Green Fluorescent Protein Intensity Research Articles

Efficient expression of heterologous protein by engineered Komagataella phaffii by harnessing a bioelectrical CO2 reduction system

Methylotrophic yeast Komagataella phaffii (syn. Pichia pastoris) is an important host for heterologous protein expression although its traditional inducer methanol is toxic and flammable. Using green fluorescent protein (GFP) as a model of heterologous protein in this research, recombinant K. phaffii was constructed through wild-type formate dehydrogenase gene knockout, intracellular and extracellular expression of Candida boidinii formate dehydrogenase gene under sorbitol dehydrogenase promoter (PSDH) respectively to reduce carbon dioxide (CO2) to formate in a two-chamber H-shaped three-electrode bioelectrical reactor equipped with a solar panel stage by stage. Generated formate in the bioelectrical reactor was used as a safe inducer of engineered K. phaffii strains expressing GFP successfully. Further, the electrical potential was optimized and three cathodes, graphite disk electrode, graphite rod electrode, and glassy carbon electrode, were used and modified subsequently with poly(neutral red) to obtain the highest volumetric GFP intensity and specific GFP intensity as 4424.5 a.u. and 384.0 a.u./OD600, which was 1.7- and 3.2- fold of that of methanol induction respectively, 3.9-fold of that formate induction. Therefore, a safe recombinant K. phaffii system was developed successfully for heterologous protein expression, and demonstrated a new approach to utilize CO2.

Heterologous protein expression enhancement of Komagataella phaffii by ammonium formate induction based on transcriptomic analysis

Heterologous protein expression by Komagataella phaffii (K. phaffii) attracts attention of researchers and engineers for high level of heterologous protein, strict pattern of alcohol oxidase 1 promoter (PAOX1), and high cell density fermentation. It is urgent to increase level of heterologous protein by K. phaffii using formate, the only substitute of methanol as inducer, because methanol is toxic and explosive, especially, in industrial scale. A recombinant K. phaffii expressing green fluorescent protein (GFP) was constructed and sequenced by RNA-seq technology based on methanol and ammonium formate (AF) optimization. The transcriptomes were analyzed using COG, GO, KEGG databases with the result of 1172 identical up-regulated genes at both conditions of methanol and AF induction. The transcription levels of 173 genes and 144 genes at AF induction were higher and lower significantly than that of methanol induction respectively within 1172 identical genes. Low energy generation pathway was considered as the reason of low GFP expression level at AF induction based on analysis of 173 and 144 genes. This result was confirmed by nutrients addition obtaining 30.6 % increment of GFP intensity at 2 g/L ATP addition. This result provided a bright future of high-level heterologous protein production by K. phaffii of AF induction.

Carbon starvation of Pseudomonas aeruginosa biofilms selects for dispersal insensitive mutants

BackgroundBiofilms disperse in response to specific environmental cues, such as reduced oxygen concentration, changes in nutrient concentration and exposure to nitric oxide. Interestingly, biofilms do not completely disperse under these conditions, which is generally attributed to physiological heterogeneity of the biofilm. However, our results suggest that genetic heterogeneity also plays an important role in the non-dispersing population of P. aeruginosa in biofilms after nutrient starvation.ResultsIn this study, 12.2% of the biofilm failed to disperse after 4 d of continuous starvation-induced dispersal. Cells were recovered from the dispersal phase as well as the remaining biofilm. For 96 h starved biofilms, rugose small colony variants (RSCV) were found to be present in the biofilm, but were not observed in the dispersal effluent. In contrast, wild type and small colony variants (SCV) were found in high numbers in the dispersal phase. Genome sequencing of these variants showed that most had single nucleotide mutations in genes associated with biofilm formation, e.g. in wspF, pilT, fha1 and aguR. Complementation of those mutations restored starvation-induced dispersal from the biofilms. Because c-di-GMP is linked to biofilm formation and dispersal, we introduced a c-di-GMP reporter into the wild-type P. aeruginosa and monitored green fluorescent protein (GFP) expression before and after starvation-induced dispersal. Post dispersal, the microcolonies were smaller and significantly brighter in GFP intensity, suggesting the relative concentration of c-di-GMP per cell within the microcolonies was also increased. Furthermore, only the RSCV showed increased c-di-GMP, while wild type and SCV were no different from the parental strain.ConclusionsThis suggests that while starvation can induce dispersal from the biofilm, it also results in strong selection for mutants that overproduce c-di-GMP and that fail to disperse in response to the dispersal cue, starvation.

Transcription factor based whole-cell biosensor for specific and sensitive detection of sodium dodecyl sulfate

Extensive use of Sodium Dodecyl Sulfate (SDS) in households, agricultural operations, and industries is leading to its subsequent disposal in waterways. There is an apprehension of the adverse effect of such detergents on various living organisms. Thus, an efficient, specific, and simple detection method to monitor SDS reliably in the environment is needed. We used sdsB1 activator protein and SDS-responsive promoter of sdsA1 gene along with Green Fluorescent Protein (GFP) to construct a novel SDS biosensor in Pseudomonas aeruginosa chassis. The GFP intensity of the biosensor showed a linear relationship (R2 = 0.99) from 0.4 to 62.5 ppm of SDS with a detection limit of 0.1 ppm. This biosensor is highly specific for SDS and has minimal interference from other detergents, metals, and inorganic ions. The biosensor showed a satisfactory and reproducible recovery rate for the detection of SDS in real samples. Overall, this is a low cost, easy-to-use, selective, and reliable biosensor for monitoring SDS in the environment.

Lack of methylation on transgene leads to high level and persistent transgene expression in induced pluripotent stem cells

Short-lived therapeutic gene expression in mammalian cells by DNA methylation is one of the major challenges in gene therapy. In this study, we assessed the implication of DNA methylation on the duration of GFP expression in mouse embryonic stem (ES) and mouse induced pluripotent stem (iPS) cells. The cells were transduced with lentivirus (LV) carrying green fluorescent protein (GFP) driven by either human elongation factor (EF1α) or cytomegalovirus (CMV) promoter. Transduced iPS cells exhibited higher percentage of GFP+ cells with persistent mean fluorescent intensity than transduced ES cells. Analysis on the integrated copy of transgene in the population of the transduced cells demonstrated similar copy number. However, significant increase in GFP intensity following 5-azaC treatment was observed in transduced ES cells only, suggesting the influence of DNA methylation in transgene silencing. Subsequent DNA methylation analysis showed that the promoter and the GFP region of the provirus in iPS cells had negligible methylation profile compared to transduced ES cells. Interestingly, sustained transgene expression was observed upon directed differentiation of transduced iPS cells towards CD34+ CD45+ cells. Hence, this study has shown that favourable transgene activity from lentiviral transduced iPS cells was due to the lack of methylation at the proviral regions.

MHC-mismatched Allotransplantation of Induced Pluripotent Stem Cell-derived Cardiomyocyte Sheets to Improve Cardiac Function in a Primate Ischemic Cardiomyopathy Model.

Although allogeneic-induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) exhibit potential in cardiomyogenesis for heart failure, whether major histocompatibility complex (MHC)-matched allogenic iPSC implantation (MMAI) minimizes immune rejection for cell survival or functional recovery remains unknown. We herein explored whether MMAI with an iPSC-CM sheet is stable for a longer period and therapeutically more effective than MHC-mismatched AI in a primate ischemic cardiomyopathy model. Green fluorescent protein-transfected iPSC-CM sheets, derived from cynomolgus macaques with homozygous MHC haplotypes ''HT1,'' were transplanted on the left ventricle, generated by ligating the left anterior descending artery for 2 weeks in an ischemic model with or without heterozygous HT1 as MMAI and MHC-mismatched AI. Sham models were made by opening the chest at 14 days after left anterior descending ligation without any treatment. Stereomicroscopy revealed that at 4 months after transplantation, green fluorescent protein intensity was higher in the MMAI group than in the MHC-mismatched AI group and the sham group. Immunohistochemistry staining revealed that host immune reaction with CD3-positive cells was stronger in MHC-mismatched AI than in MMAI at 3 months. Cardiac function improved both in MMAI and MHC-mismatched AI at 1 month after transplantation and was preserved until 6 months, whereas in the sham group, functional deterioration progressed over time. Although MHC-homo-iPSCs are preferred to avoid immune rejection, MHC-mismatched iPSC-CMs can also induce comparable cardiac functional recovery at late follow-up, suggesting that MHC-mismatched iPSC-based cardiac regenerative therapy with immunosuppressants is a feasible option for treating heart failure in clinical settings.

A Digital Method to Quantify Type I Interferon.

Interferon (IFN), the first ever-described cytokine, has a potent activity against viruses. Soon since its discovery, quantification of IFN has been an important issue. Most of the traditional methods to measure IFN biological activity rely on indirect methods that quantify dyes retained by IFN-protected cells against a lytic virus, or by techniques that indirectly quantify viral replication by measuring the expression level of viral-encoded reporter proteins such as the green fluorescent protein (GFP). In both cases, the IFN units are determined by the quantification of an effective dose 50, defined as the IFN dose that prevents 50% cell death of 50% reduction of the maximal amount of GFP intensity. In this study we propose the use of an alternative approach to measure IFN activity by calculating the minimal IFN dose 50 as the amount of IFN able to completely protect 50% of the cells from infection measured by the total absence of virus-dependent GFP signal in a cell culture plate. This sensitive approach could be used to easily quantify the Z value to determine IFN bioassay robustness. We believe that this approximation could be interesting to be considered by the IFN community.

Green Fluorescent Protein-Based Viability Assay in a Multiparametric Configuration.

Green fluorescent protein (GFP) is considered to be suitable for cell viability testing. In our study, GFP transfected A549 lung carcinoma cell line was treated with sodium fluoride (NaF), cycloheximide (CHX) and ochratoxin A (OTA). GFP fluorescence, intracellular ATP, nucleic acid and protein contents were quantified by a luminescence microplate assay developed in our laboratory. Flow cytometry was used to confirm the findings and to assess the intensity of GFP during different types of cell death. A 24 h NaF and CHX exposure caused a dramatic decrease in ATP contents (p < 0.05) compared with those of the controls. GFP fluorescence of the cells was in close correlation with total protein; however, GFP/ATP increased at NaF and decreased at CHX treatments (p < 0.05). ATP/protein and ATP/propidium iodide (PI) were largely decreased at NaF exposure in a dose-dependent manner (p < 0.05), while CHX and OTA showed markedly fewer effects. Both treatments caused apoptosis/necrosis at different rates. NaF induced mainly late apoptosis while OTA, mainly apoptosis. CHX effects varied by the incubation time with 100-fold elevation in late apoptotic cells at 24 h treatment. GFP intensity did not show a significant difference between live and apoptotic populations. Our results suggest when using GFP, a multiparametric assay is necessary for more precise interpretation of cell viability.

Engineering a yeast double-molecule carrier for drug screening

With the advantages of unicellular eukaryotic structure and easy manipulation, yeast becomes a popular tool for biochemical, genetic and medicinal studies. In order to construct an efficient anti-inflammatory drug screening platform, we engineered yeast as a double-molecule carrier, of which an inserted domain (I domain) of lymphocyte function-associated antigen 1 was displayed on yeast surface and a green fluorescent protein (GFP) was expressed inside cytosol. The I domain specifically targeted a surface marker of mammalian cells, intercellular adhesion molecule 1, whose number is correlated with the level of cellular inflammation. Examination of GFP intensity enables swift quantification of the yeast-mammalian cell binding and thus it reflects inflammatory potency, herein the inflammatory index, of a chemical imposed to cells. The inflammatory potency of a total of 1340 chemicals was indexed. Among them, 1 inflammation-inducing and 1 inflammation-reducing compounds were verified both in vitro and in vivo. Our method demonstrated a swift, facile and high-throughput screening platform at the protein level for inflammation and related diseases drug discovery without using sophisticated instruments.

Differentiation of human induced pluripotent stem cells into nucleus pulposus-like cells

BackgroundIntervertebral disc (IVD) degeneration is characterized by an early decrease in cellularity of the nucleus pulposus (NP) region, and associated extracellular matrix changes, reduced hydration, and progressive degeneration. Cell-based IVD therapy has emerged as an area of great interest, with studies reporting regenerative potential for many cell sources, including autologous or allogeneic chondrocytes, primary IVD cells, and stem cells. Few approaches, however, have clear strategies to promote the NP phenotype, in part due to a limited knowledge of the defined markers and differentiation protocols for this lineage. Here, we developed a new protocol for the efficient differentiation of human induced pluripotent stem cells (hiPSCs) into NP-like cells in vitro. This differentiation strategy derives from our knowledge of the embryonic notochordal lineage of NP cells as well as strategies used to support healthy NP cell phenotypes for primary cells in vitro.MethodsAn NP-genic phenotype of hiPSCs was promoted in undifferentiated hiPSCs using a stepwise, directed differentiation toward mesodermal, and subsequently notochordal, lineages via chemically defined medium and growth factor supplementation. Fluorescent cell imaging was used to test for pluripotency markers in undifferentiated cells. RT-PCR was used to test for potential cell lineages at the early stage of differentiation. Cells were checked for NP differentiation using immunohistochemistry and histological staining at the end of differentiation. To enrich notochordal progenitor cells, hiPSCs were transduced using lentivirus containing reporter constructs for transcription factor brachyury (T) promoter and green fluorescent protein (GFP) fluorescence, and then sorted on T expression based on GFP intensity by flow cytometry.ResultsPeriods of pellet culture following initial induction were shown to promote the vacuolated NP cell morphology and NP surface marker expression, including CD24, LMα5, and Basp1. Enrichment of brachyury (T) positive cells using fluorescence-activated cell sorting was shown to further enhance the differentiation efficiency of NP-like cells.ConclusionsThe ability to efficiently differentiate human iPSCs toward NP-like cells may provide insights into the processes of NP cell differentiation and provide a cell source for the development of new therapies for IVD diseases.

Overcoming Autofluorescence to Assess GFP Expression During Normal Physiology and Aging in Caenorhabditis elegans.

Green fluorescent protein (GFP) is widely used as a molecular tool to assess protein expression and localization. In C. elegans, the signal from weakly expressed GFP fusion proteins is masked by autofluorescence emitted from the intestinal lysosome-related gut granules. For instance, the GFP fluorescence from SKN-1 transcription factor fused to GFP is barely visible with common GFP (FITC) filter setups. Furthermore, this intestinal autofluorescence increases upon heat stress, oxidative stress (sodium azide), and during aging, thereby masking GFP expression even from proximal tissues. Here, we describe a triple band GFP filter setup that separates the GFP signal from autofluorescence, displaying GFP in green and autofluorescence in yellow. In addition, yellow fluorescent protein (YFP) remains distinguishable from both the yellowish autofluorescence and GFP with this triple band filter setup. Although some GFP intensity might be lost with the triple band GFP filter setup, the advantage is that no modification of currently used transgenic GFP lines is needed and these GFP filters are easy to install. Hence, by using this triple band GFP filter setup, the investigators can easily distinguish autofluorescence from GFP and YFP in their favorite transgenic C. elegans lines.

Dynamic and differential expression of the gonadal aromatase during the process of sexual differentiation in a novel transgenic cyp19a1a-eGFP zebrafish line

In zebrafish, there exists a clear need for new tools to study sex differentiation dynamic and its perturbation by endocrine disrupting chemicals. In this context, we developed and characterized a novel transgenic zebrafish line expressing green fluorescent protein (GFP) under the control of the zebrafish cyp19a1a (gonadal aromatase) promoter. In most gonochoristic fish species including zebrafish, cyp19a1a, the enzyme responsible for the synthesis of estrogens, has been shown to play a critical role in the processes of reproduction and sexual differentiation. This novel cyp19a1a-eGFP transgenic line allowed a deeper characterization of expression and localization of cyp19a1a gene in zebrafish gonads both at the adult stage and during development. At the adult stage, GFP expression was higher in ovaries than in testis. We showed a perfect co-expression of GFP and endogenous Cyp19a1a protein in gonads that was mainly localized in the cytoplasm of peri-follicular cells in the ovary and of Leydig and germ cells in the testis. During development, GFP was expressed in all immature gonads of 20 dpf-old zebrafish. Then, GFP expression increased in early differentiated female at 30 and 35dpf to reach a high GFP intensity in well-differentiated ovaries at 40dpf. On the contrary, males consistently displayed low GFP expression as compared to female whatever their stage of development, resulting in a clear dimorphic expression between both sexes. Interestingly, fish that undergoes ovary-to-testis transition (35 and 40dpf) presented GFP levels similar to males or intermediate between females and males. In this transgenic line our results confirm that cyp19a1a is expressed early during development, before the histological differentiation of the gonads, and that the down-regulation of cyp19a1a expression is likely responsible for the testicular differentiation. Moreover, we show that although cyp19a1a expression exhibits a clear dimorphic expression pattern in gonads during sexual differentiation, its expression persists whatever the sex suggesting that estradiol synthesis is important for gonadal development of both sexes. Monitoring the expression of GFP in control and exposed-fish will help determine the sensitivity of this transgenic line to EDCs and to refine mechanistic based-assays for the study of EDCs. In fine, this transgenic zebrafish line will be a useful tool to study physiological processes such as reproduction and sexual differentiation, and their perturbations by EDCs.

Cross-generational effects of parental low dose BPA exposure on the Gonadotropin-Releasing Hormone3 system and larval behavior in medaka (Oryzias latipes)

Growing evidence indicates that chronic exposure to Bisphenol A (BPA) may disrupt normal brain function and behavior mediated by gonadotropin-releasing hormone (GnRH) pathways. Previous studies have shown that low dose BPA (200ng/ml) exposure during embryogenesis altered development of extra-hypothalamic GnRH3 systems and non-reproductive locomotor behavior in medaka. Effects of parental low-dose BPA exposure on the development of GnRH3 systems and locomotor behavior of offspring are not well known. This study examines whether the neurophysiological and behavioral effects of BPA in parents (F0 generation) are carried over to their offspring (F1 generation) using stable transgenic medaka embryos/larvae with GnRH3 neurons tagged with green fluorescent protein (GFP). Parental fish were exposed to BPA (200ng/ml) for either life-long or different developmental time windows. Fertilized F1 eggs were collected and raised in egg/fish water with no environmental exposure to BPA. All experiments were performed on F1 embryos/larvae, which were grouped based on the following parental (F0) BPA exposure conditions – (i) Group 1 (G1): through life; (ii) G2: during embryogenesis and early larval development [1–14days post fertilization (dpf)]; (iii) G3: during neurogenesis (1–5dpf); and (iv) G4: during sex differentiation (5–14dpf). Embryos from unexposed vehicle treated parents served as controls (G0). G1 embryos showed significantly reduced survival rates and delayed hatching time compared to other groups, while G4 embryos hatched significantly earlier than all other groups. At 3 dpf, the GnRH3-GFP intensity was increased by 47% in G3 embryos and decreased in G4 embryos by 59% compared to controls. At 4dpf, G1 fish showed 42% increased intensity, while GFP intensity was reduced by 44% in G3 subjects. In addition, the mean brain size of G1, G3 and G4 embryos were smaller than that of control at 4dpf. At 20dpf, all larvae from BPA-treated parents showed significantly decreased total movement (distance covered) compared with controls, with G2 and G3 fish showing reduced velocity of movement. While at 20 dpf no group differences were seen in the soma diameter of GnRH3-GFP neurons, a 34% decrease in SV2 expression, a marker for synaptic transmission, in G1 larvae was observed. These data suggest that parental BPA exposure during critical windows of embryonic development or chronic treatment affects next-generation offspring both in embryonic and larval brain development as well as larval behavior.

Quantification of green fluorescent protein expression in mouse retinal ganglion cells following intravitreal injection of recombinant adeno‐associated virus

PurposeTo determine the transduction efficiency and spatial pattern of green fluorescent protein (GFP) expression in mouse retinal ganglion cells (RGCs) following intravitreal injection of recombinant adeno‐associated virus (rAAV2).Methods16 adult C57bL/6 mice received intravitreal injections of rAAV2‐CAG‐GFP at 3 different titres: 1 × 10E11, 1 × 10E12 or 1 × 10E13 genomic particles/ml. 2 μl of the virus was injected at each concentration. To determine the effect of volume on transduction efficiency, a subset of animals received 1 μl of the highest viral titre.Animals were sacrificed 21 days after injection. Retinal wholemounts were immunostained with Brn3a to identify RGCs and quantification carried out using imageJ software. The percentage of GFP positive RGCs was measured at each titre using Volocity software at 20× magnification. A further 20–25 images at 10× magnification were captured for each titre and merged using to reconstruct the entire wholemount to assess the spatial pattern of expression.ResultsRGC transduction rate increased with viral titre; 10% at 1 × 10E11, 53% and 1 × 10E12 and 64% at 1 × 10E13 genomic particles/ml. The volume injected did not appear to affect the transduction efficiency, with 64% of RGCs transduced at the highest titre using either 1 or 2 ul injection. GFP intensity also increased with viral titre with the spatial pattern of GFP expression more extensive at the highest titre. GFP expression at lower titres tended to localise around the injection site.ConclusionsGFP transduction efficiency of RGCs can be quantified efficiently using Volocity software. We have demonstrated an increase in GFP expression and spread at higher viral titres with similar transduction efficiency at a lower volume.

Hematopoietic stem cell-specific GFP-expressing transgenic mice generated by genetic excision of a pan-hematopoietic reporter gene.

Selective labeling of specific cell types by expression of green fluorescent protein (GFP) within the hematopoietic system would have great utility in identifying, localizing, and tracking different cell populations in flow cytometry, microscopy, lineage tracing, and transplantation assays. In this report, we describe the generation and characterization of a new transgenic mouse line with specific GFP labeling of all nucleated hematopoietic cells and platelets. This new "Vav-GFP" mouse line labels the vast majority of hematopoietic cells with GFP during both embryonic development and adulthood, with particularly high expression in hematopoietic stem and progenitor cells (HSPCs). With the exception of transient labeling of fetal endothelial cells, GFP expression is highly selective for hematopoietic cells and persists in donor-derived progeny after transplantation of HSPCs. Finally, we also demonstrate that the loxP-flanked reporter allows for specific GFP labeling of different hematopoietic cell subsets when crossed to various Cre reporter lines. By crossing Vav-GFP mice to Flk2-Cre mice, we obtained robust and highly selective GFP expression in hematopoietic stem cells (HSCs). These data describe a new mouse model capable of directing GFP labeling exclusively of hematopoietic cells or exclusively of HSCs.

Mucopolysaccharidosis IIIB confers enhanced neonatal intracranial transduction by AAV8 but not by 5, 9 or rh10.

Sanfilippo syndrome type B (mucopolysaccharidosis IIIB, MPS IIIB) is a lysosomal storage disease resulting from deficiency of N-acetyl-glucosaminidase (NAGLU) activity. To determine the possible therapeutic utility of recombinant adeno-associated virus (rAAV) in early gene therapy-based interventions, we performed a comprehensive assessment of transduction and biodistribution profiles of four central nervous system (CNS) administered rAAV serotypes, -5, -8, -9 and -rh10. To simulate optimal earliest treatment of the disease, each rAAV serotype was injected into the CNS of neonatal MPS IIIB and control animals. We observed marked differences in biodistribution and transduction profiles between the serotypes and this differed in MPS IIIB compared with healthy control mice. Overall, in control mice, all serotypes performed comparably, although some differences were observed in certain focal areas. In MPS IIIB mice, AAV8 was more efficient than AAV5, -9 and -rh10 for gene delivery to most structures analyzed, including the cerebral cortex, hippocampus and thalamus. Noteworthy, the pattern of biodistribution within the CNS varied by serotype and genotype. Interestingly, AAV8 also produced the highest green fluorescent protein intensity levels compared with any other serotype and demonstrated improved transduction in NAGLU compared with control brains. Importantly, we also show leakage of AAV8, -9 and -rh10, but not AAV5, from CNS parenchyma to systemic organs. Overall, our data suggest that AAV8 represents the best therapeutic gene transfer vector for early intervention in MPS IIIB.

PSW2, a Novel Low-Temperature-Inducible Gene Expression Vector Based on a Filamentous Phage of the Deep-Sea Bacterium Shewanella piezotolerans WP3.

A low-temperature-inducible protein expression vector (pSW2) based on a filamentous phage (SW1) of the deep-sea bacterium Shewanella piezotolerans WP3 was constructed. This vector replicated stably in Escherichia coli and Shewanella species, and its copy number increased at low temperatures. The pSW2 vector can be utilized as a complementation plasmid in WP3, and it can also be used for the production of complex cytochromes with multiple heme groups, which has the potential for application for metal ion recovery or bioremediation. Promoters of low-temperature-inducible genes in WP3 were fused into the vector to construct a series of vectors for enhancing protein expression at low temperature. The maximum green fluorescent protein intensity was obtained when the promoter for the hfq gene was used. The WP3/pSW2 system can efficiently produce a patatin-like protein (PLP) from a metagenomic library that tends to form inclusion bodies in E. coli. The yields of PLP in the soluble fraction were 8.3 mg/liter and 4.7 mg/liter of culture at 4°C and 20°C, respectively. Moreover, the pSW2 vector can be broadly utilized in other Shewanella species, such as S. oneidensis and S. psychrophila.

Linear correlation between average fluorescence intensity of green fluorescent protein and the multiplicity of infection of recombinant adenovirus.

BackgroundAdenoviral vector is an efficient tool for gene transfer. Protein expression is regulated by a number of factors, but the regulation by gene copy number remains to be investigated further.ResultsAssessed by flow cytometry, we demonstrated a significant linear correlation between average fluorescence intensity of green fluorescent protein (GFP) and a wide range of multiplicity of infection (MOI), spanning from 0.01 to 200. Average GFP intensity was calculated by mean fluorescence intensity (MFI) × percentage of infection (POI) (MFI × POI) and the correlation was observed in cells transduced with GFP-expressing adenoviral vector driven either by a cytomegalovirus (CMV) promoter for 3 to 6 h or by a human phosphoglycerate kinase (PGK) promoter for 18 to 24 h. Factors impacting this linear correlation include MOI of viral vector, strength of promoter driving GFP expression, cell type transduced and incubation time after gene transfer. We also found that weak GFP signals could be interfered by background signals, whereas strong GFP signals could overshot the detection limitation of the flow cytometer and resulted in a deviation from linearity which was prevented by adjusting the setting in flow cytometer. Moreover, we compared promoter strength as measured by MFI × POI and found that the relative activity of CMV promoter to PGK promoter was 20 to 47 folds in A549 cells and 32 to > 100 folds in H1299 cells.ConclusionsThe linear correlation between MFI × POI and a wide range of adenoviral MOI provides an efficient method to investigate factors regulating protein expression and to estimate virus titers.Electronic supplementary materialThe online version of this article (doi:10.1186/s12929-015-0137-z) contains supplementary material, which is available to authorized users.

High-Throughput Differentiation and Screening of a Library of Mutant Stem Cell Clones Defines New Host-Based Genes Involved in Rabies Virus Infection.

We used a genomic library of mutant murine embryonic stem cells (ESCs) and report the methodology required to simultaneously culture, differentiate, and screen more than 3,200 heterozygous mutant clones to identify host-based genes involved in both sensitivity and resistance to rabies virus infection. Established neuronal differentiation protocols were miniaturized such that many clones could be handled simultaneously, and molecular markers were used to show that the resultant cultures were pan-neuronal. Next, we used a green fluorescent protein (GFP) labeled rabies virus to develop, validate, and implement one of the first host-based, high-content, high-throughput screens for rabies virus. Undifferentiated cell and neuron cultures were infected with GFP-rabies and live imaging was used to evaluate GFP intensity at time points corresponding to initial infection/uptake and early and late replication. Furthermore, supernatants were used to evaluate viral shedding potential. After repeated testing, 63 genes involved in either sensitivity or resistance to rabies infection were identified. To further explore hits, we used a completely independent system (siRNA) to show that reduction in target gene expression leads to the observed phenotype. We validated the immune modulatory gene Unc13d and the dynein adapter gene Bbs4 by treating wild-type ESCs and primary neurons with siRNA; treated cultures were resistant to rabies infection/replication. Overall, the potential of such in vitro functional genomics screens in stem cells adds additional value to other libraries of stem cells. This technique is applicable to any bacterial or virus interactome and any cell or tissue types that can be differentiated from ESCs.

Gene delivery in mouse auditory brainstem and hindbrain using in utero electroporation.

BackgroundManipulation of gene expression via recombinant viral vectors and creation of transgenic knock-out/in animals has revolutionized our understanding of genes that play critical roles during neuronal development and pathophysiology of neurological disorders. Recently, target-specific genetic manipulations are made possible to perform in combination with specific Cre-lines, albeit costly, labor-intensive and time consuming. Thus, alternative methods of gene manipulations to address important biological questions are highly desirable. In this study, we utilized in utero electroporation technique which involves efficient delivery of hindbrain-specific enhancer/promoter construct, Krox20 into the third ventricle of live mouse embryo to investigate green fluorescent protein (GFP) expression pattern in mouse auditory brainstem and other hindbrain neurons.ResultsWe created a GFP/DNA construct containing a Krox20 B enhancer and β-globin promoter to drive GFP expression in the hindbrain via injection into the third ventricle of E12 to E13.5 mice. Electrical currents were applied directly to the embryonic hindbrain to allow DNA uptake into the cell. Confocal images were then acquired from fixed brain slices to analyze GFP expression in mouse whole brain at different postnatal stages (P6-P21). By using a cell-type specific enhancer as well as region specific injection and electroporation, robust GFP expression in the cerebellum and auditory brainstem but not in the forebrain was observed. GFP expression in calyx of Held terminals was more robust in <P15 compared to >P15 mice. In contrast, GFP expression in MNTB neurons was more prevalent in >P15 compared to <P15. In regards to the relative expression of GFP versus the synaptic marker Vglut1, percentage fluorescence GFP intensity in the calyx was higher in P11 to P15 than P6 to P10 and P16 to P21 groups.ConclusionsTaken together, this technique would potentially allow hindbrain-specific genetic manipulations such as knock-down, knock-in and rescue experiments to unravel critical molecular substrates underpinning functional and morphological remodeling of synapses as well as understanding the pathophysiology of certain neurological disorders targeting not only the auditory brainstem but also other parts of hindbrain, most notably the cerebellum.