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Related Concept Videos

What is Gene Expression?01:42

What is Gene Expression?

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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...
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What is Gene Expression?01:36

What is Gene Expression?

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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...
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Cell Specific Gene Expression01:58

Cell Specific Gene Expression

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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...
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Cell Specific Gene Expression

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Chromatin Position Affects Gene Expression02:35

Chromatin Position Affects Gene Expression

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Chromatin is the massive complex of DNA and proteins packaged inside the nucleus. The complexity of chromatin folding and how it is packaged inside the nucleus greatly influences  access to genetic information. Generally, the nucleus' periphery is considered transcriptionally repressive, while the cell's interior is considered a transcriptionally active area. 
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mRNA Stability and Gene Expression02:51

mRNA Stability and Gene Expression

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The structure and stability of mRNA molecules regulates gene expression, as mRNAs are a key step in the pathway from gene to protein. In eukaryotes, the half-life of mRNA varies from a few minutes up to several days. mRNA stability is essential in growth and development. The absence of the proteins regulating its stability, such as tristetraprolin in mice, can cause systemic issues, including bone marrow overgrowth, inflammation, and autoimmunity.
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A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses
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EASI Transformation: An Efficient Transient Expression Method for Analyzing Gene Function in Catharanthus roseus

Samuel Mortensen1, Diana Bernal-Franco1,2, Lauren F Cole3

  • 1Department of Biology, Northeastern University, Boston, MA, United States.

Frontiers in Plant Science
|July 3, 2019
PubMed
Summary

We developed an efficient Agrobacterium-mediated seedling infiltration (EASI) protocol for rapid gene function studies in Catharanthus roseus. This method enhances the understanding of how transcription factors regulate anticancer alkaloid biosynthesis.

Keywords:
Catharanthus roseusORCA3ZCT1agroinfiltrationmutated VirGtransactivationtransient transformationviral silencing suppressors

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Area of Science:

  • Plant biotechnology
  • Molecular biology
  • Biochemistry

Background:

  • Catharanthus roseus is the sole source of vinblastine and vincristine, crucial anticancer drugs.
  • Advancements in C. roseus genomics necessitate efficient gene function analysis methods.
  • Transient expression systems are vital for rapid gene validation in planta.

Purpose of the Study:

  • To develop and optimize an Agrobacterium-mediated transient expression system for C. roseus.
  • To enhance transformation efficiency and transgene expression levels.
  • To provide a reliable tool for studying gene function and gene regulation in C. roseus.

Main Methods:

  • Optimized Agrobacterium infiltration parameters (vacuum infiltration, seedling age, Agrobacterium density, time point).
  • Enhanced transformation by expressing virulence genes and suppressing RNA silencing.
  • Improved vector design with introns, reporter genes (luciferase, GUS), and an internal control (Renilla luciferase).

Main Results:

  • Vacuum infiltration of 10-day-old seedlings yielded 18-57 fold higher transgene expression.
  • Constitutive expression of virulence genes and silencing suppressors increased expression 2-10 fold.
  • The EASI protocol demonstrated quantitative assessment of promoter activity and gene regulation.

Conclusions:

  • The EASI protocol offers a highly efficient, reproducible, and homogenous transformation method for C. roseus.
  • This method serves as a timely tool for rapid gene function assessment in planta.
  • The developed system is particularly valuable for investigating transcription factor regulation of terpenoid indole alkaloid biosynthesis.