In Vitro Transfection
In Vitro Transfection - Related Videos
08:55
In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection
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Cited by 13 •
2012
This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.
07:28
An Introduction to Transfection
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2023
Transfection is the process of inserting genetic material, such as DNA and double stranded RNA, into mammalian cells. The insertion of DNA into a cell enables the expression, or production, of proteins using the cells own machinery, whereas insertion of RNA into a cell is used to down-regulate the production of a specific protein by stopping translation. While the site of action for transfected RNA is the cytoplasm, DNA must be transported to the nucleus for effective transfection. There, the...
06:46
Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA
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Cited by 13 •
2019
Macrophages, especially primary macrophages, are challenging to transfect as they specialize in detecting molecules of non-self origin. We describe a protocol that allows highly efficient transfection of primary macrophages with mRNA generated from DNA templates such as plasmids.
08:04
Neuronal Transfection Methods
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2023
Transfection - the process of transferring genetic material into cells - is a powerful tool for the rapid and efficient manipulation of gene expression in cells. Because this method can be used to silence the expression of specific proteins or to drive the expression of foreign or modified proteins, transfection is an extremely useful tool in the study of the cellular and molecular processes that govern neuron function. However, mature neurons have a number of properties that make them...
02:57
Transfecting Primary Macrophages with Modified mRNA
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2025
This video demonstrates the process of transfecting primary macrophages with modified mRNA encoding green fluorescent protein. These modified mRNAs, designed to reduce immunogenicity and improve its stability, ensure the successful transfection and subsequent expression of green fluorescent proteins, which is confirmed through fluorescence microscopy.
FAQs
Lipid-based carriers form complexes with DNA or RNA and help transport the cargo across the cell membrane. Electrical delivery instead uses brief pulses to transiently permeabilize that membrane, creating an opportunity for nucleic-acid entry. These mechanisms provide distinct ways to achieve intracellular delivery under controlled culture conditions and can be selected according to the experimental design.
The nucleic-acid construct determines the intended downstream effect. DNA can support protein production, whereas RNA may suppress a target gene or produce another designed response. After uptake, the construct interacts with the cell in a way that can alter gene expression, protein output, or cellular behavior, so interpretation depends on both the cargo and the biological question.
Cultured cells provide a controlled setting in which researchers can introduce a defined DNA or RNA construct and observe its consequences. This control helps connect the delivered material with changes in gene expression, protein production, or cellular behavior. Consequently, the method is useful for testing gene function while limiting variation from less defined biological conditions.
An experiment begins with cultured cells and a selected DNA or RNA construct, followed by choosing a delivery approach such as lipid-based complex formation or electrical permeabilization. Researchers then examine the resulting protein production, gene suppression, or cellular response. The workflow links a defined nucleic-acid input to a measurable biological outcome under controlled conditions.
In vitro transfection supports reporter assays, in which gene-related activity can be examined through an introduced construct, as well as gene-function studies and RNA interference experiments. It also enables protein production in cultured cells. These applications allow researchers to test how specific nucleic acids influence expression and cellular behavior in a laboratory model.
Within biology, the technique connects engineered DNA or RNA inputs with changes in cellular function, making it useful for investigating gene regulation and gene activity. Researchers can also optimize delivery approaches by comparing how nucleic acids enter cultured cells and what outcomes they produce. That optimization provides research context for developing future gene-delivery strategies.