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.

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2. 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.

3. Construction of Expression Vector
target gene
Plasmid DNA
TTTTTTTTTTT
transfection reagent
cell transfection
Cell collection
PCR
RT-PCR
Western blot
Expression detection

4. The target gene generally is the structural gene encoding the
protein. There are three ways of getting the target gene:
● obtaining from the gene library;
●amplifying the target gene by PCR technique;
●designing and synthesizing target gene.

5. 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.

6. 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.

7. After 24 to 48 hours incubation, cultured cells are collected. We
can break up the cells through ultrasound or enzymatic hydrolys, and
supernatant is obtained by centrifugation. Next, we carry out the
detection after transfection.

8. Plasmid expression should be visible or detectable 24 to 48 hours
post-transfection. Total RNA is extracted from cells with trizol protocol,
dissolved in DEPC-treated deionized water and quantified with
spectrophotometer. For RNA with polyadenylated tails, enrich with a
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.

9. 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.

10. Creative Biogene provides stable expression of a gene of
interest by plasmid transfection or lentivirus transduction. Our
scientists bring years of extensive experience in achieving gene
overexpression stable cell lines. If you have any questions or need
advice on your particular use case, feel free to contact us.

11. THANK YOU!THANK YOU!