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Related Concept Videos

What is Gene Expression?01:42

What is Gene Expression?

Overview
Gene expression is the process in which DNA directs the synthesis of functional products, that is, proteins. Cells can regulate gene expression at various stages. It allows organisms to generate different cell types and enables cells to adapt to internal and external factors.
Genetic Information Flows from DNA to RNA to Protein
A gene is a stretch of DNA that serves as the blueprint for functional RNAs and proteins. Since DNA is made up of nucleotides and proteins consist of amino...
What is Gene Expression?01:36

What is Gene Expression?

A gene is a stretch of DNA that serves as the blueprint for functional RNAs and proteins. Since DNA is comprised  of nucleotides and proteins are comprised of amino acids, a mediator is required to convert the information encoded in DNA into proteins. This mediator is the messenger RNA (mRNA). mRNA copies the blueprint from DNA by a process called transcription. In eukaryotes, transcription occurs in the nucleus by complementary base-pairing with the DNA template. The mRNA is then processed and...
Cell Specific Gene Expression01:58

Cell Specific Gene Expression

Multicellular organisms contain a variety of structurally and functionally distinct cell types, but the DNA in all the cells originated from the same parent cells. The differences in the cells can be attributed to the differential gene expression. Liver cells, whose functions include detoxification of blood, production of bile to metabolize fats, and synthesis of proteins essential for metabolism, must express a specific set of genes to perform their functions. Gene expression also varies with...
Cell Specific Gene Expression01:58

Cell Specific Gene Expression

Multicellular organisms contain a variety of structurally and functionally distinct cell types, but the DNA in all the cells originated from the same parent cells. The differences in the cells can be attributed to the differential gene expression. Liver cells, whose functions include detoxification of blood, production of bile to metabolize fats, and synthesis of proteins essential for metabolism, must express a specific set of genes to perform their functions. Gene expression also varies with...
Regulation of Expression at Multiple Steps01:23

Regulation of Expression at Multiple Steps

The gene expression in cells is regulated at different stages: (i) transcription, (ii) RNA processing, (iii) RNA localization, and (iv) translation. Transcriptional regulation is mediated by regulatory proteins such as transcription factors, activators, or repressors—these control gene expression by initiating or inhibiting the transcription of genes. Once a precursor or pre-mRNA is produced, it undergoes post-transcriptional modification, including 5' capping, splicing, and the addition of a...
Eukaryotic Compartmentalization01:37

Eukaryotic Compartmentalization

One of the distinguishing features of eukaryotic cells is that they contain membrane-bound organelles, such as the nucleus and mitochondria, that carry out specialized functions. Since biological membranes are only selectively permeable to solutes, they help create a compartment with controlled conditions inside an organelle. These microenvironments are tailored to the organelle's specific functions and help isolate them from the surrounding cytosol.
For example, lysosomes in the animal cells...

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Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach
11:20

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

Published on: October 16, 2014

Intrabody expression in eukaryotic cells.

Laurence Guglielmi1, Pierre Martineau

  • 1IRCM, Institut de Recherche en Cancérologie de Montpellier, Montpellier F-34298, France.

Methods in Molecular Biology (Clifton, N.J.)
|June 26, 2009
PubMed
Summary
This summary is machine-generated.

This study details methods for expressing single-chain variable fragments (scFv) inside eukaryotic cells. Researchers can now effectively monitor intrabody expression using immunofluorescence and FACS analysis.

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Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biotechnology

Background:

  • Single-chain variable fragments (scFv) are crucial for antibody-based research and therapeutics.
  • Intracellular expression of scFv (intrabodies) offers unique applications in studying protein function and developing novel therapies.
  • Efficient methods for producing functional intrabodies in eukaryotic systems are needed.

Purpose of the Study:

  • To establish and describe robust procedures for the intracellular expression of scFv in eukaryotic cells.
  • To facilitate the generation and monitoring of functional intrabodies for research applications.

Main Methods:

  • Cloning of scFv genes from phage-display vectors into mammalian expression vectors.
  • Transient transfection of HeLa cells with the constructed expression vectors.
  • Monitoring of intrabody expression utilizing immunofluorescence staining.
  • Quantification of intrabody expression via Fluorescence-Activated Cell Sorting (FACS) analysis.

Main Results:

  • Successful cloning of scFv genes into a mammalian expression system.
  • Demonstrated transient expression of scFv within HeLa cells.
  • Validated immunofluorescence and FACS as effective methods for detecting and quantifying intracellular scFv.

Conclusions:

  • The described procedures provide a reliable method for intracellular scFv expression in eukaryotic cells.
  • Immunofluorescence and FACS analysis are suitable techniques for monitoring intrabody expression.
  • This work supports the development of intrabody-based research tools and therapeutic strategies.