Green Fluorescent Protein Cells Research Articles

Decellularized Biohybrid Nerve Promotes Motor Axon Projections.

Developing nerve grafts with intact mesostructures, superior conductivity, minimal immunogenicity, and improved tissue integration is essential for the treatment and restoration of neurological dysfunctions. A key factor is promoting directed axon growth into the grafts. To achieve this, biohybrid nerves are developed using decellularized rat sciatic nerve modified by in situ polymerization of poly(3,4-ethylenedioxythiophene) (PEDOT). Nine biohybrid nerves are compared with varying polymerization conditions and cycles, selecting the best candidate through material characterization. These results show that a 1:1 ratio of FeCl3 oxidant to ethylenedioxythiophene (EDOT) monomer, cycled twice, provides superior conductivity (>0.2mScm-1), mechanical alignment, intact mesostructures, and high compatibility with cells and blood. To test the biohybrid nerve's effectiveness in promoting motor axon growth, human Spinal Cord Spheroids (hSCSs) derived from HUES 3 Hb9:GFP cells are used, with motor axons labeled with green fluorescent protein (GFP). Seeding hSCS onto one end of the conduit allows motor axon outgrowth into the biohybrid nerve. The construct effectively promotes directed motor axon growth, which improves significantly after seeding the grafts with Schwann cells. This study presents a promising approach for reconstructing axonal tracts in humans.

Comparative Approaches for Quantification of Product Yield in a Model Recombinant Green Fluorescent Protein Expressed in E. coli.

Process Analytical Technologies (PAT) used to monitor and control manufacturing processes are crucial for efficient and automated bioprocessing, which is in congruence with lights-off-manufacturing and Industry 4.0 initiatives. As biomanufacturing seeks to realize more high-throughput and automated operation, an increasing need for multimodal analysis of process metrics becomes essential. Herein, we detail a series of methods for analyzing product yield from a bioreactor and how to conduct cross-method comparisons. We employ a model system of Escherichia coli (E. coli) expression of green fluorescent protein (GFP), which is a simple, cost effective model for students and educators to replicate at different scales. GFP is an ideal analytical marker as it is easy to visualize due to its fluorescence which indicates cellular protein expression, cell localization and physiological changes of the cell population. In this study, samples from a 300 L bioreactor with GFP-expressing E. coli are analyzed to improve product yield and bioprocessing efficiency. Utilizing a fed-batch process for enhanced cell density and product titer, this bioreactor runs on a 24-hour schedule from inoculation to GFP induction and final harvest. To reliably quantify relative GFP expression and E. coli proliferation, we provide simple protocols and example results for comparing three different analytical methods: (1) in-line bioreactor measurements, (2) plate reader assays, and (3) microscopy. The GFP and cell density results follow similar trends based on the various inline and offline analytical methods and show a peak of GFP expression and cell density between 12.5 and 18 hours post inoculation.

Selective radiosensitization by nitazoxanide of quiescent clonogenic colon cancer tumour cells.

Nitazoxanide is a Food and Drug Administration-approved antiprotozoal drug recently demonstrated to be selectively active against quiescent and glucose-deprived tumour cells. This drug also has several characteristics that suggest its potential as a radiosensitizer. The present study aimed to investigate the interaction between nitazoxanide and radiation on human colon cancer cells cultured as monolayers, and to mimic key features of solid tumours in patients, as spheroids, as well as in xenografts in mice. In the present study, colon cancer HCT116 green fluorescent protein (GFP) cells were exposed to nitazoxanide, radiation or their combination. Cell survival was analysed by using total cell kill and clonogenic assays. DNA double-strand breaks were evaluated in the spheroid experiments, and HCT116 GFP cell xenograft tumours in mice were used to investigate the effect of nitazoxanide and radiation in vivo. In the clonogenic assay, nitazoxanide synergistically and selectively sensitized cells grown as spheroids to radiation. However, this was not observed in cells cultured as monolayers, as demonstrated in the total cell kill assays, and much less with the clinically established sensitizer 5-fluorouracil. The sensitizing effect from nitazoxanide was confirmed via spheroid γ-H2A histone family member X staining. Nitazoxanide and radiation alone similarly inhibited the growth of HCT116 GFP cell xenograft tumours in mice with no evidence of synergistic interaction. In conclusion, nitazoxanide selectively targeted quiescent glucose-deprived tumour cells and sensitized these cells to radiation in vitro. Nitazoxanide also inhibited tumour growth in vivo. Thus, nitazoxanide is a candidate for repurposing into an anticancer drug, including its use as a radiosensitizer.

Coaxially Bioprinted Cell-Laden Tubular-Like Structure for Studying Glioma Angiogenesis.

Glioblastomas are the most frequently diagnosed and one of the most lethal primary brain tumors, and one of their key features is a dysplastic vascular network. However, because the origin of the tumor blood vessels remains controversial, an optimal preclinical tumor model must be established to elucidate the tumor angiogenesis mechanism, especially the role of tumor cells themselves in angiogenesis. Therefore, shell-glioma cell (U118)-red fluorescent protein (RFP)/core-human umbilical vein endothelial cell (HUVEC)-green fluorescent protein (GFP) hydrogel microfibers were coaxially bioprinted. U118–RFP and HUVEC–GFP cells both exhibited good proliferation in a three-dimensional (3D) microenvironment. The secretability of both vascular endothelial growth factor A and basic fibroblast growth factor was remarkably enhanced when both types of cells were cocultured in 3D models. Moreover, U118 cells promoted the vascularization of the surrounding HUVECs by secreting vascular growth factors. More importantly, U118–HUVEC-fused cells were found in U118–RFP/HUVEC–GFP hydrogel microfibers. Most importantly, our results indicated that U118 cells can not only recruit the blood vessels of the surrounding host but also directly transdifferentiate into or fuse with endothelial cells to participate in tumor angiogenesis in vivo. The coaxially bioprinted U118–RFP/HUVEC–GFP hydrogel microfiber is a model suitable for mimicking the glioma microenvironment and for investigating tumor angiogenesis.

A Toxic Synergy between Aluminium and Amyloid Beta in Yeast.

Alzheimer’s disease (AD), the most prevalent, age-related, neurodegenerative disease, is associated with the accumulation of amyloid beta (Aβ) and oxidative stress. However, the sporadic nature of late-onset AD has suggested that other factors, such as aluminium may be involved. Aluminium (Al3+) is the most ubiquitous neurotoxic metal on earth, extensively bioavailable to humans. Despite this, the link between Al3+ and AD has been debated for decades and remains controversial. Using Saccharomyces cerevisiae as a model organism expressing Aβ42, this study aimed to examine the mechanisms of Al3+ toxicity and its interactions with Aβ42. S. cerevisiae cells producing Aβ42 treated with varying concentrations of Al3+ were examined for cell viability, growth inhibition, and production of reactive oxygen species (ROS). Al3+ caused a significant reduction in cell viability: cell death in yeast producing green fluorescent protein tagged with Aβ42 (GFP–Aβ42) was significantly higher than in cells producing green fluorescent protein (GFP) alone. Additionally, Al3+ greatly inhibited the fermentative growth of yeast producing GFP–Aβ42, which was enhanced by ferric iron (Fe3+), while there was negligible growth inhibition of GFP cells. Al3+- induced ROS levels in yeast expressing native Aβ42 were significantly higher than in empty vector controls. These findings demonstrate Al3+ has a direct, detrimental toxic synergy with Aβ42 that can be influenced by Fe3+, causing increased oxidative stress. Thus, Al3+ should be considered as an important factor, alongside the known characteristic hallmarks of AD, in the development and aetiology of the disease.

Cuboplex-Mediated Nonviral Delivery of Functional siRNA to Chinese Hamster Ovary (CHO) Cells.

Lipid nanoparticles of internal cubic symmetry, termed cuboplexes, are potential nonviral delivery vehicles for gene therapy due to their "topologically active" nature, which may enhance endosomal escape and improve delivery outcomes. In this study, we have used cationic cuboplexes, based on monoolein (MO) doped with a cationic lipid, for the encapsulation and delivery of antisense green fluorescent protein (GFP)-small interfering RNA (siRNA) into Chinese Hamster Ovary (CHO)-GFP cells. Agarose gel electrophoresis has confirmed the successful encapsulation of siRNA within cationic cubosomes, while synchrotron small-angle X-ray scattering (SAXS) demonstrated that the underlying cubic nanostructure of the particles was retained following encapsulation. The cationic cubosomes were shown to be reasonably nontoxic against the CHO-GFP cell line. Fluorescence-activated cell sorting (FACS) provided evidence of the successful transfection to CHO-GFP cells. Knockdown efficiency was strongly linked to the type of cationic lipid used, although all cubosomes had essentially the same internal nanostructure. The gene knockdown efficiency for some cationic cubosomes was shown to be higher than lipofectamine, which is a commercially available liposome-based formulation, while the controlled release of the siRNA from the cubosomes over a 72 h period was observed using confocal microscopy. This combination exemplifies the potential of cationic cuboplexes as a novel, nonviral, controlled-release delivery vector for siRNA.

Improved cell-specificity of adeno-associated viral vectors for medullary thyroid carcinoma using calcitonin gene regulatory elements.

Targeted gene therapy using recombinant adeno-associated virus (rAAV) vectors is a potential therapeutic strategy for treating cancer, and tissue-specific promoters may help with tissue targeting. Medullary thyroid carcinoma (MTC) is a disease of the calcitonin secreting thyroid C cells, and calcitonin is highly expressed in MTC tumors compared to other cells. To target MTC cells, we evaluated an rAAV serotype 2 vector (rAAV2-pM+104-GFP) containing a modified calcitonin/calcitonin gene related peptide promoter (pM+104) and a green fluorescent protein (GFP) reporter gene. In vitro transduction experiments comparing the MTC TT cell line with non-MTC cell lines demonstrated that rAAV2-pM+104-GFP infection yielded significantly (p < 0.05) higher GFP expression in TT cells than in non-MTC cell lines (HEK293 and HeLa), and significantly higher expression than in TT cells infected with the positive control rAAV2-pCBA-GFP vector. The rAAV2-pCBA-GFP control vector included a well-characterized, ubiquitously expresses control promoter, the chicken beta actin promoter with a cytomegalovirus enhancer (pCBA). In vivo experiments using a TT cell xenograft tumor mouse model showed that tumors directly injected with 2 x 1010 vg of rAAV2-pM+104-GFP vector resulted in GFP expression detected in 21.7% of cells, 48 hours after the injection. Furthermore, GFP expression was significantly higher for rAAV-pM+104-GFP treatments with a longer vector treatment duration and higher vector dose, with up to 52.6% (q < 0.05) GFP cells detected 72 hours after injecting 1x 1011 vg/tumor. These data show that we have developed an rAAV vector with improved selectivity for MTC.

Differentiation and roles of bone marrow-derived cells on the tumor microenvironment of oral squamous cell carcinoma.

The stroma affects the properties and dynamics of the tumor. Previous studies have demonstrated that bone marrow-derived cells (BMDCs) possess the capability of differentiating into stromal cells. However, the characteristics and roles of BMDCs in oral squamous cell carcinoma remain unclear. The current study therefore investigated their locations and features by tracing green fluorescent protein (GFP)-labeled BMDCs in a transplantation mouse model. After irradiation, BALB-c nu-nu mice were injected with bone marrow cells from C57BL/6-BALB-C-nu/nu-GFP transgenic mice. These recipient mice were then injected subcutaneously in the head with human squamous cell carcinoma-2 cells. Immunohistochemistry for GFP, Vimentin, CD11b, CD31 and α-smooth muscle actin (SMA), and double-fluorescent immunohistochemistry for GFP-Vimentin, GFP-CD11b, GFP-CD31 and GFP-α-SMA was subsequently performed. Many round-shaped GFP-positive cells were observed in the cancer stroma, which indicated that BMDCs served a predominant role in tumorigenesis. Vimentin(+) GFP(+) cells may also be a member of the cancer-associated stroma, originating from bone marrow. Round or spindle-shaped CD11b(+) GFP(+) cells identified in the present study may be macrophages derived from bone marrow. CD31(+)GFP(+) cells exhibited a high tendency towards bone marrow-derived angioblasts. The results also indicated that spindle-shaped α-SMA(+) GFP(+) cells were not likely to represent bone marrow-derived cancer-associated fibroblasts. BMDCs gathering within the tumor microenvironment exhibited multilineage potency and participated in several important processes, such as tumorigenesis, tumor invasion and angiogenesis.

Fetal Cell Microchimerism; Normal and Immunocompromised Gestations in Mice

Objective: To compare fetal cell microchimerism in normal and immunocompromised gestations. Materials and methods: The study consists of two groups of mature female mice. In the control group and the immunocompromised study group, 5 mg of saline and cyclosporine were injected intraperitoneally, respectively. In the second step, all female mice were mated with “Actine-Luc (+) green fluorescent protein (GFP)” transgenic male mice. Immunohistochemical studies (ALPL-antiluciferase, cytokeratin-antiluciferase, and CD 105-antiluciferase) were carried out on maternal liver, skin, and lung tissues at 6–7th and 14–15th gestational days, and postpartum 3–4th, 12th, and 18–24 months. Results: GFP (+) cells were detected in maternal liver and skin but not in lung tissue. Liver was the most affected tissue. GFP was found to be more intense in the immunocompromised group. Conclusion: Fetal microchimerism was demonstrated in maternal liver and skin and found to be more intensive in the immunocompromised group.

Translation enhancement by a Dictyostelium gene sequence in Escherichia coli

Methods for heterologous protein production in Escherichia coli have revolutionized biotechnology and the bioindustry. It is ultimately important to increase the amount of protein product from bacteria. To this end, a variety of tools, such as effective promoters, have been developed. Here, we present a versatile molecular tool based on a phenomenon termed "translation enhancement by a Dictyostelium gene sequence" ("TED") in E. coli. We found that protein expression was increased when a gene sequence of Dictyostelium discoideum was placed upstream of the Shine-Dalgarno sequence located between the promoter and the initiation codon of a target gene. The most effective sequence among the genes examined was mlcR, which encodes the myosin regulatory light chain, a subunit of myosin II. Serial deletion analysis revealed that at least 10 bases of the 3' end of the mlcR gene enhanced the production of green fluorescent protein in cells. We applied this tool to a T7 expression system and found that the expression level of the proteins tested was increased when compared with the conventional method. Thus, current protein production systems can be improved by combination with TED.

Bone Marrow Mesenchymal Stem Cells (BMSCs) Restore Functional Endometrium in the Rat Model for Severe Asherman Syndrome.

To investigate the feasibility to restore functional endometrium using bone marrow mesenchymal stem cells (BMSCs) in the Sprague Dawley (SD["SD" has been defined as both "Sprague Dawley" and "standard deviation." Please clarify which one is to be followed.]) rat model for Asherman syndrome (AS). Basic research on treatment of AS utilizing an optimized rat model. University research laboratories. Sprague Dawley rat model in which AS was induced in accordance to an optimized protocol. Bone marrow mesenchymal stem cells were harvested from the rat's bone marrow and labeled with green fluorescent protein (GFP) in the second passage of BMSCs. The fifth passage of GFP-labeled BMSCs was injected systemically through the tail vein in the optimized AS rat model. We examined the reproliferation of the endometrial lining and the expression of markers for endometrium and endometrial receptivity. The localization of engrafted GFP-labeled BMSCs was determined by a laser scanning confocal microscope and a fluorescence microscope. The number of pregnant rats and implanted embryos in each uterus was recorded to evaluate the function of endometrium. We had demonstrated that in the in vivo experiments on our rat model for AS, the group which received BMSC injection had significantly improved reproductive outcomes-70% of these rats conceived, whereas none of the rats in the control group got pregnant ( P < .01, χ2 test). The mean number of embryos undergoing implantation was 14 ± 1.24 in the sham group and 7 ± 5.70 in the BMSC group (Levene test, P = .001). There was no significant difference between the groups from the time of coitus to conception. To further determine how BMSC injection could have resulted in the improved reproductive outcomes in rats with AS, we employed immunohistochemical techniques to examine the endometrium of these treated rats. On hematoxylin-eosin staining, we noted the reproliferation of all layers of the endometrium and with Masson staining, we noted significant reduction in fibrosis in the damaged endometrium of rats treated with BMSCs. Counterstaining for GFP and cytokeratin-positive cells was noted in the endometrial lining of treated rats, which might suggest the action of BMSCs in regenerating the damaged endometrial lining. The expression of the endometrial receptivity marker, Leukemia inhibitory factor (LIF), in this regenerated endometrial lining could have resulted in the improved reproductive outcomes observed in the AS rat model treated with BMSCs. Bone marrow mesenchymal stem cells were likely to play an important role in the reconstruction of the injured endometrium and improvement of reproductive outcomes in the optimized AS rat model.

In Vivo Fluorescence Microendoscopic Monitoring of Stent-Induced Fibroblast Cell Proliferation in an Esophageal Mouse Model

PurposeTo evaluate the feasibility of self-expanding metal stent (SEMS) placement and fluorescence microendoscopic monitoring for determination of fibroblast cell proliferation after stent placement in an esophageal mouse model. Materials and MethodsTwenty fibroblast-specific protein (FSP)-1 green fluorescent protein (GFP) transgenic mice were analyzed. Ten mice (Group A) underwent SEMS placement, and fluoroscopic and fluorescence microendoscopic images were obtained biweekly until 8 weeks thereafter. Ten healthy mice (Group B) were used for control esophageal values. ResultsSEMS placement was technically successful in all mice. The relative average number of fibroblast GFP cells and the intensities of GFP signals in Group A were significantly higher than in Group B after stent placement. The proliferative cellular response, including granulation tissue, epithelial layer, submucosal fibrosis, and connective tissue, was increased in Group A. FSP-1-positive cells were more prominent in Group A than in Group B. ConclusionsSEMS placement was feasible and safe in an esophageal mouse model, and proliferative cellular response caused by fibroblast cell proliferation after stent placement was longitudinally monitored using a noninvasive fluorescence microendoscopic technique. The results have implications for the understanding of proliferative cellular response after stent placement in real-life patients and provide initial insights into new clinical therapeutic strategies for restenosis.

Molecular and immunohistochemical analysis of the urethra of female rats after induced trauma and intravenous therapy with muscle derived stem cells.

To identify the urethral migration of muscle derived stem cells (MDSCs) after intravenous (IV) injection in rats that underwent vaginal distension (VD) and to analyze the effects of MDSC in the urethra of rats after trauma in regards to: (1) mRNA expression of collagens, Vegf, Ngf, Ki67, Myh11, and Myh2; (2) expression of smooth and striated muscle proteins. MDSCs expressing green fluorescent protein (GFP) were injected into the tail vein of rats 3 days after VD. The location of GFP cells was verified at 2 h and at 7 days following IV injection. Urethras of three groups were analyzed: Control, Trauma 7D, and MDSC 7D. Real-time RT-qPCR and immunohistochemistry were performed. MDSCs were identified only after 2 h of the procedure in the urethra. Myh11 gene was overexpressed in the Trauma group in relation to Control. Ki67 gene expression was increased in the MDSC group relative to Trauma and Control. Col1a1 and Col3a1 genes expression were increased in the MDSC group relative to Control. Ngf mRNA level was decreased in the MDSC group in relation to Trauma. Protein expression of Mhy11, Myh2, and Desmin were increased in the MDSC group in relation to Trauma and decreased in the Trauma in relation to Control. MDSCs migrated early to the traumatized urethra, but did not integrate into the tissue. MDSC alters the expression of genes related to cell proliferation, neural growth factor and extracellular matrix and the expression of smooth and striated muscle proteins in the traumatized rat urethra.

Neuronal regeneration in injured rat spinal cord after human dental pulp derived neural crest stem cell transplantation.

This study aimed to analyze the effect of human Dental Pulp-Neural Crest Stem Cells (hDP-NCSCs) delivery on lesion site after spinal cord injury (SCI), and to observe the functional recovery after transplantation. Neural Crest Stem Cells (NCSCs) were isolated from human Dental Pulp (hDP). The experimental rat population was divided into four groups (n = 6/24). Their behavioral motility was scored regularly. After 4-weeks, rats were sacrificed, and their spinal cords were examined for Green Fluorescent Protein (GFP) labeled hDP-NCSCs by immunofluorescence (IF) staining. In early post-injury (p.i) period, the ultrastructure of spinal cord tissue was preserved in Group 4. The majority of cells forming the ependymal region around the central canal were found to be hDP-NCSCs. While the grey-and-white-matter around the ependymal region was composed of e.g. GFP cells, with astrocytic-like appearance. The scores showed significant motor recovery in hind limb functions in Group 4. However, no obvious change was observed in other groups. Cells e.g., mesenchymal (Vimentin+) which express GFP+ cells in the gray-and-white-matter around the ependymal region could indicate the potential to self-renewal and plasticity. Thus, transplantation of hDP-NCSCs might be an effective strategy to improve functional recovery following spinal cord trauma (Fig. 10, Ref. 32).

Tropism of engineered and evolved recombinant AAV serotypes in the rd1 mouse and ex vivo primate retina

There is much debate on the adeno-associated virus (AAV) serotype that best targets specific retinal cell types and the route of surgical delivery—intravitreal or subretinal. This study compared three of the most efficacious AAV vectors known to date in a mouse model of retinal degeneration (rd1 mouse) and macaque and human retinal explants. Green fluorescent protein (GFP) driven by a ubiquitous promoter was packaged into three AAV capsids: AAV2/8(Y733F), AAV2/2(quad Y-F) and AAV2/2(7m8). Overall, AAV2/2(7m8) transduced the largest area of retina and resulted in the highest level of GFP expression, followed by AAV2/2(quad Y-F) and AAV2/8(Y733F). AAV2/2(7m8) and AAV2/2(quad Y-F) both resulted in similar patterns of transduction whether they were injected intravitreally or subretinally. AAV2/8(Y733F) transduced a significantly smaller area of retina when injected intravitreally compared with subretinally. Retinal ganglion cells, horizontal cells and retinal pigment epithelium expressed relatively high levels of GFP in the mouse retina, whereas amacrine cells expressed low levels of GFP and bipolar cells were infrequently transduced. Cone cells were the most frequently transduced cell type in macaque retina explants, whereas Müller cells were the predominant transduced cell type in human retinal explants. Of the AAV serotypes tested, AAV2/2(7m8) was the most effective at transducing a range of cell types in degenerate mouse retina and macaque and human retinal explants.

Establishment of A Myelodysplastic Syndrome Transformed to Leukemia Cell Line Stably Expressing Green Fluorescent Protein

To establish a myelodysplastic syndrome transformed to leukemia cell line stably expressing green fluorescent protein (GFP), and to evaluate its biological characteristics and applications. SKM-1 cells were transfected by lentiviral particles with vector of GFP. The GFP positive single cell clone was isolated by limiting dilution and continued being cultured. The cells were injected into mice subcutaneously and were screened in vivo. Then SKM-1/GFP cells were obtained after tumour plaque was separated and cultivated. The cell morphology was observed by fluorescence microscopy. The GFP expression was further detected by flow cytometry. The cell proliferation was analysed by CCK-8 assay. SKM-1/GFP cells were inoculated to subcutaneous tissue of the immunodeficiency mice. The growth and invasion of the tumour were observed after tumour formation. No differences in cell morphology and growth characteristics were observed between SKM-1 cells and SKM-1/GFP cells. The rate of GFP expression was 100%. No differences in cell proliferation were observed between SKM-1 cells and SKM-1/GFP cells. The tumour mass was observed after 14 days of subcutaneous vaccination in NOD/SCID mice. Spontaneous fluorescence from plaque was observed by living fluorescence microscopy at 30th day after vaccination. Homogenous GFP positive cells were observed by fluorescence microscopy in the frozen section of tumour mass. The invasion of SKM-1/GFP cells was also detected in heart, liver, stomach and kidney of mice. A myelody-splastic syndrome transformed to leukemia cell line stably expressing green fluorescent protein has been established successfully, which can track tumor cell sensitively and can be applied to the research of minimal residual leukemia. The establishment of SKM-1/GFP cells may serve as a powerful means for studing myelodysplastic syndrome transformation.

Tetraploid embryonic stem cells can contribute to the development of chimeric fetuses and chimeric extraembryonic tissues

Our study examined the in vivo chimeric and survival capacities of chimeras created by injecting tetraploid embryonic stem cells (ESCs) expressing green fluorescent protein (GFP) into diploid embryos. At 3.5 days post-coitum (dpc) and 4.5 dpc, the tetraploid ESCs were able to contribute to the inner cell mass (ICM) just as diploid ESCs tagged with GFP. At 6.5 dpc, 8.0 dpc and 10.5 dpc, the tetraploid ESCs manifested in the same location as the diploid ESCs. The GFP cells in the extraembryonic tissues and fetuses of tetraploid ESC chimeras were tetraploid as determined by fluorescence activated cell sorting (FACS). Furthermore, tetraploid ESCs contributed to the development of the placenta, embryolemma and umbilical cord at 13.5 dpc and 16.5 dpc; however, very less GFP cells were found in the fetuses of tetraploid ESC chimeras. We further found that the proliferation of tetraploid ESCs was slower than that of diploid ESCs. In addition, the relative mRNA expression in the three germ layers and the trophoblast was abnormal in the EBs of tetraploid ESCs compared with diploid ESCs. In short, slower proliferation and abnormal differentiation potential of tetraploid ESCs might be two of the reasons for their poor survival and chimeric capacities.

Stealth magnetic nanocarriers of siRNA as platform for breast cancer theranostics

The endogenous mechanism of RNA interference is more and more used in research to obtain specific down-regulation of gene expression in diseases such as breast cancer. Currently, despite the new fields of study open up by RNA interference, the rapid degradation of siRNA by nucleases and their negative charges prevent them from crossing cell membranes. To overcome these limitations, superparamagnetic iron oxide nanoparticles (SPIONs) represent a promising alternative for nucleic acid delivery. Previously, we reported the magnetic siRNA nanovectors (MSN) formulation using electrostatic assembly of (1) SPIONs, also able to act as contrast agents for magnetic resonance imaging (MRI), (2) siRNA and (3) chitosan aiming at their protection and enhancing their transfection efficacy. However, these nanoparticles displayed low stability in biological suspensions and inefficient transfection of active siRNA.This work aimed at upgrading MSN to Stealth MSN (S-MSN) by adding a polyethylene glycol coating to ensure colloidal stability and stealth properties. Furthermore, another polymer (poly-L-arginine) was added for efficient siRNA transfection and the quantitative composition of the formulation was adapted for biological purposes. Results showed that S-MSN provide high siRNA complexation and protection against enzymatic degradation. Green fluorescent protein (GFP) specific down-regulation on MDA-MB231/GFP cells was comparable to that of commercially available reagents, without observable cytotoxicity. According to our works, S-MSN appears as an effective formulation for in vitro siRNA specific delivery.

The effect of the hepatitis C virus (HCV) NS3 protein on the expression of miR-150, miR-199a, miR-335, miR-194 and miR-27a

Hepatitis C virus (HCV) infection is considered one of the most important causes of chronic liver diseases. Many reports have shown that the proteins of the HCV via interactions with gene expression regulatory networks such as cellular pathways and microRNAs can contribute to the development of chronic liver diseases. The present study aimed to investigate the effects of the HCV NS3 protein on the expression of miR-150 miR-199a, miR-335, miR-194, miR-27a in a cell culture model. Plasmids expressing the full length of the HCV NS3 protein were transfected into the LX-2 cell line, while at the same time a plasmid expressing empty GFP (green fluorescent protein) was used as a negative control group. Subsequently, total RNA was extracted and real-time PCR was performed to measure microRNA expression levels. Additionally, the trypan blue exclusion test was performed to examine the effect of the expressing NS3 protein plasmid on cellular viability. The analysis of microRNA gene expression in LX-2 cells indicated that the NS3 protein, which is endogenous to HCV, can significantly upregulate the expression of miR-27a and downregulate the expression of miR-335 and miR-150 in comparison with the control plasmid expressing GFP and normal cells (p < 0.01). These results suggest that the HCV NS3 protein may play a role in the pathogenesis of chronic hepatic diseases such as liver fibrosis via interaction with cellular microRNAs and modulation of microRNA gene expressions.

Spectral Unmixing Plate Reader: High-Throughput, High-Precision FRET Assays in Living Cells

We have developed a microplate reader that records a complete high-quality fluorescence emission spectrum on a well-by-well basis under true high-throughput screening (HTS) conditions. The read time for an entire 384-well plate is less than 3 min. This instrument is particularly well suited for assays based on fluorescence resonance energy transfer (FRET). Intramolecular protein biosensors with genetically encoded green fluorescent protein (GFP) donor and red fluorescent protein (RFP) acceptor tags at positions sensitive to structural changes were stably expressed and studied in living HEK cells. Accurate quantitation of FRET was achieved by decomposing each observed spectrum into a linear combination of four component (basis) spectra (GFP emission, RFP emission, water Raman, and cell autofluorescence). Excitation and detection are both conducted from the top, allowing for thermoelectric control of the sample temperature from below. This spectral unmixing plate reader (SUPR) delivers an unprecedented combination of speed, precision, and accuracy for studying ensemble-averaged FRET in living cells. It complements our previously reported fluorescence lifetime plate reader, which offers the feature of resolving multiple FRET populations within the ensemble. The combination of these two direct waveform-recording technologies greatly enhances the precision and information content for HTS in drug discovery.