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Methods in Molecular Biology (Clifton,... 2024RNA interference (RNAi) serves as an indispensable tool for gene function studies and has been substantiated through extensive research for its practical applications in...
RNA interference (RNAi) serves as an indispensable tool for gene function studies and has been substantiated through extensive research for its practical applications in the baculovirus expression vector system (BEVS). This chapter expands the RNAi toolkit in insect cell culture by including small interfering RNA (siRNA) in the protocol, in addition to the conventional use of double-stranded RNA (dsRNA). This chapter also brings attention to key design and reporting considerations, based on Minimum Information About an RNAi Experiment (MIARE) guidelines. Recommendations regarding online tools for dsRNA and siRNA design are provided, along with guidance on choosing suitable methods for measuring silencing outcomes.
Topics: Animals; RNA Interference; Baculoviridae; RNA, Double-Stranded; RNA, Small Interfering; Genetic Vectors; Insecta; Cell Line; Sf9 Cells
PubMed: 38951329
DOI: 10.1007/978-1-0716-3961-0_7 -
Methods in Molecular Biology (Clifton,... 2024Adaptive laboratory evolution (ALE) is a powerful tool for enhancing the fitness of cell lines in specific applications, including recombinant protein production....
Adaptive laboratory evolution (ALE) is a powerful tool for enhancing the fitness of cell lines in specific applications, including recombinant protein production. Through adaptation to nonstandard culture conditions, cells can develop specific traits that make them high producers. Despite being widely used for microorganisms and, to lesser extent, for mammalian cells, ALE has been poorly leveraged for insect cells. Here, we describe a method for adapting insect High Five and Sf9 cells to nonstandard culture conditions via an ALE approach. Aiming to demonstrate the potential of ALE to improve productivity of insect cells, two case studies are demonstrated. In the first, we adapted insect High Five cells from their standard pH (6.2) to neutral pH (7.0); this adaptation allowed to improve production of influenza virus-like particles (VLPs) by threefold, using the transient baculovirus expression vector system. In the second, we adapted insect Sf9 cells from their standard culture temperature (27 °C) to hypothermic growth (22 °C); this adaptation allowed to improve production of influenza VLPs by sixfold, using stable cell lines. These examples demonstrate the potential of ALE for enhancing productivity within distinct insect cell hosts and expression systems by manipulating different culture conditions.
Topics: Animals; Recombinant Proteins; Cell Line; Sf9 Cells; Baculoviridae; Cell Culture Techniques; Insecta; Directed Molecular Evolution; Hydrogen-Ion Concentration; Temperature
PubMed: 38951328
DOI: 10.1007/978-1-0716-3961-0_6 -
Methods in Molecular Biology (Clifton,... 2024This chapter outlines the use of TOPO cloning for streamlined generation of a recombinant plasmid containing your gene of interest for use in the Bac-to-Bac Baculovirus...
This chapter outlines the use of TOPO cloning for streamlined generation of a recombinant plasmid containing your gene of interest for use in the Bac-to-Bac Baculovirus Expression System.
Topics: Plasmids; Cloning, Molecular; Genetic Vectors; Baculoviridae; Chromosomes, Artificial, Bacterial
PubMed: 38951327
DOI: 10.1007/978-1-0716-3961-0_5 -
Methods in Molecular Biology (Clifton,... 2024This chapter outlines the workflow using the ExpiSf Expression System designed for high-density infection of suspension ExpiSf9 cells. The system utilizes a chemically...
This chapter outlines the workflow using the ExpiSf Expression System designed for high-density infection of suspension ExpiSf9 cells. The system utilizes a chemically defined, serum-free, protein-free, and animal origin free medium, making it suitable for recombinant protein expression experiments. The ExpiSf chemically defined medium allows efficient transfection and baculovirus production directly within the same culture medium. The ExpiSf Expression System Starter Kit provides all necessary components, including cells, culture medium, and reagents needed to infect one (1) liter of cell culture. The system's versatility and animal origin free nature make it a valuable tool for various protein expression studies and biotechnological applications.
Topics: Animals; Workflow; Recombinant Proteins; Baculoviridae; Transfection; Culture Media; Cell Culture Techniques; Cell Line; Gene Expression
PubMed: 38951326
DOI: 10.1007/978-1-0716-3961-0_4 -
Methods in Molecular Biology (Clifton,... 2024The baculovirus expression vector system (BEVS) is recognized as a powerful platform for producing challenging proteins and multiprotein complexes both in academia and...
The baculovirus expression vector system (BEVS) is recognized as a powerful platform for producing challenging proteins and multiprotein complexes both in academia and industry. Since a baculovirus was first used to produce heterologous human IFN-β protein in insect cells, the BEVS has continuously been developed and its applications expanded. We have recently established a multigene expression toolbox (HR-bac) composed of a set of engineered bacmids expressing a fluorescent marker to monitor virus propagation and a library of transfer vectors. Unlike platforms that rely on Tn7-medidated transposition for the construction of baculoviruses, HR-bac relies on homologous recombination, which allows to evaluate expression constructs in 2 weeks and is thus perfectly adapted to parallel expression screening. In this chapter, we detail our standard operating procedures for the preparation of the reagents, the construction and evaluation of baculoviruses, and the optimization of protein production for both intracellularly expressed and secreted proteins.
Topics: Baculoviridae; Animals; Recombinant Proteins; Genetic Vectors; Sf9 Cells; Gene Expression; Humans; Insecta; Spodoptera; Cell Line; Homologous Recombination; Cost-Benefit Analysis
PubMed: 38951325
DOI: 10.1007/978-1-0716-3961-0_3 -
Methods in Molecular Biology (Clifton,... 2024The success of using the insect cell-baculovirus expression technology (BEST) relies on the efficient construction of recombinant baculovirus with genetic stability and...
The success of using the insect cell-baculovirus expression technology (BEST) relies on the efficient construction of recombinant baculovirus with genetic stability and high productivity, ideally within a short time period. Generation of recombinant baculoviruses requires the transfection of insect cells, harvesting of recombinant baculovirus pools, isolation of plaques, and the expansion of baculovirus stocks for their use for recombinant protein production. Moreover, many options exist for selecting the genetic elements to be present in the recombinant baculovirus. This chapter describes the most commonly used homologous recombination systems for the production of recombinant baculoviruses, as well as strategies to maximize generation efficiency and recombinant protein or baculovirus production. The key steps for generating baculovirus stocks and troubleshooting strategies are described.
Topics: Baculoviridae; Animals; Recombinant Proteins; Genetic Vectors; Transfection; Homologous Recombination; Sf9 Cells; Cell Line; Spodoptera; Insecta
PubMed: 38951324
DOI: 10.1007/978-1-0716-3961-0_2 -
Methods in Molecular Biology (Clifton,... 2024Healthy insect cell cultures are critical for any method described in this book, including making productive baculovirus banks, protein or AAV expression, and...
Healthy insect cell cultures are critical for any method described in this book, including making productive baculovirus banks, protein or AAV expression, and determining viral titers. This chapter describes cell maintenance in shake flasks using serum-free conditions and the expansion of virus stocks from a single plaque purified virus. Insect cells can be passaged over multiple generations, but as the cells may undergo changes over multiple passages, limiting the use of your cells to a defined number of passages such as 50 passages is recommendable. Baculovirus stocks once created using serum-free media are not very stable at 4-8 °C. This chapter also includes a simple method to store cells from an early cell passage and your virus stock in liquid nitrogen.
Topics: Animals; Baculoviridae; Cell Culture Techniques; Insecta; Cell Line
PubMed: 38951323
DOI: 10.1007/978-1-0716-3961-0_1 -
Mikrochimica Acta Jul 2024A stable DNA signal amplification sensor was developed on account of rolling circle amplification (RCA). This sensor includes target DNA-controlled rolling circle...
A stable DNA signal amplification sensor was developed on account of rolling circle amplification (RCA). This sensor includes target DNA-controlled rolling circle amplification technology and locking probe DNA replacement technology, which can be used to detect DNA fragments with genetic information, thus constructing a biosensor for universal detection of DNA. This study takes the homologous DNA of human immunodeficiency virus (HIV) and let-7a as examples to describe this biosensor. The padlock probe is first cyclized by T4 DNA ligase in response to the target's reaction with it. Then, rolling cycle amplification is initiated by Phi29 DNA polymerase, resulting in the formation of a lengthy chain with several triggers. These triggers can open the locked probe LP1 with the fluorescence signal turned off, so that it can continue to react with H2 to form a stable H1-H2 double strand. This regulates the distance between B-DNA modified by the quenching group and H1 modified by fluorescent group, and the fluorescence signal is recovered.
Topics: Biosensing Techniques; Nucleic Acid Amplification Techniques; Humans; DNA Probes; Fluorescent Dyes; DNA, Viral; DNA; Spectrometry, Fluorescence; Fluorescence; DNA-Directed DNA Polymerase; Limit of Detection; HIV
PubMed: 38951284
DOI: 10.1007/s00604-024-06501-2 -
Zhonghua Nei Ke Za Zhi Jul 2024A 19-year-old male patient with high-risk acute B-cell lymphoblastic leukemia received haploidentical stem cell transplantation. He developed anemia repeatedly and...
A 19-year-old male patient with high-risk acute B-cell lymphoblastic leukemia received haploidentical stem cell transplantation. He developed anemia repeatedly and parvovirus B19 nucleic acid was positive in blood plasma. The patient was diagnosed with cold agglutinin syndrome and multiple organ dysfunction including respiratory failure and hepatitis. In the conflict between viral infection and the treatment of cold agglutinin syndrome, we provided supportive treatment, complement inhibitors to control hemolysis, and antiviral therapy. After timely glucocorticoid and immunosuppressant therapy, the patient had achieved a good response.
Topics: Humans; Male; Parvovirus B19, Human; Young Adult; Multiple Organ Failure; Parvoviridae Infections; Anemia, Hemolytic; Anemia, Hemolytic, Autoimmune
PubMed: 38951100
DOI: 10.3760/cma.j.cn112138-20231210-00376 -
Molecular Metabolism Jun 2024Aberrant glucolipid metabolism in the heart is a characteristic factor in diabetic cardiomyopathy (DbCM). Super-enhancers-driven noncoding RNAs (seRNAs) are emerging as...
OBJECTIVE
Aberrant glucolipid metabolism in the heart is a characteristic factor in diabetic cardiomyopathy (DbCM). Super-enhancers-driven noncoding RNAs (seRNAs) are emerging as powerful regulators in the progression of cardiac diseases. However, the functions of seRNAs in DbCM have not been fully elucidated.
METHODS
Super enhancers and their associated seRNAs were screened and identified by H3K27ac ChIP-seq data in the Encyclopedia of DNA Elements (ENCODE) dataset. A dual-luciferase reporter assay was performed to analyze the function of super-enhancers on the transcription of peroxisome proliferator-activated receptor α-related seRNA (PPARα-seRNA). A DbCM mouse model was established using db/db leptin receptor-deficient mice. Adeno-associated virus serotype 9-seRNA (AAV9-seRNA) was injected via the tail vein to evaluate the role of seRNA in DbCM. The underlying mechanism was explored through RNA pull-down, RNA and chromatin immunoprecipitation, and chromatin isolation by RNA purification.
RESULTS
PPARα-seRNA was regulated by super-enhancers and its levels were increased in response to high glucose and palmitic acid stimulation in cardiomyocytes. Functionally, PPARα-seRNA overexpression aggravated lipid deposition, reduced glucose uptake, and repressed energy production. In contrast, PPARα-seRNA knockdown ameliorated metabolic disorder in vitro. In vivo, overexpression of PPARα-seRNA exacerbated cardiac metabolic disorder and deteriorated cardiac dysfunction, myocardial fibrosis, and hypertrophy in DbCM. Mechanistically, PPARα-seRNA bound to the histone demethylase KDM4B (Lysine-specific demethylase 4B) and decreased H3K9me3 levels in the promoter region of PPARα, ultimately enhancing its transcription.
CONCLUSIONS
Our study revealed the pivotal function of a super-enhancer-driven long noncoding RNA (lncRNA), PPARα-seRNA, in the deterioration of cardiac function and the exacerbation of metabolic abnormalities in diabetic cardiomyopathy, which recruited KDM4B to the promoter region of PPARα and repression of its transcription. This suggests a promising therapeutic strategy for the treatment of DbCM.
PubMed: 38950776
DOI: 10.1016/j.molmet.2024.101978