explain how gene expression may be confirmed by the use of marker genes coding for fluorescent products

Principles of Genetic Technology

Objective

Explain how gene expression may be confirmed by the use of marker genes coding for fluorescent products.

What is a Marker Gene?

A marker gene is a DNA sequence that encodes a protein whose presence can be easily detected. In molecular biology, fluorescent proteins such as Green Fluorescent Protein (GFP) are commonly used as markers because they emit visible light when excited by specific wavelengths.

How Fluorescent Marker Genes Confirm Gene Expression

1. Construction of a Fusion Gene

   A DNA fragment containing the promoter of the gene of interest is ligated upstream of a fluorescent‑protein coding sequence. The resulting construct is:

   \$\text{Promoter}_{\text{target}} \;-\; \text{ATG} \;-\; \text{Fluorescent‑protein coding region}\$

2. Introduction into Host Cells

   The construct is introduced into the host (bacterial, yeast, plant or animal cells) using transformation, transfection or microinjection.

3. Transcription and Translation

   If the promoter is active, the host cell transcribes the construct into mRNA:

   \$\text{DNA} \xrightarrow{\text{RNA polymerase}} \text{mRNA}_{\text{fusion}}\$

   and translates it into a fluorescent protein:

   \$\text{mRNA}_{\text{fusion}} \xrightarrow{\text{ribosome}} \text{Fluorescent protein}\$

4. Detection of Fluorescence

   The cells are examined under a fluorescence microscope or a plate reader. Emission of light at the characteristic wavelength indicates that the fluorescent protein has been produced, confirming that the promoter (and therefore the gene of interest) is active.

Experimental Workflow

• Design primers to amplify the promoter region.
• Clone the promoter upstream of the fluorescent‑protein gene in a suitable vector.
• Verify the construct by restriction digestion and sequencing.
• Introduce the construct into the target cells.
• Incubate cells under appropriate conditions to allow expression.
• Observe fluorescence using the correct excitation/emission filters.
• Quantify fluorescence intensity if required.

Common Fluorescent Marker Genes

Advantages of Using Fluorescent Marker Genes

• Non‑destructive: live cells can be observed in real time.
• Quantitative: fluorescence intensity correlates with expression level.
• Versatile: multiple colours allow simultaneous monitoring of several promoters.
• High sensitivity: detectable even at low expression levels.

Limitations and Considerations

• Fluorescent proteins may affect the function or localisation of the fused protein.
• Autofluorescence from host cells can give background signal.
• Photobleaching reduces fluorescence over prolonged exposure.
• Expression of the marker gene must not be toxic to the host.

Key Points to Remember

• A fluorescent marker gene provides a visual read‑out of promoter activity.

• The presence of fluorescence confirms that transcription and translation have occurred.

• Choice of fluorescent protein depends on experimental needs such as colour, brightness and compatibility with other markers.