GFP
 |
Fig.1 |
GFP (Green Fluorescent Protein) and its variants (YFP, CFP, and BFP) are in vogue in various fields of the life sciences including medicine and biology. Search in the MEDLINE : the keyword "GFP" shows dozens more reports every month. GFP is found in the jellyfish and is a special protein that emits fluorescence when exposed to excitation light. Its primary importance for current research lies in the ability of the isolated jellyfish GFP gene to express the fluorescent protein in other living organisms. When the GFP gene is introduced in a culture cell, the entire cytoplasm will emit a green fluorescence (Fig. 1).
GFP consists of 238 amino acids arranged in a basket shape. Three amino acids form an annular structure inside the basket by special bonding; it is this part that emits fluorescence.
Link to GFP STRUCTURE (Lab. of Dr. Roger Y. Tsien),
 |
 |
Fig.2 |
Fig.3 |
It is possible to make a fusion gene combining the GFP with another protein (for example the alpha-tubulin, which is one of proteins comprising a microtubule, shown in red in Figure 2), and to express it inside a cell. When such a fusion gene is introduced into a cell, the alpha-tubulin tagged with the GFP is produced inside the cell and, after a while, it is incorporated into the microtubule to form a tubular structure that emits green fluorescence (Fig. 3). This technique makes it possible to visualize the localization of a specific gene product (i.e. a protein) in cells. It enables the researcher to observe the localization of proteins in the living state while the fluorescence antibody technique fixes the cell (causing its death).
We will show how to express a fused protein inside a cell in two steps.
1. Preparation of a fusion gene
2. Introduction of a gene into the cell
Preparation of a fusion gene
The alpha-tubulin gene was prepared from human brain cDNA libraries purchased from TAKARA BIO Inc., Otsu, Japan. A cDNA library is synthesized by templating the sequences of mRNA that are expressed in the cells. This cDNA is integrated in annular DNA, called plasmids, for storage. Some of the plasmids integrate the alpha-tubulin gene and these plasmids are used as templates for amplifying alpha-tubulin by means of PCR. At present, the GFP vectors can be purchased from BD Biosciences Clontech, Palo Alto, CA, USA or Qbiogene, Inc. (Quantum Biotechnologies Inc.), Illkirch Cedex, France. Selection of the GFP vector depends on whether the gene of interest is linked at the 3' or 5' end of the GFP gene.
Introduction of a gene in the cell
To express the GFP-tubulin in the cell, it is first required to introduce the fusion gene in the cell using one of the following techniques.
- Microinjection:
Plasmids can be injected directly into the cell nucleus. This method is unsuitable for expressing genes in large numbers of cells, but is used in the case of Nematoda or when particular cells are the targets. - Lipofectamine transfection:
When a mixture of positively charged lipids and neutral lipids is blended with DNA, a complex is formed by an electrical interaction and caught inside the cell by phagocytosis or membrane fusion. Lipofectamine transfection has high efficiency and low toxicity. - Calcium phosphate precipitation:
When calcium phosphate is mixed with DNA, a complex is precipitated. When this precipitate is applied to the cell, part of it is caught inside the nucleus. - Electroporation:
A high-voltage pulse is applied to the cell to make pores on the cell membrane. The DNA enters the cell through these pores.
The fusion protein will be expressed several hours after the gene introduction, but the culture should be continued for about two days until the fluorescent tubulin is fully integrated in the microtubule.
It is also possible to apply double staining with two proteins emitting different fluorescent colors. Besides GFP, the available fluorescent proteins include BFP (Blue Fluorescent Protein), CFP (Cyan Fluorescent Protein) and YFP (Yellow Fluorescent Protein). Recently, a red fluorescent protein has been introduced by Clontech .
