Transfect commonly used reporter genes-Gene Transfection

A reporter gene is a gene that encodes a detectable protein or enzyme, and its expression product is easily identifiable. Fusing its coding sequence with gene expression regulatory sequence to form a chimeric gene, or fusing it with other target genes, and expressing it under the control of regulatory sequence, so as to use its expression product to calibrate the expression regulation of target genes and screen for transformants.

As a reporter gene, the following conditions must be met in genetic selection and screening testing:

(1) Cloned and fully sequenced;

(2) The expression product does not exist in the recipient cells, i.e. there is no background, and there are no similar endogenous expression products in the transfected cells;

(3) Its expression product can be quantitatively measured.

In the field of plant genetic engineering research, the main reported genes used are as follows:

Cochineal synthase gene (nos), octopusine synthase gene (ocs)

The genes nos and ocs are unique to the Ti plasmid of Agrobacterium tumefaciens, which is responsible for tumor causing soil. When the Ti plasmid is modified and the corresponding Agrobacterium tumefaciens is used to transform the plant body, if the exogenous gene is transferred into the plant body, these two reporter genes can be expressed in the roots, stems, and leaves of the plant without developmental regulation. The detection can be directly performed by paper electrophoresis using the transformed body extract, staining, and observing fluorescence under ultraviolet light.

Neomycin phosphotransferase gene (npt Ⅱ), chloramphenicol acetyltransferase gene (cat)
　

The npt II, cat, and gentamicin transferase genes are all antibiotic screening genes, and related enzymes can modify substrates (phosphorylation, acetylation, etc.) to make these antibiotics lose their inhibitory effect on plant growth, allowing transformants containing these resistance genes to grow normally on screening media containing these antibiotics. Alternatively, transformants can be extracted with liquid, labeled with isotopes, and screened by autoradiography. Chloramphenicol acetyltransferase gene testing can be observed by autoradiography.

Luciferase Gene

The enzyme was cloned from the cDNA libraries of North American firefly and kowtow in 1985. It emits fluorescence in the presence of ATP, Mg2+, O2, and fluorescein, making it suitable for direct detection of whole or partial transgenic plants using X-rays or specialized instruments. It has the advantages of fast detection speed, sensitivity 30-1000 times higher than cat genes, low cost, and no need to use radioactive isotopes, and has been widely .

β - D-glucosidase gene

This enzyme catalyzes the formation of β - D-glucosinolate from substrates, and it has almost no background in plants. Histochemical detection is very stable and can be detected by spectrophotometry, fluorescence, and other methods.

In addition, there are several reported genes used in animal gene expression regulation research, such as the gentamicin transferase gene. The following are the main reported genes used:

Green fluorescent protein (GFP) gene, etc.

Green fluorescent protein is derived from marine organisms such as jellyfish, and its genes can be expressed and produce fluorescence in heterologous tissues. The GFP Cdnad open reading framework is approximately 740bp long, encoding 238 amino acid residues. The serine dehydrotyrosine glycine at positions 65-67 inside its peptide chain forms a chromogenic gene through self cyclization and oxidation, which emits green fluorescence under long ultraviolet wavelength or blue light irradiation. The transfected cells can be directly observed for gene expression under fluorescence microscopy or flow cytometry (FACS).

In addition, there are genes for β - galactosidase, dihydrofolate reductase, chloramphenicol acetyltransferase (cat), etc

previous item: Comparison of various transfection methods for gene transfection

About Sunma

Product Center

Technical Data

News Center

Transfect commonly used reporter genes-Gene Transfection

Transfect commonly used reporter genes-Gene Transfection