of enhanced green fluorescent protein (eGFP), and human ovarian tissue was grafted using a previously described two-step procedure. In the control group, ovarian tissue was transplanted using the standard one-step approach. Samples were collected and analyzed after 7 days. Detection of the eGFP antigen by immunofluorescence showed ASCs surrounding and infiltrating ovarian tissue grafts. Significantly higher vessel density was observed in the ASC group (P = 0.0182 versus control) on Day 7. Co-expression of eGFP, CD34 and CD31 was demonstrated in human vessels, confirming ASC differentiation into human endothelial cell lineages. Increased gene expression of vascular endothelial growth factor (VEGF) was also shown in the ASC group (P = 0.0182 versus control). Immunohistochemistry targeting anti-human VEGF revealed significantly higher expression levels in the ASC group (P = 0.033 versus control), while VEGF and eGFP immunofluorescence showed greater growth factor expression in areas surrounding ASCs. In conclusion, ASCs differentiate into human vessels and promote secretion of VEGF when transplanted together with human ovarian tissue to SCID mouse peritoneum using a two-step ovarian tissue grafting procedure. This is a promising step towards potentially improving ovarian tissue quality and lifespan. Long-term studies should be conducted to investigate ASC safety and efficacy in the context of ovarian tissue transplantation.

488. In Vitro and In Vivo Genome Editing of the RHO Gene to Downregulate Dominant Mutations

Although progresses have been made in the understanding of the genetic basis for Retinitis Pigmentosa (RP), the development of therapeutic intervention is still lagging behind. Gene therapy was successfully applied to retina degeneration but only to recessive mutations. Rhodopsin (RHO) mutations represent a common cause of RP, accounting for 25% of adRP and 8 to 10% of all RP (Hartong et al., 2006) with more that 100 different mutations identified so far. RHO is a G-protein coupled receptor with localization restricted to rod outer segments where the phototransduction cascade initiates. Data on the pathogenic mechanism of mutant RHO are still controversial. Accumulation of mutant RHO in different subcellular compartments, and among those the ER, may trigger unfolded protein response (UPR) devoted to cytoprotective outputs to permit cells to reduce protein synthesis and up-regulate chaperons to cope with stress (Lin et al., 2007). In the autosomal dominant RP the mutation in only one allele is sufficient to the onset of retinal degeneration. To treat this kind of disorder a gene addition therapy is not suitable, as the mutant allele, mRNA, or protein product must be silenced beforehand. We aimed at developing genome editing tools to knock out the RHO defective alleles by introducing a double strand breaks (DSB) into the target gene. Two gRNAs were designed in the first exon on the RHO gene encompassing the P23H mutation. The two gRNAs were tested singularly or together in vitro on HeLa clones stably expressing P23H RHO. We demonstrated insertions or deletions (indels) in the genomic DNA specifically in the RHO gene by Cel I assay, TIDE and sequencing. Indels caused strong reduction of the RHO mRNA and of RHO protein up to 90%. The higher effect was obtained with the two gRNAs together. The two gRNAs were then in vivo expressed with Cas9 in photoreceptors of transgenic mice expressing the human P23H Rho gene by electroporation. Targeted cells were tracked by co-expression of EGFP. EGFP+ cells were FACS-sorted and indels in the human P23H RHO gene were analyzed by sequencing. We were able to detect up to 30% of genome editing in vivo. No editing was scored on murine Rho allele. We also detected reduction of RHO mRNA expression as well as RHO protein. Finally, we developed new tools to downregulate mutant RHO in dominant forms of RP. The CRISPR/Cas9 system reveled a high efficiency and should be tested for knock-down followed by gene replacement approaches.

Evidence for proteolytic processing and stimulated organelle redistribution of iPLA 2β

Over the past decade, important roles for the 84–88 kDa Group VIA Ca 2+-independent phospholipase A 2 (iPLA 2β) in various organs have been described. We demonstrated that iPLA 2β participates in insulin secretion, insulinoma cells and native pancreatic islets express full-length and truncated isoforms of iPLA 2β, and certain stimuli promote perinuclear localization of iPLA 2β. To gain a better understanding of its mobilization, iPLA 2β was expressed in INS-1 cells as a fusion protein with EGFP, enabling detection of subcellular localization of iPLA 2β by monitoring EGFP fluorescence. Cells stably-transfected with fusion protein expressed nearly 5-fold higher catalytic iPLA 2β activity than control cells transfected with EGFP cDNA alone, indicating that co-expression of EGFP does not interfere with manifestation of iPLA 2β activity. Dual fluorescence monitoring of EGFP and organelle Trackers combined with immunoblotting analyses revealed expression of truncated iPLA 2β isoforms in separate subcellular organelles. Exposure to secretagogues and induction of ER stress are known to activate iPLA 2β in β-cells and we find here that these stimuli promote differential localization of iPLA 2β in subcellular organelles. Further, mass spectrometric analyses identified iPLA 2β variants from which N-terminal residues were removed. Collectively, these findings provide evidence for endogenous proteolytic processing of iPLA 2β and redistribution of iPLA 2β variants in subcellular compartments. It might be proposed that in vivo processing of iPLA 2β facilitates its participation in multiple biological processes.

Membrane targeted horseradish peroxidase as a marker for correlative fluorescence and electron microscopy studies

Synaptic dynamics and reorganization are fundamental features of synaptic plasticity both during synaptic circuit development and in the mature CNS underlying learning, memory, and experience-dependent circuit rearrangements. Combining in vivo time-lapse fluorescence imaging and retrospective electron microscopic analysis provides a powerful technique to decipher the rules governing dynamics of neuronal structure and synaptic connections. Here we have generated a membrane-targeted horseradish peroxidase (mHRP) that allows identification of transfected cells without obscuring the intracellular ultrastructure or organelles and in particular allows identification of synaptic sites using electron microscopy. The expression of mHRP does not affect dendritic arbor growth or dynamics of transfected neurons. Co-expression of EGFP and mHRP was used to study neuronal morphology at both the light and electron microscopic levels. mHRP expression greatly facilitates 3D reconstruction based on serial EM sections. We expect this reagent will be valuable for studying the mechanisms that guide construction of neuronal networks.

Regulation of Hypocretin (Orexin) Expression in Embryonic Zebrafish

Hypocretins/orexins are neuropeptides involved in the regulation of sleep and energy balance in mammals. Conservation of gene sequence, hypothalamic localization of cell bodies, and projection patterns in adult zebrafish suggest that the architecture and function of the hypocretin system are conserved in fish. We report on the complete genomic structure of the zebrafish and Tetraodon hypocretin genes and the complete predicted hypocretin protein sequences from five teleosts. Using whole mount in situ hybridization, we have traced the development of hypocretin cells in zebrafish from onset of expression at 22 h post-fertilization through the first week of development. Promoter elements of similar size from zebrafish and Tetraodon were capable of driving efficient and specific expression of enhanced green fluorescent protein in developing zebrafish embryos, thus defining a minimal promoter region able to accurately mimic the native hypocretin pattern. This enhanced green fluorescent protein expression also revealed a complex pattern of projections within the hypothalamus, to the midbrain, and to the spinal cord. To further analyze the promoter, a series of deletion and substitution constructs were injected into embryos, and resulting promoter activity was monitored in the first week of development. A critical region of 250 base pairs was identified containing a core 13-base pair element essential for hypocretin expression.

Differential Role of Actin, Clathrin, and Dynamin in Fcγ Receptor-mediated Endocytosis and Phagocytosis

Clustering of macrophage Fc gamma receptors by multimeric immunoglobulin complexes leads to their internalization. Formation of small aggregates leads to endocytosis, whereas large particulate complexes induce phagocytosis. In RAW-264.7 macrophages, Fc gamma receptor endocytosis was found to be dependent on clathrin and dynamin and insensitive to cytochalasin. Clathrin also associates with nascent phagosomes, and earlier observations suggested that it plays an essential role in phagosome formation. However, we find that phagocytosis of IgG-coated large (> or =3 microm) particles was unaffected by inhibition of dynamin or by reducing the expression of clathrin using antisense mRNA but was eliminated by cytochalasin, implying a distinct mechanism dependent on actin assembly. The uptake of smaller particles (< or =1 microm) was only partially blocked by cytochalasin. Remarkably, the cytochalasin-resistant component was also insensitive to dominant-negative dynamin I and to clathrin antisense mRNA, implying the existence of a third internalization mechanism, independent of actin, dynamin, and clathrin. The uptake of small particles occurred by a process distinct from fluid phase pinocytosis, because it was not inhibited by dominant-negative Rab5. The insensitivity of phagocytosis to dominant-negative dynamin I enabled us to test the role of dynamin in phagosomal maturation. Although internalization of receptors from the plasma membrane was virtually eliminated by the K44A and S45N mutants of dynamin I, clearance of transferrin receptors and of CD18 from maturing phagosomes was unaffected by these mutants. This implies that removal of receptors from the phagosomal membrane occurs by a mechanism that is different from the one mediating internalization of the same receptors at the plasma membrane. These results imply that, contrary to prevailing notions, normal dynamin and clathrin function is not required for phagocytosis and reveal the existence of a component of phagocytosis that is independent of actin and Rab5.