Higher Green Fluorescent Protein Expression Research Articles

Impact of cell wall polysaccharide modifications on the performance of Pichia pastoris: novel mutants with enhanced fitness and functionality for bioproduction applications

BackgroundPichia pastoris is a widely utilized host for heterologous protein expression and biotransformation. Despite the numerous strategies developed to optimize the chassis host GS115, the potential impact of changes in cell wall polysaccharides on the fitness and performance of P. pastoris remains largely unexplored. This study aims to investigate how alterations in cell wall polysaccharides affect the fitness and function of P. pastoris, contributing to a better understanding of its overall capabilities.ResultsTwo novel mutants of GS115 chassis, H001 and H002, were established by inactivating the PAS_chr1-3_0225 and PAS_chr1-3_0661 genes involved in β-glucan biosynthesis. In comparison to GS115, both modified hosts exhibited a looser cell surface and larger cell size, accompanied by faster growth rates and higher carbon-to-biomass conversion ratios. When utilizing glucose, glycerol, and methanol as exclusive carbon sources, the carbon-to-biomass conversion rates of H001 surpassed GS115 by 10.00%, 9.23%, and 33.33%, respectively. Similarly, H002 exhibited even higher increases of 32.50%, 12.31%, and 53.33% in carbon-to-biomass conversion compared to GS115 under the same carbon sources. Both chassis displayed elevated expression levels of green fluorescent protein (GFP) and human epidermal growth factor (hegf). Compared to GS115/pGAPZ A-gfp, H002/pGAPZ A-gfp showed a 57.64% higher GFP expression, while H002/pPICZα A-hegf produced 66.76% more hegf. Additionally, both mutant hosts exhibited enhanced biosynthesis efficiencies of S-adenosyl-L-methionine and ergothioneine. H001/pGAPZ A-sam2 synthesized 21.28% more SAM at 1.14 g/L compared to GS115/pGAPZ A-sam2, and H001/pGAPZ A-egt1E obtained 45.41% more ERG at 75.85 mg/L. The improved performance of H001 and H002 was likely attributed to increased supplies of NADPH and ATP. Specifically, H001 and H002 exhibited 5.00-fold and 1.55-fold higher ATP levels under glycerol, and 6.64- and 1.47-times higher ATP levels under methanol, respectively, compared to GS115. Comparative lipidomic analysis also indicated that the mutations generated richer unsaturated lipids on cell wall, leading to resilience to oxidative damage.ConclusionsTwo novel P. pastoris chassis hosts with impaired β-1,3-D-glucan biosynthesis were developed, showcasing enhanced performances in terms of growth rate, protein expression, and catalytic capabilities. These hosts exhibit the potential to serve as attractive alternatives to P. pastoris GS115 for various bioproduction applications.

The power of sulfhydryl groups: Thiolated lipid-based nanoparticles enhance cellular uptake of nucleic acids

AimThe aim of the study was to evaluate the effect of thiolation of lipid-based nanoparticles (LNPs) on cellular uptake of nucleic acids. MethodsA thiolated surfactant was synthesized by binding palmitic acid covalently to cysteine. Green fluorescent protein (GFP) encoding plasmid DNA (pDNA) was used as model nucleic acid and incorporated via hydrophobic ion-pairing with a cationic cholesterol derivate (DcCholesterol) in LNPs that were prepared by solvent injection method using the thiolated surfactant for surface decoration. LNPs were characterized regarding size, polydispersity index, zeta potential and stability in biorelevant media. The endosomal escape properties of LNPs were evaluated by erythrocytes interaction studies. Cell viability and transfection efficiency on HEK293 cells were investigated. ResultsThe structure of the thiolated surfactant was confirmed by 1H NMR and FT-IR. LNPs containing the nucleic acid-DcCholesterol complex with and without the thiolated surfactant were developed and displayed sizes in the range from 173 nm to 233 nm with a narrow size distribution (PDI < 0.3) and a negative zeta potential. LNPs showed no significant increase in size after 4 h of incubation in artificial body fluids. Erythrocytes interaction studies revealed enhanced endosomal escape properties for thiolated LNPs compared to non-thiolated LNPs. LNPs showed in concentrations lower than 0.74 mg/mL cell viability ≥ 80 %. Transfection studies on HEK cells with thiolated LNPs compared to non-thiolated LNP showed a 4.6-fold higher expression of GFP. ConclusionSurface thiolation of LNPs represents a promising tool for enhancing intracellular nucleic acid delivery of LNPs.

Performance of abiotic stress-inducible synthetic promoters in genetically engineered hybrid poplar (Populus tremula × Populus alba).

Abiotic stresses can cause significant damage to plants. For sustainable bioenergy crop production, it is critical to generate resistant crops to such stress. Engineering promoters to control the precise expression of stress resistance genes is a very effective way to address the problem. Here we developed stably transformed Populus tremula × Populus alba hybrid poplar (INRA 717-1B4) containing one-of-six synthetic drought stress-inducible promoters (SDs; SD9-1, SD9-2, SD9-3, SD13-1, SD18-1, and SD18-3) identified previously by transient transformation assays. We screened green fluorescent protein (GFP) induction in poplar under osmotic stress conditions. Of six transgenic lines containing synthetic promoter, three lines (SD18-1, 9-2, and 9-3) had significant GFP expression in both salt and osmotic stress treatments. Each synthetic promoter employed heptamerized repeats of specific and short cis-regulatory elements (7 repeats of 7-8 bases). To verify whether the repeats of longer sequences can improve osmotic stress responsiveness, a transgenic poplar containing the synthetic promoter of the heptamerized entire SD9 motif (20 bases, containing all partial SD9 motifs) was generated and measured for GFP induction under osmotic stress. The heptamerized entire SD9 motif did not result in higher GFP expression than the shorter promoters consisting of heptamerized SD9-1, 9-2, and 9-3 (partial SD9) motifs. This result indicates that shorter synthetic promoters (~50 bp) can be used for versatile control of gene expression in transgenic poplar. These synthetic promoters will be useful tools to engineer stress-resilient bioenergy tree crops in the future.

Rescue of the first alphanucleorhabdovirus entirely from cloned complementary DNA: An efficient vector for systemic expression of foreign genes in maize and insect vectors.

Recent reverse genetics technologies have enabled genetic manipulation of plant negative-strand RNA virus (NSR) genomes. Here, we report construction of an infectious clone for the maize-infecting Alphanucleorhabdovirus maydis, the first efficient NSR vector for maize. The full-length infectious clone was established using agrobacterium-mediated delivery of full-length maize mosaic virus (MMV) antigenomic RNA and the viral core proteins (nucleoprotein N, phosphoprotein P, and RNA-directed RNA polymerase L) required for viral transcription and replication into Nicotiana benthamiana. Insertion of intron 2 ST-LS1 into the viral L gene increased stability of the infectious clone in Escherichia coli and Agrobacterium tumefaciens. To monitor virus infection in vivo, a green fluorescent protein (GFP) gene was inserted in between the N and P gene junctions to generate recombinant MMV-GFP. Complementary DNA (cDNA) clones of MMV-wild type (WT) and MMV-GFP replicated in single cells of agroinfiltrated N.benthamiana. Uniform systemic infection and high GFP expression were observed in maize inoculated with extracts of the infiltrated N.benthamiana leaves. Insect vectors supported virus infection when inoculated via feeding on infected maize or microinjection. Both MMV-WT and MMV-GFP were efficiently transmitted to maize by planthopper vectors. The GFP reporter gene was stable in the virus genome and expression remained high over three cycles of transmission in plants and insects. The MMV infectious clone will be a versatile tool for expression of proteins of interest in maize and cross-kingdom studies of virus replication in plant and insect hosts.

Reducing phenotypic instabilities of a microbial population during continuous cultivation based on cell switching dynamics.

Predicting the fate of individual cells among a microbial population (i.e., growth and gene expression) remains a challenge, especially when this population is exposed to very dynamic environmental conditions, such as those encountered during continuous cultivation. Indeed, the dynamic nature of a continuous cultivation process implies the potential diversification of the microbial population resulting in genotypic and phenotypic heterogeneity. The present work focused on the induction of the arabinose operon in Escherichia coli as a model system to study this diversification process in continuous cultivations. As a preliminary step, the green fluorescent protein (GFP) level triggered by an arabinose-inducible ParaBAD promoter was tracked by flow cytometry in chemostat cultivations with glucose-arabinose co-feeding. For a wide range of glucose-arabinose co-feeding concentrations in the chemostats, the simultaneous occurrence of GFP positive and negative subpopulation was observed. In the second set of experiments, continuous cultivation was performed by adding glucose continuously and arabinose based on the capability of individual cells to switch from low GFP to high GFP expression states, performed with a technology setup called segregostat. In the segregostat cultivation mode, on-line flow cytometry analysis was used for adjusting the arabinose/glucose transitions based on the phenotypic switching profiles of the microbial population. This strategy allowed finding an appropriate arabinose pulsing frequency, leading to prolonged maintenance of the induction level with a limited increase in the phenotypic diversity for more than 60 generations. The results suggest that the steady forcing of individual cells into a given phenotypic trajectory may not be the best strategy for controlling cell populations. Instead, allowing individual cells to switch periodically around a predefined threshold seems to be a more robust strategy leading to oscillations, but within a predictable cell population behavior range.

Identification of oxygen-sensitive neuroepithelial cells through an endogenous reporter gene in larval and adult transgenic zebrafish.

In teleost fish, specialized oxygen (O2) chemoreceptors, called neuroepithelial cells (NECs), are found in the gill epithelium in adults. During development, NECs are present in the skin before the formation of functional gills. NECs are known for retaining the monoamine neurotransmitter, serotonin (5-HT) and are conventionally identified through immunoreactivity with antibodies against 5-HT or synaptic vesicle protein (SV2). However, identification of NECs in live tissue and isolated cell preparations has been challenging due to the lack of a specific marker. The present study explored the use of the transgenic zebrafish, ETvmat2:GFP, which expresses green fluorescent protein (GFP) under the control of the vesicular monoamine transporter 2 (vmat2) regulatory element, to identify NECs. Using immunohistochemistry and confocal microscopy, we confirmed that the endogenous GFP in ETvmat2:GFP labelled serotonergic NECs in the skin of larvae and in the gills of adults. NECs of the gill filaments expressed a higher level of endogenous GFP compared with other cells. The endogenous GFP also labelled intrabranchial neurons of the gill filaments. Flow cytometric analysis demonstrated that filamental NECs could be distinguished from other dissociated gill cells based on high GFP expression alone. Acclimation to 2weeks of severe hypoxia (PO2=35mmHg) induced an increase in filamental NEC frequency, size and GFP gene expression. Here we present for the first time a transgenic tool that labels O2 chemoreceptors in an aquatic vertebrate and its use in high-throughput experimentation.

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.

Application of polybrene to increase transfection efficiency of lentiviral vector in bone marrow derived dendritic cells

Objective To investigate the toxicity and optimal concentration of polybrene on lentiviral vector transduction of bone marrow derived dendritic cells (BMDCs). Methods The BMDCs suspensions in mice were prepared and randomly divided into experimental and control groups. The BMDCs in experimental groups were treated with different concentrations of polybrene, and those in control groups were added with phosphate buffer (PBS). Cell counting kit-8 (CCK-8) assay and annexin V-fluoresceine isothiocyanate (FITC)/propidium iodide (PI) staining were performed to detect cells viability and apoptosis. Add to BMDCs suspensions were added with lentiviral vector [multiplicity of infection (MOI)=10] and the mixture was divided into experimental and control groups. The mixture in experimental groups was treated with different concentrations of polybrene, and that in control groups was given PBS. The expression of green fluorescent protein (GFP) was observed by fluorescence microscope every day. After the BMDCs were stimulated to mature, the GFP expression was detected using flow cytometry. The t test was used for comparison between two groups. Results The number of living cells in experimental group was significantly less than in control group when polybrene ≥11 mg/L [The absorbance (A) values were 0.881±0.007 vs. 1.031±0.017, t=7.832, P<0.05]. The GFP expression in experimental groups was significantly higher than in control groups. The highest GFP expression was observed when polybrene at 8 mg/L. Conclusion Polybrane shows dose-dependent toxicity on BMDCs, and in the safe range of concentration, polybrane can significantly increase the transfection efficacy of lentiviral vector to BMDCs and the optimal concentration is 8 mg/L. Key words: Polybrene; Lentiviral vector; Bone marrow derived dendritic cells

Modulation of mRNA Translation and Cell Viability by Influenza A Virus Derived Nonstructural Protein 1.

Translation of in vitro transcribed messenger RNA (mRNA) is known to be compromised by cell's innate immune responses. Herein we show that when mRNA encoding nonstructural protein 1 (NS1), an immune evasion gene derived from influenza A virus, is co-delivered with mRNA encoding green fluorescent protein (GFP), higher GFP expression can be observed in four different interferon competent cell types within 6 h, indicating NS1's wide host range property and rapid counter response to the cells' innate immune response. Enhanced mRNA translation correlates with reduced interferon production in all tested cell types and substituting a small portion of luciferase mRNA with NS1 mRNA enhances luciferase production compared to the same dose composing of only luciferase mRNA although in a cell type specific manner. Toxicity caused by transfection of unmodified mRNA is mitigated with the delivery of NS1 mRNA and is observed only in NS1 without cleavage and polyadenylation specificity factor 30 kda (CPSF30) inhibition function. Conversely, delivery of mRNA encoding NS1 with CPSF30 inhibition function aggravated toxicity. Overall, we demonstrate that NS1 enhanced mRNA transfection through active evasion of innate immune responses and modulated cellular viability during mRNA transfection.

Real - time monitoring of circulation tumor cells in a surgical orthotopic implantation model of human metastatic prostate cancer

Objective To study the relationship of circulation tumor cells (CTCs) and metastatic process, we use subcutaneous model and surgical orthotopic implantation model of human metastatic prostate cancer and detected by in vivo flow cytometry (IVFC). Methods The human prostate cancer cell line PC3 cells were transduced with pLVX-EGFP-3FLAG lentivirus vector containing the green fluorescent protein (GFP) and puromycin resistance gene. PC3 cells, high-expression of GFP, were obtained by screening of stably transfected clones. PC3-GFP cells or unlabeled tumor cells were implanted into the right armpit of 10 nude mice to build up subcutaneous xenograft prostate cancer mode1, which provided tumor fragments to implant the orthotopic prostate tumor mode1. Tumor tissue with high fluorescent from the subcutaneous tumor was harvested and cut into pieces of 1 mm3, and grafted into the anterior prostates of 10 nude mice. The number of CTCs monitored by IVFC real-time detecting fluorescent signal noninvasively at 5 d, 10 d and 15 d after tumor established. Results A PC3-GFP cell line was established which had the same growth pattern as well as tumorigenesis ability as mock-transfected PC3 cells. IVFC detected 2.37, 6.25, 99.75 CTCs/h at 5, 10, and 15 days. Conclusion The orthotopic prostate cancer models are better to assess the morphology, growth, metastatic pattern and development characteristics of clinical disease. They are also more representative of a primary tumor with respect to tumor site and metastasis, especially CTCs dynamics. The in vivo flow cytometry technique combined with orthotopic tumor models might provide insights into tumor hematogenous metastasis and guidance to cancer therapy. Key words: Prostate cancer; Metastasis; Circulating tumor cells; In vivo flow cytometry

Generation of a novel transgenic rat model for tracing extracellular vesicles in body fluids.

Extracellular vesicles (EVs) play an important role in the transfer of biomolecules between cells. To elucidate the intercellular transfer fate of EVs in vivo, we generated a new transgenic (Tg) rat model using green fluorescent protein (GFP)-tagged human CD63. CD63 protein is highly enriched on EV membranes via trafficking into late endosomes and is often used as an EV marker. The new Tg rat line in which human CD63-GFP is under control of the CAG promoter exhibited high expression of GFP in various body tissues. Exogenous human CD63-GFP was detected on EVs isolated from three body fluids of the Tg rats: blood serum, breast milk and amniotic fluid. In vitro culture allowed transfer of serum-derived CD63-GFP EVs into recipient rat embryonic fibroblasts, where the EVs localized in endocytic organelles. These results suggested that this Tg rat model should provide significant information for understanding the intercellular transfer and/or mother-child transfer of EVs in vivo.

Vector serotype screening for use in ovine perinatal lung gene therapy

PurposeSuccessful in utero or perinatal gene therapy for congenital lung diseases, such as cystic fibrosis and surfactant protein deficiency, requires identifying clinically relevant viral vectors that efficiently transduce airway epithelial cells. The purpose of the current preclinical large animal study was to evaluate lung epithelium transduction of adeno-associated viral (AAV) vector serotypes following intratracheal delivery. MethodsSix different AAV vector serotypes (AAV1, AAV5, AAV6, AAV8, AAV9, and AAVrh10) expressing the green fluorescent protein (GFP) as the transgene were injected into the right upper lobe of perinatal sheep via bronchoscopy. At 1 week, samples were harvested, analyzed by fluorescent stereomicroscopy and immunohistochemistry, and quantified using a radial grid and quantitative real-time polymerase chain reaction (qPCR). ResultsFluorescent stereomicroscopy demonstrated GFP expression in the right upper lobe following injection of all AAV serotypes assessed except AAV5. Immunohistochemistry analysis confirmed GFP expression in small- and medium-sized airways following intratracheal injection of AAV1, 6, 8, 9, and rh10. However, only AAV8 and AAVrh10 resulted in transgene expression in large airways. These results were confirmed by qPCR, yet, after 40 cycles, AAV1 did not show GFP gene amplification. ConclusionAdeno-associated viral vector serotypes 6, 8, 9, and rh10 demonstrated efficient GFP transgene expression at early time points, and AAV8 demonstrated efficient transduction of all airway sizes with high pulmonary GFP expression tested using qPCR.

Sex differences in cells expressing green fluorescent protein under the control of the estrogen receptor-α promoter in the hypothalamus of mice

Estradiol that originates from testicular testosterone and binds to estrogen receptor-α (ERα) during developing period acts to organize the male-type brain in mice. Here, we examined transgenic mice expressing green fluorescent protein (GFP) under the control of the ERα promoter, in which ERα-expressing cells in the brain can be visualized by GFP. Fluorescence microscopy revealed the existence of many GFP-expressing cells in the medial preoptic area, medial preoptic nucleus (MPN), bed nucleus of the stria terminalis (BNST), and striohypothalamic nucleus (StHy) of adult transgenic mice. Neuronal nuclear antigen, a neuron marker, but not glial fibrillary acidic protein, an astrocyte marker, was mostly expressed by GFP-expressing cells. Analysis of GFP expression area showed that adult females had higher GFP expression in a region including the ventral part of the BNST, StHy, and dorsal part of the MPN than in adult males. Such female-biased sex difference was also found in transgenic pups on postnatal day 5 and 8. The GFP expression area of adult females was decreased by postnatal treatment with testosterone or estradiol. These results indicate that GFP visualizes a sex difference of ERα-expressing neurons. The transgenic mice may be useful for the analysis of the sexual differentiation of the brain.

In vivo tracking of adipose-derived stem cells labeled with green fluorescent protein and luciferase dual reporter gene

Objective T o explore the feasibility of in vivo tracking of adipose-derived stem cells( ADSC s) by using lentiviral vectors containing green fluorescent protein( GFP) and luciferase( Luc) dual reporter gene transfection and bioluminescent imaging techniques. Methods T he ADSC s were isolated from human adipose tissue samples of cosmetic subdermal liposuction. T he ADSC s were transfected by lentiviral vectors containing GFP and Luc. T he labeling efficiency was evaluated by fluorescence microscope and flow cytometry in vitro. T he transfected ADSC s were subcutaneously injected into BALB /C mice, and then a non-invasive bioluminescence imaging procedure was performed 1 h, 1, 2, 4, 6 and 8 weeks after transplantation. T he DAPI staining of skin tissues was performed to identify the survival and the location of transplanted ADSC s. Results High GFP expression was observed in transfected ADSC s by lentiviral vector under fluorescence microscope. T he transfection efficiency of lentiviral vectors containing GFP and Luc was 87. 3%. T ransfected ADSC s can express GFP and Luc after 25 successive generations, and the transduction of ADSC s with lentiviral bioluminescence reporter vectors had no effect on cell proliferation. T he bioluminescence of ADSC s in vivo can be detectable as long as 8 weeks after transplantion, and the ADSC s mainly survive in the inoculating site of the thigh.Histological analysis of sections showed that the labeled ADSC s located in the subcutaneous layer of the implantation site. Conclusion U sing lentiviral vectors containing GFP and Luc dual reporter gene transfection and bioluminescent imaging techniques can monitor ADSC s in vivo.

Optimization of particle bombardment parameters for DNA delivery into the male flowers of banana

The possibility of increasing the efficiency of banana transformation was investigated by particle bombardment of the male flowers of banana plants for constitutive expression of gfp gene. The effects of particle bombardment parameters, such as acceleration pressure, bombardment distance, chamber vacuum pressure, gold microcarrier size, gold quantity, DNA quantity, number of bombardments and pre-culture were examined. Single cauliflower-like bodies (CLBs) clusters, induced from meristemic parts of Musa sapientum cv. Nangka (AAB) male flowers, were bombarded by pCambia1304 plasmid carrying gfp gene driven by the CaMV 35S promoter. Optimal transient expression of green-fluorescent protein (GFP) was obtained when the three-day old cultured tissues were bombarded two times at 1100 psi helium pressure. However, the highest GFP expression was observed when 9 cm was applied as bombardment distance with 28 mmHg chamber vacuum pressure. Gold particle with 1 μm diameter at 60 μg/μL concentrations coated with 1.5 μg/μL of DNA have been used as the optimum bombardment parameter since GFP expression was significantly different compared to other conditions. Application of optimized condition proved effective for the generation of stable transgenic banana plants. PCR and southern blot analyses confirmed the presence and integration of gfp gene in genomic DNA of transformed plants. Transformation frequency achieved with the optimized protocol was 7.5% which was significantly higher than the conventional protocol.

Novel anti‐tumor mechanism of galanin receptor type 2 in head and neck squamous cell carcinoma cells

Galanin and its receptors, GALR1 and GALR2, are known tumor suppressors and potential therapeutic targets in head and neck squamous cell carcinoma (HNSCC). Previously, we demonstrated that, in GALR1-expressing HNSCC cells, the addition of galanin suppressed tumor proliferation via upregulation of ERK1/2 and cyclin-dependent kinase inhibitors, whereas, in GALR2-expressing cells, the addition of galanin not only suppressed proliferation, but also induced apoptosis. In this study, we first transduced HEp-2 and KB cell lines using a recombinant adeno-associated virus (rAAV)-green fluorescent protein (GFP) vector and confirmed a high GFP expression rate (>90%) in both cell lines at the standard vector dose. Next, we demonstrated that GALR2 expression in the presence of galanin suppressed cell viability to 40–60% after 72 h in both cell lines. Additionally, the annexin V-positive rate and sub-G0/G1 phase population were significantly elevated in HEp-2 cells (mock vs GALR2: 12.3 vs 25.0% (P < 0.01) and 9.1 vs 32.0% (P < 0.05), respectively) after 48 h. These changes were also observed in KB cells, although to a lesser extent. Furthermore, in HEp-2 cells, GALR2-mediated apoptosis was caspase-independent, involving downregulation of ERK1/2, followed by induction of the pro-apoptotic Bcl-2 protein, Bim. These results illustrate that transient GALR2 expression in the presence of galanin induces apoptosis via diverse pathways and serves as a platform for suicide gene therapy against HNSCC.

Baculovirus-mediated gene transfer in butterfly wings in vivo: an efficient expression system with an anti-gp64 antibody

BackgroundCandidate genes for color pattern formation in butterfly wings have been known based on gene expression patterns since the 1990s, but their functions remain elusive due to a lack of a functional assay. Several methods of transferring and expressing a foreign gene in butterfly wings have been reported, but they have suffered from low success rates or low expression levels. Here, we developed a simple, practical method to efficiently deliver and express a foreign gene using baculovirus-mediated gene transfer in butterfly wings in vivo.ResultsA recombinant baculovirus containing a gene for green fluorescent protein (GFP) was injected into pupae of the blue pansy butterfly Junonia orithya (Nymphalidae). GFP fluorescence was detected in the pupal wings and other body parts of the injected individuals three to five days post-injection at various degrees of fluorescence. We obtained a high GFP expression rate at relatively high virus titers, but it was associated with pupal death before color pattern formation in wings. To reduce the high mortality rate caused by the baculovirus treatment, we administered an anti-gp64 antibody, which was raised against baculovirus coat protein gp64, to infected pupae after the baculovirus injection. This treatment greatly reduced the mortality rate of the infected pupae. GFP fluorescence was observed in pupal and adult wings and other body parts of the antibody-treated individuals at various degrees of fluorescence. Importantly, we obtained completely developed wings with a normal color pattern, in which fluorescent signals originated directly from scales or the basal membrane after the removal of scales. GFP fluorescence in wing tissues spatially coincided with anti-GFP antibody staining, confirming that the fluorescent signals originated from the expressed GFP molecules.ConclusionsOur baculovirus-mediated gene transfer system with an anti-gp64 antibody is reasonably efficient, and it can be an invaluable tool to transfer, express, and functionally examine foreign genes in butterfly wings and also in other non-model insect systems.

Transformation of Upland Cotton (Gossypium hirsutum L.) with gfp Gene as a Visual Marker

The green-fluorescent protein (gfp) gene was evaluated as a screening marker during cotton (Gossypium hirsutum L.) transforming and plant regeneration. High expression of GFP (green-fluorescent protein) was observed in transgenic cells as early as 42 h after co-culture with Agrobacterium. Most of the stable transformation events were detected in the cells of primary vascular tissue. GFP transient expression could be detected on all the explants after co-culturing for 4 d, however, the highest GFP stable expression was recorded when the explants were co-cultured for 3 d. We believe the transient and stable expression of a foreign gene in genetic transformation were two relative but different events, because high transient expression did not surely lead to high stable transformation. Under the same conditions of in vitro culture, transgenic and non-transgenic calli exhibited different morphological characters on different stages of development. High concentration of plant growth regulators (PGRs) was efficient for somatic embryogenesis of the transgenic calli, which means that the transgenic calli need relatively higher dose of hormone for further growth and somatic embryogenesis than non-transgenic ones. Strong GFP-expression was observed in leaf, stem, petioles, floral tissues, and seedlings of T1 progeny. Segregation ratios of eight transgenic lines were scored for expression of GFP in the T1 progeny that providing further evidence of stable transformation. These results proved that GFP is a powerful reporter gene for protocol optimization, selection, and monitioring in whole transformation events.

English

&nbsp; Constructs carrying visual reporter genes coupled with efficient promoters could facilitate the process of identification and selection of stable transformants in recalcitrant crops.&nbsp;Here,&nbsp;a&nbsp;novel construct utilizing a ribulose-1,5-bisphosphate carboxylase (RbcS) promoter combined with the green fluorescent protein&nbsp;(GFP)reporter gene to initiate very high expression of GFP in florist&#39;s chrysanthemum(Chrysanthemum morifolium&nbsp;Ramat.)&nbsp;was&nbsp;described.&nbsp;Based on this expression cassette,&nbsp;a new regeneration protocol using leaf discs as explants was developed for the&nbsp;Agrobacterium-mediated transformation of&nbsp;Chrysanthemum&nbsp;genotype &lsquo;1581&rsquo;, and a transformation efficiency of 7% was obtained. The expression of two different GFP constructs targeted to either cytosol or plastids was compared in transgenic lines.&nbsp;Both GFP constructs were expressed at such a high level that the green fluorescence dominated red fluorescence in the leaf tissues, allowing easy observation and microdissection of transformed tissues even without a GFP filter.Under normal light,&nbsp;plants with GFP targeted to plastids had a light green phenotype deriving from the high GFP expression. Quantitative reverse transcriptional PCR analysis showed that&nbsp;the plastid targeted construct&nbsp;with intron had significantly higher steady state transcript levels of GFP mRNA. This novel expression cassettemay allow&nbsp;direct visual selection of transformed tissues independent of antibiotic selection in a wide range of plant species. &nbsp; Key words:&nbsp;Florist&#39;s chrysanthemum&nbsp;(Chrysanthemum morifolium), genetic transformation, GFP gene, RbcS promoter, RbcS targeting signal.

Assessment of receptor-dependent Ad5 & Ad5F35 adenovirai vector mediated gene transfection in lung cancer cells

Objective To investigate the expression of coxsackie adenoviris receptor (CAR) and CD46, making efforts to provide evidence to explore the relationship between them and the efficiency of adenovirus gene transfer. Methods CAR and CD46 expression in the lung cancer lines (A549, Z793,QG56 and NCI-H520) and primary lung cancer cells from 10 patients were assayed by flow cytometric analysis. The lung cancer cell lines and primary lung cancer cells were all infected with Ad5-GFP or Ad5F35-GFP adenoviral vector at MOI 10 or 50 labeled by green fluorescent protein (GFP) protein. Fortyeight h after transfection, the GFP-positive cell percentage in the lung cancer cells was measured by flow cytometry to evaluate the transfection efficiency of Ad5-GFP or Ad5F35-GFP adenoviral vector. Results The expression of CD46 was higher than the CAR expression both in the lung cancer lines ( A549, Z793,QG56 and NCI-H520) and primary lung cancer cells in 10 cases. Consistently, a significantly higher GFP expression 48 h post-transfection with Ad5F35-GFP (MOI = 50) was obtained, and GFP-positive cell percentage was 78%, 75%, 75% and 54% in A549, Z793, QG56 and NCI-H520, respectively, and that was 39%, 37%, 45% and 4%, respectively after transfection with Ad5-GFP. The same results were obtained in the primary lung cancer cells notwithstanding the different percents of GFP-positive cells in 10 patients. Conclusion The efficiency of Ad5 and Ad5F35 adenovirus gene transfection is closely correlated to the levels of CAR and CD46 expression in the lung cancer cells. The expression of CD46 is higher than that of CAR and the gene transfer efficacy of Ad5F35 is prior to the Ad5 in lung cancer cells. Key words: Lung carcinoma; Adenovirus; CD46; Gene transfer