Constructing an efficient and easy-to-operate multigene expression system is currently a crucial part of plant genetic engineering. In this study, a fragment carrying three independent gene expression cassettes and the expression unit of the gene-silencing suppressor protein(RNA silencing suppressor 19 kDa protein, P19) simultaneously was designed and constructed. This fragment was cloned into the commonly used plant expression vector pCAMBIA300, and the plasmid pC1300-TP2-P19 was obtained. Each gene expression cassette consists of different promoters, fusion tags, and terminators. The target gene can be flexibly inserted into the corresponding site through enzymatic digestion and ligation or recombination and fused with different protein tags, which provides great convenience for subsequent detection. The enhanced green fluorescent protein(eGFP) reporter gene was individually constructed into each expression cassette to verify the feasibility of this vector system. The results of tobacco transient expression and laser-confocal microscopy showed that each expression cassette presented independent and normal expression. Meanwhile, the three key enzyme genes in the betanin synthesis pathway, BvCYP76AD, BvDODA1, and DbDOPA5GT, were constructed into the three expression cassettes. The results of tobacco transient expression phenotype, protein immunoblotting(Western blot), and chemical detection of product demonstrated that the three exogenous genes were highly expressed, and the target compound betanin was successfully produced. The above results indicated that the constructed multigene expression system for plants in this study was efficient and reliable and can achieve the co-transformation of multiple plant genes. It can provide a reliable vector platform for the analysis of plant natural product synthesis pathways, functional verification, and plant metabolic engineering.
Nicotiana/metabolism* ; Genetic Vectors/metabolism* ; Gene Expression Regulation, Plant ; Plant Proteins/metabolism* ; Plants, Genetically Modified/metabolism* ; Genetic Engineering/methods* ; Green Fluorescent Proteins/metabolism* ; Gene Expression

The advancement of tissue clearing technology has significantly propelled neuroscience research. Nevertheless, the fluorescent proteins used in traditional transgenic mouse strains were not specifically optimized for tissue clearing procedures, resulting in a substantial decrease in fluorescent intensity after clearing. In this study, we developed the Ci1 reporter mouse strain (where Ci stands for the Chinese Institute for Brain Research, CIBR) based on the bright red fluorescent protein mScarlet. The Ci1 reporter exhibits no fluorescence leakage in various organs or tissue types and can be readily crossed with multiple tissue-specific Cre lines. Compared to the Ai14 mouse strain, the Ci1 reporter strain demonstrates lower non-specific leakage, stronger fluorescence intensity in different tissues, and better preservation of fluorescence following tissue clearing treatment. The creation of the Ci1 reporter provides a more effective tool for both neuroscience and other biomedical research applications.
Animals ; Luminescent Proteins/metabolism* ; Mice, Transgenic ; Mice ; Red Fluorescent Protein ; Brain/metabolism* ; Genes, Reporter ; Fluorescence

Biosensors have become powerful tools for real-time monitoring of specific small molecules and precise control of gene expression in biological systems. High-throughput sensors for 1, 4-butanediamine biosynthesis can greatly improve the screening efficiency of high-yielding 1, 4-butanediamine strains. However, the strategies for adapting the characteristics of biosensors are still rarely studied, which limits the applicability of 1, 4-butanediamine biosensors. In this paper, we propose the development of a 1, 4-butanediamine biosensor based on the transcriptional regulator PuuR, whose homologous operator puuO is installed in the constitutive promoter P_gapA of Escherichia coli to control the expression of the downstream superfolder green fluorescent protein (sfGFP) as the reporter protein. Finally, the biosensor showed a stable linear relationship between the GFP/OD₆₀₀ value and the concentration of 1, 4-butanediamine when the concentration of 1, 4-butanediamine was 0-50 mmol/L. The promoters with different strengths in the E. coli genome were used to modify the 1, 4-butanediamine biosensor, and the functional properties of the PuuR-based 1, 4-butanediamine biosensor were explored and improved, which laid the groundwork for high-throughput screening of engineered strains highly producing 1, 4-butanediamine.
Biosensing Techniques/methods* ; Escherichia coli/metabolism* ; Promoter Regions, Genetic/genetics* ; Green Fluorescent Proteins/metabolism* ; Transcription Factors/genetics* ; Escherichia coli Proteins/genetics* ; Diamines/metabolism* ; Gene Expression Regulation, Bacterial

In this study, high-throughput promoter screening was employed to optimize the heterologous expression of Mesorhizobium loti carbonic anhydrase (MlCA) in order to reduce the costs associated with carbon capture and storage (CCS). To simplify the complexity of traditional vectors, a fusion protein expression system was constructed using superfolder green fluorescent protein (sfGFP) and MlCA. The synthetic promoter library in Escherichia coli was utilized for efficient one-step screening. Based on fluorescence intensity on agar plates, a total of 143 monoclonal colonies were identified, forming a library with varying expression levels. The top four recombinants with the highest fluorescence intensity were selected, among which MlCA driven by the promoter 342042/+ exhibited the highest enzymatic activity, with a specific activity of the 34.6 Wilbur-Anderson units (WAU)/mg. Optimization experiments revealed that MlCA exhibited the best performance when cultured for 4 days under pH 7.0 and 40 ℃ conditions. The Michaelis constant (K_m·hdy) and maximum reaction rate (V_max·hdy) for CO₂ hydration were determined to be 62.46 mmol/L and 0.164 mmol/(s·L), respectively. For esterase hydrolysis, MlCA showed the K_m and V_max of 639.8 mmol/L and 0.035 mmol/(s·L), respectively. MlCA accelerated the CO₂ hydration process, promoting CO₂ mineralized into CaCO₃ within 9 min at low pH and room temperature conditions. Scanning electron microscopy (SEM) and X-ray diffraction (XRD) analyses confirmed that the precipitated product was calcite. This study provides a low-cost and environmentally friendly alternative for future CCS applications.
Carbonic Anhydrases/biosynthesis* ; Promoter Regions, Genetic/genetics* ; Escherichia coli/metabolism* ; Carbon Sequestration ; Carbon Dioxide/metabolism* ; Green Fluorescent Proteins/metabolism*

Objective To investigate the relationship between interleukin-1β (IL-1β) and miR-185-5p in the process of joint injury in acute gouty arthritis (AGA). Methods The serum miR-185-5p levels of 89 AGA patients and 91 healthy volunteers were detected by real-time quantitative PCR. The correlation between miR-185-5p expression level and VAS score or IL-1β expression level was evaluated by Pearson correlation coefficient method. Receiver operating characteristic (ROC) curve was used to evaluate the diagnostic value of miR-185-5p in AGA. THP-1 cells were induced by sodium urate (MSU) to construct an in vitro acute gouty inflammatory cell model. After the expression level of miR-185-5p in THP-1 cells was upregulated or downregulated by transfection of miR-185-5p mimics or inhibitors in vitro, inflammatory cytokines of THP-1 cells, such as IL-1β, IL-8 and tumor necrosis factor α (TNF-α), were detected by ELISA. The luciferase reporter gene assay was used to determine the interaction between miR-185-5p and the 3'-UTR of IL-1β. Results Compared with the healthy control group, the expression level of serum miR-185-5p in AGA patients was significantly reduced. The level of serum miR-185-5p was negatively correlated with VAS score and IL-1β expression level. The area under the curve (AUC) was 0.905, the sensitivity was 80.17% and the specificity was 83.52%. Down-regulation of miR-185-5p significantly promoted the expression of IL-1β, IL-8 and tumor necrosis factor (TNF-α), while overexpression of miR-185-5p showed the opposite results. Luciferase reporter gene assay showed that IL-1β was the target gene of miR-185-5p, and miR-185-5p negatively regulated the expression of IL-1β. Conclusion miR-185-5p alleviates the inflammatory response in AGA by inhibiting IL-1β.
Humans ; 3' Untranslated Regions ; Arthritis, Gouty/genetics* ; Interleukin-1beta/genetics* ; Interleukin-8 ; Luciferases ; MicroRNAs/genetics* ; Tumor Necrosis Factor-alpha

To investigate the mechanism of the major capsid protein VP5 (encoded by the UL19 gene) of oncolytic herpes simplex virus type Ⅱ (oHSV2) in regulating the antitumor function of immune cells, we constructed a mouse breast cancer cell line 4T1-iRFP-VP5-GFP stably expressing VP5 protein, near-infrared fluorescent protein (iRFP), and green fluorescent protein (GFP) by using the PiggyBac transposon system. Flow cytometry and Western blotting were employed to screen the monoclonal cell lines expressing both GFP and VP5 and examine the expression stability of UL19 in the constructed cell line. The results of SYBR Green I real-time PCR and Western blotting showed that the copies of UL19 and the expression level of VP5 protein in the 15th passage of 4T1-iRFP-VP5-GFP cells were significantly higher than those in the 4T1 cells transiently transfected with UL19, demonstrating the stable insertion of UL19 into the 4T1 cell genome. The real-time cell analysis (RTCA) was employed to monitor the proliferation of 4T1-iRFP-VP5-GFP cells, which showed similar proliferation activity to their parental 4T1 cells. Further studies confirmed that NK92 cells exhibited stronger cytotoxicity against 4T1-iRFP-VP5-GFP cells than against 4T1 cells. This study layed a foundation for elucidating the role of VP5 protein in regulating immune cells, including T cells and NK cells, via HLA-E in 4T1 cells to exert the anti-tumor function.
Animals ; Mice ; DNA Transposable Elements/genetics* ; Cell Line, Tumor ; Capsid Proteins/biosynthesis* ; Transfection ; Green Fluorescent Proteins/metabolism* ; Oncolytic Viruses/genetics* ; Female ; Simplexvirus/genetics*

The purpose of this study is to construct a muscle-specific synthetic promoter library, screen out muscle-specific promoters with high activity, analyze the relationship between element composition and activity of highly active promoters, and provide a theoretical basis for artificial synthesis of promoters. In this study, 19 promoter fragments derived from muscle-specific elements, conserved elements, and viral regulatory sequences were selected and randomLy connected to construct a muscle-specific synthetic promoter library. The luciferase plasmids pCMV-Luc and pSPs-Luc were constructed and transfected into the myoblast cell line C2C12. The activities of the synthesized promoters were evaluated by the luciferase activity assay. Two non-muscle-derived cell lines HeLa and 3T3 were used to verify the muscle specificity of the highly active promoters. The sequences of promoters with high activity, good muscle specificity, and correct sequences were analyzed to explore the relationship between the element composition and activity of promoters. We successfully constructed a muscle-specific promoter library and screened out 321 effective synthetic promoter plasmids. Among them, the activity of SP-301 promoter was 5.63 times that of CMV. The 15 promoters with high activity were muscle-specific. In the promoters with high activity and correct sequences, there was a relationship between their element composition and activity. Muscle-specific elements accounted for a high proportion in the promoters, while they had weak correlations with the promoter activity, being tissue-specific determinants. Viral elements accounted for no less than 20% in highly active promoters, which may be the key elements for the promoter activity. The content of conserved elements was proportional to the promoter activity. This study lays a theoretical foundation for the synthesis of tissue-specific efficient promoters and provides a new idea for the construction and application of in-situ gene delivery systems.
Promoter Regions, Genetic ; Humans ; Animals ; Mice ; Gene Library ; Cell Line ; Transfection ; HeLa Cells ; Luciferases/metabolism* ; Muscle, Skeletal/metabolism* ; Plasmids/genetics* ; Myoblasts/cytology*

In order to develop a transgenic zebrafish line with green fluorescent protein (enhanced green fluorescent protein, EGFP) expressed specifically in muscle and heart, the recombinant expression vector constructed using the zebrafish ttn.2 gene promoter fragment and EGFP gene coding sequence and the capped mRNA of Tol2 transposase were co-injected into the zebrafish 1-cell stage embryos. The stable genetic Tg (ttn.2: EGFP) transgenic zebrafish line was successfully developed by fluorescence detection, followed by genetic hybridization screening and molecular identification. Fluorescence signals and whole-mount in situ hybridization showed that EGFP expression was located in muscle and heart, the specificity of which was consistent with the expression of ttn.2 mRNA. Inverse PCR showed that EGFP was integrated into chromosomes 4 and 11 of zebrafish in No. 33 transgenic line, while integrated into chromosome 1 in No. 34 transgenic line. The successful construction of this fluorescent transgenic zebrafish line, Tg (ttn.2: EGFP), laid a foundation for the research of muscle and heart development and related diseases. In addition, the transgenic zebrafish lines with strong green fluorescence can also be used as a new ornamental fish.
Animals ; Zebrafish/genetics* ; Animals, Genetically Modified/genetics* ; Green Fluorescent Proteins/metabolism* ; Zebrafish Proteins/genetics* ; Promoter Regions, Genetic

OBJECTIVE: To investigate the regulatory effects of miR-30e-5p on biological behaviors of colorectal cancer cells and the role of PTEN/CXCL12 axis in mediating these effects. METHODS: Bioinformatic analysis was performed to explore the differential expression of miR-30e-5p between colorectal cancer tissues and normal tissues. RT-qPCR was used to detect the differential expression of miR-30e-5p in intestinal epithelial cells and colorectal cancer cells. Bioinformatics and dual luciferase assay were used to predict and validate the targeting relationship between miR-30e-5p and PTEN. Human and murine colorectal cancer cell lines were transfected with miR-30e-5p mimics, miR-30e-5p inhibitor, miR-30e-5p mimics+LV-PTEN, or miR-30e-5p inhibitor + si-PTEN. The changes in biological behaviors of the cells were detected using plate clone formation assay, CCK-8 assay, flow cytometry, scratch healing and Transwell assays. PTEN and CXCL12 expressions in the cancer cells were detected by Western blotting. The effects of miR-30e-5p inhibitor on colorectal carcinogenesis and development were observed in nude mice. RESULTS: Bioinformatic analysis showed that miR-30e-5p expression was significantly elevated in colorectal cancer tissues compared with the adjacent tissue (P < 0.01). Higher miR-30e-5p expression was detected in colorectal cancer cell lines than in intestinal epithelial cells (P < 0.01). Dual luciferase assay confirmed the targeting relationship between miR-30e-5p and PTEN (P < 0.05). Transfection with miR-30e-5p mimics significantly enhanced proliferation and metastasis and inhibited apoptosis of the colorectal cancer cells (P < 0.05), and co-transfection with LV-PTEN obviously reversed these changes (P < 0.05). MiR-30e-5p mimics significantly inhibited PTEN expression and enhanced CXCL12 expression in the cancer cells (P < 0.01), and miR-30e-5p inhibitor produced the opposite effect. Transfection with miR-30e-5p inhibitor caused cell cycle arrest in the cancer cells, which was reversed by co-transfection with si-PTEN (P < 0.05). In the in vivo experiments, the colorectal cancer cells transfected with miR-30e-5p inhibitor showed significantly lowered tumorigenesis. CONCLUSION Overexpression of miR-30e-5p promotes the malignant behaviors of colorectal cancer cells by downregulating PTEN to activate the CXCL12 axis.
Humans ; Animals ; Mice ; MicroRNAs/metabolism* ; Cell Line, Tumor ; Cell Proliferation/physiology* ; Mice, Nude ; Cell Movement/physiology* ; Colorectal Neoplasms/pathology* ; Luciferases/metabolism* ; Gene Expression Regulation, Neoplastic ; PTEN Phosphohydrolase/metabolism* ; Chemokine CXCL12/metabolism*

BACKGROUND: Steroid receptor-associated and regulated protein (SRARP) suppresses tumor progression and modulates steroid receptor signaling by interacting with estrogen receptors and androgen receptors in breast cancer. In endometrial cancer (EC), progesterone receptor (PR) signaling is crucial for responsiveness to progestin therapy. The aim of this study was to investigate the role of SRARP in tumor progression and PR signaling in EC. METHODS: Ribonucleic acid sequencing data from the Cancer Genome Atlas, Clinical Proteomic Tumor Analysis Consortium, and Gene Expression Omnibus were used to analyze the clinical significance of SRARP and its correlation with PR expression in EC. The correlation between SRARP and PR expression was validated in EC samples obtained from Peking University People's Hospital. SRARP function was investigated by lentivirus-mediated overexpression in Ishikawa and HEC-50B cells. Cell Counting Kit-8 assays, cell cycle analyses, wound healing assays, and Transwell assays were used to evaluate cell proliferation, migration, and invasion. Western blotting and quantitative real-time polymerase chain reaction were used to evaluate gene expression. The effects of SRARP on the regulation of PR signaling were determined by co-immunoprecipitation, PR response element (PRE) luciferase reporter assay, and PR downstream gene detection. RESULTS: Higher SRARP expression was significantly associated with better overall survival and disease-free survival and less aggressive EC types. SRARP overexpression suppressed growth, migration, and invasion in EC cells, increased E-cadherin expression, and decreased N-cadherin and Wnt family member 7A ( WNT7A ) expression. SRARP expression was positively correlated with PR expression in EC tissues. In SRARP -overexpressing cells, PR isoform B (PRB) was upregulated and SRARP bound to PRB. Significant increases in PRE-based luciferase activity and expression levels of PR target genes were observed in response to medroxyprogesterone acetate. CONCLUSIONS This study illustrates that SRARP exerts a tumor-suppressive effect by inhibiting the epithelial-mesenchymal transition via Wnt signaling in EC. In addition, SRARP positively modulates PR expression and interacts with PR to regulate PR downstream target genes.
Female ; Humans ; Receptors, Progesterone/metabolism* ; Proteomics ; Cell Line, Tumor ; Endometrial Neoplasms/metabolism* ; Cell Proliferation/genetics* ; Luciferases/pharmacology* ; Gene Expression Regulation, Neoplastic/genetics*