Green Fluorescent Protein Gene Research Articles

3'UTR of tobacco vein mottling virus regulates downstream GFP expression and changes in host gene expression.

Tobacco vein mottling virus (TVMV) is a member of the family Potyviridae. The 3' untranslated region (3'UTR) of viral genomic RNA has been reported to significantly impact viral infection. Nevertheless, the role of the TVMV 3'UTR during viral infection remains unknown. Here, a 3'UTR-GFP expression vector was transiently expressed in Nicotiana benthamiana, in which the 3'UTR of TVMV was introduced upstream of the green fluorescent protein (GFP) gene. Transcriptome sequencing was performed to analyze the genes associated with plant resistance. The effect of the TVMV 3'UTR on GFP expression was studied using an Agrobacterium-mediated transient expression assay, revealing that the TVMV 3'UTR significantly inhibited GFP expression. Transcriptome analysis of differentially expressed genes in 3'UTR-GFP in N. benthamiana was performed to elucidate the why the TVMV 3'UTR inhibited GFP expression. Eighty genes related to plant disease resistance were differentially expressed, including 29 upregulated and 51 downregulated genes. Significantly upregulated genes included those encoding the calcium-binding protein CML24, leucine-rich repeat receptor-like tyrosine-protein kinase, and respiratory burst oxidase homolog protein E. The significantly downregulated genes included calcium-binding protein 7, ethylene-responsive transcription factor 10, endoglucanase 5, and receptor-like protein kinase. These findings indicate that the 3'UTR of TVMV may inhibit the expression of GFP gene by inducing the expression of plant resistance genes. This study provides a theoretical basis for further research on the function and mechanism of the TVMV 3'UTR.

RNA lipid nanoparticles as efficient in vivo CRISPR-Cas9 gene editing tool for therapeutic target validation in glioblastoma cancer stem cells

In vitro and ex-vivo target identification strategies often fail to predict in vivo efficacy, particularly for glioblastoma (GBM), a highly heterogenous tumor rich in resistant cancer stem cells (GSCs). An in vivo screening tool can improve prediction of therapeutic efficacy by considering the complex tumor microenvironment and the dynamic plasticity of GSCs driving therapy resistance and recurrence. This study proposes lipid nanoparticles (LNPs) as an efficient in vivo CRISPR-Cas9 gene editing tool for target validation in mesenchymal GSCs. LNPs co-delivering mRNA (mCas9) and single-guide RNA (sgRNA) were successfully formulated and optimized facilitating both in vitro and in vivo gene editing. In vitro, LNPs achieved up to 67 % reduction in green fluorescent protein (GFP) expression, used as a model target, outperforming a commercial transfection reagent. Intratumoral administration of LNPs in GSCs resulted in ∼80 % GFP gene knock-out and a 2-fold reduction in GFP signal by day 14. This study showcases the applicability of CRISPR-Cas9 LNPs as a potential in vivo screening tool in GSCs, currently lacking effective treatment. By replacing GFP with a pool of potential targets, the proposed platform presents an exciting prospect for therapeutic target validation in orthotopic GSCs, bridging the gap between preclinical and clinical research.

Development and characterization of reverse genetics systems of feline infectious peritonitis virus for antiviral research

Feline infectious peritonitis (FIP) is a lethal, immune-mediated disease in cats caused by feline infectious peritonitis virus (FIPV), a biotype of feline coronavirus (FCoV). In contrast to feline enteric coronavirus (FECV), which exclusively infects enterocytes and causes diarrhea, FIPV specifically targets macrophages, resulting in the development of FIP. The transmission and infection mechanisms of this complex, invariably fatal disease remain unclear, with no effective vaccines or approved drugs for its prevention or control. In this study, a full-length infectious cDNA clone of the wild-type FIPV WSU79-1149 strain was constructed to generate recombinant FIPV (rFIPV-WT), which exhibited similar growth kinetics and produced infectious virus titres comparable to those of the parental wild-type virus. In addition, the superfold green fluorescent protein (msfGFP) and Renilla luciferase (Rluc) reporter genes were incorporated into the rFIPV-WT cDNA construct to generate reporter rFIPV-msfGFP and rFIPV-Rluc viruses. While the growth characteristics of the rFIPV-msfGFP virus were similar to those of its parental rFIPV-WT, the rFIPV-Rluc virus replicated more slowly, resulting in the formation of smaller plaques than did the rFIPV-WT and rFIPV-msfGFP viruses. In addition, by replacing the S, E, M, and ORF3abc genes with msfGFP and Rluc genes, the replicon systems repFIPV-msfGFP and repFIPV-Rluc were generated on the basis of the cDNA construct of rFIPV-WT. Last, the use of reporter recombinant viruses and replicons in antiviral screening assays demonstrated their high sensitivity for quantifying the antiviral effectiveness of the tested compounds. This integrated system promises to significantly streamline the investigation of virus replication within host cells, enabling efficient screening for anti-FIPV compounds and evaluating emerging drug-resistant mutations within the FIPV genome.

Agrobacterium-mediated transient expression in Torreya grandis cones: A simple and rapid tool for gene expression and functional gene assay

Agrobacterium-mediated transient gene expression is a powerful tool for characterizing gene function in plants, particularly in fruit trees with long juvenile periods. However, few studies have systematically explored transient expression in nut trees, such as T. grandis. In this study, we developed a rapid and easily manipulated transient transformation system using Agrobacterium-mediated injection infiltration in T. grandis cones. Cones at the enlargement stage (30 days after aril protrusion) were used, and the bacterial solution was gently injected into the kernel at a depth of 1 cm, filling nearly the entire cone, as indicated by a red dye. Using the green fluorescent protein (GFP) gene as a reporter for Agrobacterium-mediated transformation, we confirmed GFP expression in T. grandis cone successfully through confocal microscopy, western blot, and PCR. Transformation conditions, including bacterial density and days after agroinfiltration, were optimized for higher transformation efficiency, with the optimal conditions being an OD600 of 0.6 and an agroinfiltration period of 6 - 9 days. Transient overexpression of TgWRKY47, a cell size regulator, altered cell size in T. grandis cones, indicating that this Agrobacterium infiltration method can be used for gene functional analysis. Furthermore, confocal microscopy revealed that TgWRKY47-GFP was localized in the nucleus along with a nuclear marker, demonstrating its applicability for protein subcellular localization. In summary, this method presents a versatile tool for efficient and targeted transient gene functional analysis and subcellular localization in T. grandis cones, offering new insights into nut tree biology research.

A Novel Transgenic Sf9 Cell Line for Quick and Easy Virus Quantification.

The following study was conducted to generate a transgenic Sf9 cell line for rapid and easy virus quantification in the baculovirus expression system (BES). The hr3 (homologous region 3) and 39K and p10 promoters were used as the expression structures to induce rapid and intense expression of the enhanced green fluorescent protein gene in cells in response to viral infection. Of 20 transgenic Sf9 cell lines generated using the piggyBac system, the cell line that showed the highest fluorescence expression in the shortest time in response to viral infection was selected and named Sf9-QE. The average diameter of the Sf9-QE cells was around 16 μm, which is 2 μm smaller than the average diameter of Sf9 cells, whereas the rate of cell proliferation was around 1.6 times higher in the Sf9-QE cells. Virus quantification using the Sf9-QE cell line did not produce significantly different results compared to the other cell lines; however, the time required for complete virus quantification was approximately 5.3 to 6.0 days for the Sf9-QE cells, which is around 4 to 6 days shorter than the time required for the other cell lines, enabling convenient and accurate virus quantification via fluorescence photometry within around 6.0 to 6.3 days. The properties of the Sf9-QE cells were stable for up to at least 100 passages.

Coat Proteins of the Novel Victoriviruses FaVV1 and FaVV2 Suppress Sexual Reproduction and Virulence in the Pathogen of Fusarium Head Blight.

Fusarium head blight (FHB), a disease inflicted by Fusarium graminearum and F. asiaticum, poses a growing threat to wheat in China, particularly in the face of climate change and evolving agricultural practices. This study unveiled the discovery of the victorivirus FgVV2 from the F. asiaticum strain F16176 and comprehensively characterized the function of the two victoriviruses FaVV1 and FaVV2 in virulence. Through comparative analysis with a virus-free strain, we established that these mycoviruses markedly repress the sexual reproduction and pathogenicity of their fungal hosts. Furthermore, we synthesized the coat protein (CP) genes CP1 from FaVV1 and CP2 from FaVV2, which were fused with the green fluorescent protein (GFP) gene and successfully expressed in Fusarium strains in wild-type isolates of F. asiaticum and F. graminearum. Similar to virus-infected strains, the transformed strains expressing CPs showed a significant decrease in perithecia formation and pathogenicity. Notably, CP2 exhibited a stronger inhibitory effect than CP1, yet the suppression of sexual reproduction in F. graminearum was less pronounced than that in F. asiaticum. Additionally, the pathogenicity of the F. asiaticum and F. graminearum strains expressing CP1 or CP2 was substantially diminished against wheat heads. The GFP-tagged CP1 and CP2 revealed distinct cellular localization patterns, suggesting various mechanisms of interaction with the host. The findings of this study provide a significant research foundation for the study of the interaction mechanisms between FaVV1 and FaVV2 with their hosts, as well as for the exploration and utilization of fungal viral resources.

Construction and optimization of a genetic transformation system for efficient expression of human insulin-GFP fusion gene in flax

The human insulin gene modified with a C-peptide was synthesized according to the plant-preferred codon, and a fusion gene expression vector of insulin combined with green fluorescent protein (GFP) was constructed. The optimization of the flax callus culturing was undertaken, and a more efficient Agrobacterium-mediated genetic transformation of the flax hypocotyls was achieved. The critical concentration values of hygromycin on the flax hypocotyl development, as well as on its differentiated callus, were explored by the method of antibiotic gradient addition, and the application of antibiotic screening for the verification of positive calluses was assessed. The fusion gene of insulin and GFP was successfully inserted into the flax genome and expressed, as confirmed through polymerase chain reaction and Western blotting. In conclusion, we have established a flax callus culture system suitable for insulin expression. By optimizing the conditions of the flax callus induction, transformation, screening, and verification of a transgenic callus, we have provided an effective way to obtain insulin. Moreover, the herein-employed flax callus culture system could provide a feasible, cheap, and environmentally friendly platform for producing bioactive proteins.Graphical

High production of recombinant protein using geminivirus-based deconstructed vectors in Nicotiana benthamiana.

We focused on the geminiviral vector systems to develop an efficient vector system for plant biotechnology. Begomoviruses and curtoviruses, which belong to the Geminiviridae family, contain an intergenic region (IR) and four genes involved in replication, including replication-associated protein (Rep, C1), transcriptional activator (TrAP, C2), and replication enhancer (REn, C3). Geminiviruses can amplify thousands of copies of viral DNA using plant DNA polymerase and viral replication-related enzymes and accumulate viral proteins at high concentrations. In this study, we optimized geminiviral DNA replicon vectors based on tomato yellow leaf curl virus (TYLCV), honeysuckle yellow vein virus (HYVV), and mild curly top virus (BMCTV) for the rapid, high-yield plant-based production of recombinant proteins. Confirmation of the optimal combination by co-delivery of each replication-related gene and each IR harboring the Pontellina plumata-derived turbo green fluorescence protein (tGFP) gene via agroinfiltration in Nicotiana benthamiana leaves resulted in efficient replicon amplification and robust protein production within 3 days. Co-expression with the p19 protein of the tomato bush stunt virus, a gene-silencing suppressor, further enhanced tGFP accumulation by stabilizing mRNA. With this system, tGFP protein was produced at 0.7-1.2 mg/g leaf fresh weight, corresponding to 6.9-12.1% in total soluble protein. These results demonstrate the advantages of rapid and high-level production of recombinant proteins using the geminiviral DNA replicon system for transient expression in plants.

3'-UTR Sequence of Exosomal NANOGP8 DNA as an Extracellular Vesicle-Localization Signal.

Extracellular vesicles (EVs) are garnering attention as a safe and efficient biomolecule delivery system. EVs intrinsically play a crucial role in intercellular communication and pathophysiology by transporting functionally active DNA molecules. The internalized DNA pleiotropically affects the recipient cells. Considering these salient features, an intentional incorporation of specific DNA gene cassettes into EVs and their subsequent delivery to the target cells has potential applications in genetic engineering. Moreover, efficient ways to insert the DNA into EVs during their biogenesis is valuable. Our current research is a step in the development of this technology. As such, cancer cells are known to secrete exosomes containing increased amounts of double-stranded DNA than normal cells. The clonal analysis in our previously published data revealed that exosomes released from various cancer cells contained a significantly larger population of NANOGP8 DNA with a 22-base pair insertion in the 3'-untranslated region (UTR) compared to those secreted by normal cells. This finding led us to hypothesize that the 22-base pair insertion may act as a signal to facilitate the incorporation of NANOGP8 DNA into the exosomes. To test this hypothesis, we compared the EV localization of an Enhanced Green Fluorescent Protein (EGFP) gene fused with the NANOGP8 3'-UTR, with and without the 22-base pair insertion. The quantitative PCR analysis showed a significantly higher EGFP DNA accumulation in exosomes released from cells transfected with the gene cassette containing the 3'-UTR with the 22-base pair insertion. The discovery of a DNA localization signal in exosomal DNA's 3'-UTR could pave the way for the development of an EV-based DNA delivery system. This technology will open new possibilities in genetic engineering and innovative therapies using nucleic acid medicine.

Characterization of an envelope protein 118L in invertebrate iridescent virus 6 (IIV6).

Invertebrate iridescent virus 6 (IIV6) is a nucleocytoplasmic insect virus and a member of the family Iridoviridae. The IIV6 genome consists of 212,482bp of linear dsDNA with 215 non-overlapping and putative protein-encoding ORFs. The IIV6 118L ORF is conserved in all sequenced members of the Iridoviridae and encodes a 515 amino acid protein with three predicted transmembrane domains and several N-glycosylation/N-myristoylation sites. In this study, we characterized the 118L ORF by both deleting it from the viral genome and silencing its expression with dsRNA in infected insect cells. The homologous recombination method was used to replace 118L ORF with the green fluorescent protein (gfp) gene. Virus mutants in which the 118L gene sequence had been replaced with gfp were identified by fluorescence microscopy but could not be propagated separately from the wild-type virus in insect cells. Unsuccessful attempts to isolate the mutant virus with the 118L gene deletion suggested that the protein is essential for virus replication. To support this result, we used dsRNA to target the 118L gene and showed that treatment resulted in a 99% reduction in virus titer. Subsequently, we demonstrated that 118L-specific antibodies produced against the 118L protein expressed in the baculovirus vector system were able to neutralize the virus infection. All these results indicate that 118L is a viral envelope protein that is required for the initiation of virus replication.

Peptosome: A New Efficient Transfection Tool as an Alternative to Liposome.

Gene therapy is one of the most promising techniques for treating genetic diseases and cancer. The current most important problem in gene therapy is gene delivery. Viral and non-viral vectors like liposomes, used for gene delivery, have many limitations. We have developed new hybrid peptides by combining cell-penetrating peptides (CPPs) with the DNA-binding domain of the human histone H4 protein. These small peptides bind to DNA molecules through their histone domain, leaving the CPP part free and available for binding and penetration into cells, forming complexes that we named "peptosomes". We evaluated the transfection efficiency of several hybrid peptides by delivering a plasmid carrying the green fluorescent protein gene and following its expression by fluorescent microscopy. Among several hybrid peptides, TM3 achieved a gene delivery efficiency of 76%, compared to 52% for Lipofectamine 2000. TM3 peptosomes may become important gene delivery tools with several advantages over current gene delivery agents.

Customizable and stable multilocus chromosomal integration: a novel glucose-dependent selection system in Aureobasidium spp.

BackgroundNon-conventional yeasts hold significant potential as biorefinery cell factories for microbial bioproduction. Currently, gene editing systems used for these yeasts rely on antibiotic and auxotrophic selection mechanisms. However, the drawbacks of antibiotics, including high costs, environmental concerns, and the dissemination of resistance genes, make them unsuitable for large-scale industrial fermentation. For auxotrophic selection system, the engineered strains harboring auxotrophic marker genes are typically supplemented with complex nutrient-rich components instead of precisely defined synthetic media in large-scale industrial fermentations, thus lack selection pressure to ensure the stability of heterologous metabolic pathways. Therefore, it is a critical to explore alternative selection systems that can be adapted for large-scale industrial fermentation.ResultsHere, a novel glucose-dependent selection system was developed in a high pullulan-producing non-conventional strain A. melanogenum P16. The system comprised a glucose-deficient chassis cell Δpfk obtained through the knockout of the phosphofructokinase gene (PFK) and a series of chromosomal integration plasmids carrying a selection marker PFK controlled by different strength promoters. Utilizing the green fluorescent protein gene (GFP) as a reporter gene, this system achieved a 100% positive rate of transformation, and the chromosomal integration numbers of GFP showed an inverse relationship with promoter strength, with a customizable copy number ranging from 2 to 54. More importantly, the chromosomal integration numbers of target genes remained stable during successive inoculation and fermentation process, facilitated simply by using glucose as a cost-effective and environmental-friendly selectable molecule to maintain a constant and rigorous screening pressure. Moreover, this glucose-dependent selection system exhibited no significant effect on cell growth and product synthesis, and the glucose-deficient related selectable marker PFK has universal application potential in non-conventional yeasts.ConclusionHere, we have developed a novel glucose-dependent selection system to achieve customizable and stable multilocus chromosomal integration of target genes. Therefore, this study presents a promising new tool for genetic manipulation and strain enhancement in non-conventional yeasts, particularly tailored for industrial fermentation applications.

BZIP60 and Bax inhibitor 1 contribute IRE1-dependent and independent roles to potexvirus infection.

IRE1, BI-1, and bZIP60 monitor compatible plant-potexvirus interactions though recognition of the viral TGB3 protein. This study was undertaken to elucidate the roles of three IRE1 isoforms, the bZIP60U and bZIP60S, and BI-1 roles in genetic reprogramming of cells during potexvirus infection. Experiments were performed using Arabidopsis thaliana knockout lines and Plantago asiatica mosaic virus infectious clone tagged with the green fluorescent protein gene (PlAMV-GFP). There were more PlAMV-GFP infection foci in ire1a/b, ire1c, bzip60, and bi-1 knockout than wild-type (WT) plants. Cell-to-cell movement and systemic RNA levels were greater bzip60 and bi-1 than in WT plants. Overall, these data indicate an increased susceptibility to virus infection. Transgenic overexpression of AtIRE1b or StbZIP60 in ire1a/b or bzip60 mutant background reduced virus infection foci, while StbZIP60 expression influences virus movement. Transgenic overexpression of StbZIP60 also confers endoplasmic reticulum (ER) stress resistance following tunicamycin treatment. We also show bZIP60U and TGB3 interact at the ER. This is the first demonstration of a potato bZIP transcription factor complementing genetic defects in Arabidopsis. Evidence indicates that the three IRE1 isoforms regulate the initial stages of virus replication and gene expression, while bZIP60 and BI-1 contribute separately to virus cell-to-cell and systemic movement.

Construction of ATMT system of Fusarium oxysporum and evaluation of its promoting effect on Glycyrrhiza uralensis

This study aims to evaluate the in vivo function of Fusarium oxysporum in Glycyrrhiza uralensis by salt tolerance,indoleacetic acid(IAA) production capacity, phosphate-dissolving capacity, and iron carrier production capacity. The stable genetic transformation system of the F. oxysporum was established by Agrobacterium tumefaciens-mediated genetic transformation( ATMT)technology, and the stability and staining efficiency of transformants were detected by the cloning of the marker gene green fluorescent protein(GFP) and the efficiency of β-glucuronidase staining(GUS). Efficient and stable transformants were selected for restaining G. uralensis and evaluating its influence on the growth of the G. uralensis seedlings. The results show that F. oxysporum has good salt tolerance and could still grow on potato glucose agar(PDA) medium containing 7% sodium chloride, but the growth rate slows down with the increase in sodium chloride content in PDA medium. F. oxysporum has the function of producing indoleacetic acid, and the concentration of IAA in its fermentation broth is about 3. 32 mg · m L~(-1). In this study, the genetic transformation system of F. oxysporum is successfully constructed, and the ATMT system is efficient and stable. One transformant with both high staining efficiency and genetic stability is selected, and the restaining rate of the transformant in G. uralensis is 76. 92%, which could significantly improve the main root length of one-month-old G. uralensis seedlings and promote the growth and development of G. uralensis seedlings. The results of this study can lay the foundation for the development of biological bacterial fertilizer and the growth regulation of high-quality G. uralensis.

Long-duration leptin transgene expression in dorsal vagal complex does not alter bone parameters in female Sprague Dawley rats

The hypothalamus and dorsal vagal complex (DVC) are both important for integration of signals that regulate energy balance. Increased leptin transgene expression in either the hypothalamus or DVC of female rats was shown to decrease white adipose tissue and circulating levels of leptin and adiponectin. However, in contrast to hypothalamus, leptin transgene expression in the DVC had no effect on food intake, circulating insulin, ghrelin and glucose, nor on thermogenic energy expenditure. These findings imply different roles for hypothalamus and DVC in leptin signaling. Leptin signaling is required for normal bone accrual and turnover. Leptin transgene expression in the hypothalamus normalized the skeletal phenotype of leptin-deficient ob/ob mice but had no long-duration (≥10 weeks) effects on the skeleton of leptin-replete rats. The goal of this investigation was to determine the long-duration effects of leptin transgene expression in the DVC on the skeleton of leptin-replete rats. To accomplish this goal, we analyzed bone from three-month-old female rats that were microinjected with recombinant adeno-associated virus encoding either rat leptin (rAAV-Leptin, n = 6) or green fluorescent protein (rAAV-GFP, control, n = 5) gene. Representative bones from the appendicular (femur) and axial (3rd lumbar vertebra) skeleton were evaluated following 10 weeks of treatment. Selectively increasing leptin transgene expression in the DVC had no effect on femur cortical or cancellous bone microarchitecture. Additionally, increasing leptin transgene expression had no effect on vertebral osteoblast-lined or osteoclast-lined bone perimeter or marrow adiposity. Taken together, the findings suggest that activation of leptin receptors in the DVC has minimal specific effects on the skeleton of leptin-replete female rats.

Enabling Non-invasive Tracking of Vascular Endothelial Cells Derived from Induced Pluripotent Stem Cells Using Nuclear Imaging.

The absence of clinically applicable imaging techniques for continuous monitoring of transplanted cells poses a significant obstacle to the clinical translation of stem cell-based therapies for vascular regeneration. This study aims to optimize a clinically applicable, non-invasive imaging technique to longitudinally monitor vascular endothelial cells (ECs) for vascular regeneration in peripheral artery disease (PAD). Human induced pluripotent stem cells (HiPSCs) were employed to generate ECs (HiPSC-ECs). Lentiviral vectors encoding human sodium iodide symporter (hNIS) and enhanced green fluorescent protein (eGFP) genes were introduced to HiPSCs and HiPSC-ECs at varying multiplicities of infection (MOI). Through a combination of fluorescence microscopy and flow cytometry, an optimized transduction technique for introducing hNIS-eGFP into HiPSC-ECs was established. Subsequently, single-photon emission computed tomography (SPECT) was utilized for imaging of the transduced cells in vitro and in vivo after transplantation into the gastrocnemius muscle of nude mice. Lentiviral transduction resulted in sustained co-expression of hNIS and eGFP in HiPSC-ECs when transduced post-endothelial differentiation. An optimal MOI of five yielded over 90% hNIS-eGFP expression efficiency without compromising cell viability. hNIS-eGFP+ HiPSC-ECs exhibited 99mTc uptake and were detectable through SPECT in vitro. Additionally, intramuscular injection of hNIS-eGFP+ HiPSC-ECs with MatrigelTM into the hindlimbs of nude mice enabled real-time SPECT/CT tracking, from which a reduction in signal exceeding 80% was observed within 7days. This study establishes an optimized cell modification and imaging protocol for tracking transplanted cells. Future efforts will focus on enhancing cell survival and integration via improved delivery systems, thereby advancing the potential of cell-based therapies for PAD.

Establishment and characterization of a kidney cell line from hybrid snakehead (male Channa argus × female Channa maculata) and its susceptibility to hybrid snakehead rhabdovirus (HSHRV)

Hybrid snakehead (male Channa argus × female Channa maculata) is an emerging fish breed with increasing production levels. However, infection with hybrid snakehead rhabdovirus (HSHRV) critically affects hybrid snakehead farming. In this study, a fish cell line called CAMK, derived from the kidneys of hybrid snakehead, was established and characterized. CAMK cells exhibited the maximum growth rate at 28 °C in Leibovitz's-15 medium supplemented with 10% fetal bovine serum(FBS). Karyotyping revealed diploid chromosomes in 54% of the cells at the 50th passage (2n = 66), and 16S rRNA sequencing validated that CAMK cells originated fromhybrid snakehead, and the detection of kidney-specific antibodies suggested that it originated from kidney. .The culture was free from mycoplasma contamination, and the green fluorescent protein gene was effectively transfected into CAMK cells, indicating their potential use for in vitro gene expression investigations. Furthermore, qRT-PCR and immunofluorescence analysis revealed that HSHRV could replicate in CAMK cells, indicating that the cells were susceptible to the virus. Transmission electron microscopy revealed that the viral particles had bullet-like morphology. The replication efficiency of HSHRV was 107.33 TCID50/mL. Altogether, we successfully established and characterized a kidney cell line susceptible to the virus. These findings provide a valuable reference for further genetic and virological studies.

An in vitro validation system for chicken bioreactors using immortalized chicken oviductal epithelial cells

The utilization of chicken oviductal epithelial cells (OECs) as a bioreactor to produce therapeutic proteins has shown promise, but the time taken to obtain transgenic offspring impedes efficient validation of protein production. To overcome this barrier, we focused on the immortalization of chicken OECs (cOECs) using retroviral vector-mediated c-MYC oncogene expression to establish an in vitro pre-validation system for chicken bioreactors. The resulting immortalized cOECs exhibited sustained proliferation, maintained a normal diploid chicken karyotype, and expressed key oviduct-specific genes (OVA, OVM, LYZ, AVD, and ESR1). Notably, hormonal administration of diethylstilbestrol (DES) or progesterone (P4) upregulated oviduct-specific genes in these cells. To enhance the utility of these immortalized cOECs as an in vitro validation system for chicken bioreactors, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology was employed to knock-in (KI) an enhanced green fluorescence protein (EGFP) gene at the ovalbumin (OVA) locus. The resulting OVA EGFP KI immortalized cOECs secreted both EGFP and OVA proteins into the culture medium, with secretion enhanced under DES treatment. This successful integration of an exogenous gene into cOECs enhances their potential as a versatile in vitro validation system for chicken bioreactors. The established immortalized cOECs overcome previous challenges associated with long-term culture and maintenance, providing a reliable platform for efficient protein production validation. This study presents a comprehensive characterization of the immortalized cOECs, addressing critical limitations associated with in vivo systems and laying a foundation for the development of a streamlined and effective chicken bioreactor model.

Development of an improved reverse genetics system for avian metapneumovirus (aMPV): A novel vaccine vector protects against aMPV and infectious bursal disease virus

Avian metapneumovirus (aMPV), a paramyxovirus, causes acute respiratory diseases in turkeys and swollen head syndrome in chickens. This study established a reverse genetics system for aMPV subtype B LN16-A strain based on T7 RNA polymerase. Full-length cDNA of the LN16-A strain was constructed by assembling five cDNA fragments between the T7 promoter and hepatitis delta virus ribozyme. Transfection of this plasmid, along with the supporting plasmids encoding the N, P, M2-1, and L proteins of LN16-A into BSR-T7/5 cells, resulted in the recovery of aMPV subtype B. To identify an effective insertion site, the enhanced green fluorescent protein (EGFP) gene was inserted into different sites of the LN16-A genome to generate recombinant LN16-As. The results showed that the expression levels of EGFP at the site between the G and L genes of LN16-A were significantly higher than those at the other two sites (between the leader and N genes or replacing the SH gene). To verify the availability of the site between G and L for foreign gene expression, the VP2 gene of very virulent infectious bursal disease virus (vvIBDV) was inserted into this site, and recombinant LN16-A (rLN16A-vvVP2) was successfully rescued. Single immunization of specific-pathogen-free chickens with rLN16A-vvVP2 induced high levels of neutralizing antibodies and provided 100% protection against the virulent aMPV subtype B and vvIBDV. Establishing a reverse genetics system here provides an important foundation for understanding aMPV pathogenesis and developing novel vector vaccines.

Expression of modified enhanced green fluorescent polyarginine protein in Saccharomyces cerevisiae INVSc1

Background The enhanced green fluorescent protein (EGFP) gene is a reporter gene that can be used to optimize protein isolation procedures and the functional working of a transduction protein. EGFP, with the addition of eleven arginine residues, has been engineered to functionally improve the protein transduction process, which can later be used for cell reprogramming like induced pluripotent stem cells. The addition of six histidine amino acid residues at its C-terminal is intended for the protein isolation process using the His-tag antibody. Methods The study aimed to investigate the optimization of the EGFP polyarginine protein expression in Saccharomyces cerevisiae in sufficient quantities for the protein isolation stage. This study also analyzed EGFP expression without polyarginine to analyze the polyarginine addition effect on expression processes. Protein expression was qualitatively measured by looking at expression fluorescence and protein levels of EGFP and EGFP - PolyR proteins. Results The addition of a PolyR group to the C-terminal of EGFP carrying C-terminal 6×His-tag showed similar fluorescence expression levels compare to EGFP without addition of PolyR as well. Moreover, yeast with plasmid insertion showed decrease S. cerevisiae growth curve but still preserving the fluorescence EGFP. Conclusions The expression of the EGFP modified protein in S. cerevisiae is not affected by the addition of arginine.