The future prospect or potential to fully integrate genes to the full DNA sequence of a mammalian cell has a significant impact on recent biomedical researches which includes the development and improvement of novel medicines and pharmaceutical products. Although the transient transfection is more beneficial for fast and quicker analysis of genes and relatively smaller scale protein production, still the stable transfection guarantees long-term, consistent and accurately stated gene expression. Important applications for stable transfections are drug discovery and development of gene therapy, large scale of protein production and the analytical study of gene functions and regulations. Development of cell lines with stable expression is a commercially available service offered by Altogen Labs.
Stable transfection can be achieved by the integration and consolidation of the gene of interest into the desired target chromosome of the cell. At the beginning, the gene of interest has to be presented and introduced into the (A) cell, afterwards into the (B) nucleus and finally, it has to be incorporated into (C) chromosomal DNA.
Furthermore, this stable transfection can be greatly influenced by two factors: the method of transfection used and the transmitter vector which contains the gene of interest.
The method of transfection defines which of the cell type can be aimed as a target for stable integration. Although many lipofection chemical agents and transfection kits to transfect DNA at a definite quantity into a sticking fast cell lines, efficient transfer of DNA into an extremely difficult-to-transfect cell lines including the primary cells can happen mostly with electroporation and viral methods. Unluckily, these viral methods are subjected to many limitations which includes the time-consuming processing of vectors and concerns related to safety.
The kind of vector that will be utilized for stable transfection specifies the mechanism of the integration, the regulation of the transgene expression and the condition of selections for stable expressing cells. Afterwards the integration, the time and level of expression of the gene largely depends on the promoter copied on the expression vector and of the specific integration site. For organic expression, CMV promoters are preferred. For regulated expression, inducible promoters are accessible.
Moreover, the site of integration has an effect on the rate of transcription of the gene of interest. Commonly, a plasmid of regular expression is integrated into the full DNA sequence of the target cell randomly. There is a little or no transgene expression at all if integration into non-active heterochromatin happened, whereas integration into an active euchromatin oftentimes permits transgene expression. Still, random integration frequently leads to shutting up of the transgene. Various strategies have been discovered to compensate the negative effects of this random integration. Some of these strategies used were site-specific homologous and transposed mediated integration, but needs additional plasmid sequence.
The precise and accurate mechanism in which the plasmid DNA is incorporated is still on its development stage and still remains a research matter.
Generation of Stable Cell Line in 28 Days – is a commercially available service provided by Altogen Labs, as well as stable RNAi knockdown cell line development and other laboratory services including pharm/tox IND studies, over 50 cancer xenograft models, ELISA, IC-50 assays.