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Gene overexpression is the process which leads to the abundant target protein expression subsequently. The process may be in the cell where the gene is originally located or in other expression systems. The fundamental principle is to add regulatory elements to the upstream of the target gene through artificial construction, so that genes can be transcribed and translated efficiently under controlled conditions. There are two general purposes of gene overexpression. On the one hand, we can obtain a large number of target gene products. The target gene products can be used in research or production, such as studying information of 3D structure of proteins and preparing insulin by fermentation technology. On the other hand, we can study the biological function of target gene products through gene overexpression.
1. Obtain target gene
The target gene generally is the structural gene encoding the protein. There are three ways of getting the target gene: (1) obtaining from the gene library; (2) amplifying the target gene by PCR technique; (3) designing and synthesizing target gene.
2. Construction of Expression Vector
In order to express stably the definite gene in the cell and transmit genetic information to the next generation, we need to connect target gene with vector. Firstly, we select the suitable expression vector according to make gene expression with high efficiency and play a role in the cell. The expression vector should contain promoter, terminator and marker gene. Then the target gene is cloned into vector by enzyme digestion and ligation. Finally, by PCR, gel electrophoresis and sequencing, clones with correct sequence can be obtained.
3. Cell transfection
Through selecting appropriate cell line, we can make transgene with high expression level. The transfection efficiency is affected by many factors, including cell type, cell culture condition, cell density at the time of transfection and the method of transfection. If plated >1 day prior to transfection, the transfection efficiency may decrease. And transfection complexes can be added to cells in media containing antibiotics and serum without impacting the transfection efficiency. There are four main methods for introducing foreign DNA into cells: electric shock, calcium phosphate method, liposome-mediated and virus-mediated. For many ordinary cell lines, the methods of transient transfection were mostly liposome-mediated. Transfection reagents should be mixed with cells at an appropriate proportion.
4. Cell collection
After 24 to 48 hours incubation, cultured cells are collected. We can break up the cells through ultrasound or enzymatic hydrolysis, and supernatant is obtained by centrifugation. Next, we carry out the detection after transfection.
5. Characterization of mRNA expression level
Plasmid expression should be visible or detectable 24 to 48 hours post-transfection. Total RNA is extracted from cells with the help of Trizol, dissolved in DEPC-treated deionized water and quantified with spectrophotometer. For RNA with polyadenylated tails, enrich them with mRNA Purification Kit. RNA of cells turn into its DNA complement by using the reverse transcriptase. The cDNA need further analyses with realtime-PCR or reverse transcription PCR.
6. Characterization of protein expression level
Protein expression should be detectable 48 hours post-transfection. Cells are collected with centrifugation. And add lysate which contains PMSF to disintegrate protein. In order to determine whether target genes are translated into proteins, the protein are extract for western blot analysis. Through protein electrophoresis, transmembrane, antibody incubation and colour-reaction of thorium, we can get fragments that have blot signal if target gene is expressed in protein level.