Transient (siRNA) and Stable (shRNA) Transfection Services
Transfection is the process of introducing purified nucleic acids [deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)] into cultured eukaryotic cells via viral or nonviral methods. Through the process of transfection, genes can be manipulated to be over-expressed (by transfection of DNA-encoding plasmid constructs) or silenced based on sequence specific targeting of genes (siRNA induced gene silencing and RNAi techniques). This application allows researchers to analyze gene function, determine disease pathways and identify potential drug targets. As a result, transfection has become an important experimental tool to the study of genetics, molecular and cell biology research.
Altogen Labs offers transfection services for the delivery of siRNA, microRNA, shRNA, and plasmid DNA into the cell line of choice. The standard service includes the optimization of transfection of DNA or RNA (at least 20 µg should be provided by the client) into the cell line of choice and final validation of construct expression by real-time qRT-PCR (mRNA expression) and/or Western blot (protein expression).
Transient Transfection of siRNA, microRNA, mRNA, or plasmid DNA
Transient transfection occurs when the transfected nucleic acids are introduced into the cell but are not permanently expressed or incorporated into the host cell genome. Subsequently, the nucleic acids are expressed for a short period of time but are eventually recognized as foreign genetic material and are degraded or diluted through mitosis and cell division. The main advantage of transient transfection is the rapid production of recombinant proteins that are of high quality, already activated and fully post-translationally modified. Although this mechanism of protein production is short lived (3 – 5 days), it is of benefit to basic biological studies.
Gene Knockdown: Stable RNAi Cell Line Development
Stable RNAi cell lines, also known as knockdown stable cell lines, are cell lines that have integrated shRNA or a plasmid DNA construct into the cellular genome to permanently silence target genes. Additional biological mechanisms for the regulation of this gene silencing can also be incorporated into the plasmid construct to only silence genes upon induction by chemical or environmental events (for example, Tetracycline-induced stable cell lines).
Although only a few of the transfected cells will actually incorporate the foreign nucleic acids into their genome, co-transfection of marker genes expressing antibiotic resistance genes allows drug selection of stable knockdown cells. Those cells that gain the ability to proliferate in the presence of an antibiotic selecting agent (such as Geneticin) can then be isolated and characterized. The development of stable cell line is an important gene expression research tool that provides valuable information on gene function, pathway signaling, and long-term silencing effects, however generation of stable RNAi -expressing cell line is a long and complex process.
Standard service includes plasmid DNAs encoding antibiotic-resistant gene, synthesis and cloning of shRNA constructs targeting the gene of interest into plasmid DNA, plasmid DNA amplification and purification, stable transfection of plasmid DNAs into the cell line of choice, drug selection of clonal cells, colony isolation, characterization of clonal cell lines, validation of shRNA construct expression and RNAi gene silencing by real-time qRT-PCR (mRNA expression) and/or Western blot (protein expression).
Altogen Labs offers in vitro and in vivo transfection services our expertise and extensive experience to develop stable cell lines in as little as 28 days (read more about Development of Stable Cell Line in 28 Days). Assistance with experimental design, cell line choice and synthesis of shRNA constructs is also available.
Once we know the details of your project, we can provide you an immediate price quote (contact e-mail: firstname.lastname@example.org or call Altogen Labs technical support at 512-433-6177). Please note that experimental details will help us to provide an accurate quote and timeline estimate.