Cancer Cell Lines and Primary Cells
Primary cell cultures are used in biological and gene therapy studies and serve as important model systems that may more accurately represent the biology of normal cells. Many cultured cell lines, as well as the majority of primary cell cultures are able to be transfected with exogenous nucleic acids when appropriate transfection approaches are employed. Since the majority of transfection methods causes significant toxicity in primary cell cultures, optimizing this procedure, specifically the protocol and reagents to be utilized, is essential for developing an effective transfection strategies for a given cell type. ALTOGEN® Kits for primary cells and sensitive cell lines have been designed to have significantly lower cytotoxicity than classical alternatives.

siRNA Transfection
Gene silencing by RNA Interference (RNAi) is a powerful research tool for studying gene function in mammalian cells. RNAi is a biological phenomenon by which double stranded RNA (dsRNA) specifically reduces gene expression of its corresponding gene. Potent inhibition of specific gene expression is experimentally achieved by the transfection of small interfering RNA (siRNA), which binds RISC complex and cause degradation of target complementary mRNA molecules in the cell. Therefore, successful, potent RNAi experimentation is dependent upon the highly efficient delivery of the siRNA into cells by transfection of stable and functional siRNA molecules.
Most DNA transfection reagents are incompatible with siRNA, sensitive to serum, or are capable of inducing cytotoxicity, which negatively affects gene expression studies. ALTOGEN® Transfection Reagents have been developed and optimized specifically for use with siRNA transfection and allow transfection in the presence of serum without lowering transfection efficiency or reducing cell viability. Ready-to-run transfection protocols eliminate the need for extensive siRNA optimization experiments. Altogen Biosystems kits are also compatible for transfection of microRNA (miRNA) molecules.

Gene Silencing (RNAi) in Neuronal Cells
A number of studies have demonstrated the successful application of RNAi in primary cell cultures and non-neuronal cell lines; however the use of gene silencing in neuronal cell types has been notoriously difficult because of several technical and biological limitations. Neurons have been considered the most resistant to RNAi. Methods to consider when performing nucleic acid delivery experiments with neuronal cultures are: electroporation, microinjection, viral-based methods, and certain chemical transfection reagents. Although physical delivery methods appear to be efficient for siRNA-induced studies where transient modification of gene expression is sufficient, these methods do not appear to be adequate for behavioral studies where long term alteration of gene expression may be necessary. The viral-based siRNA expression systems appear to be most applicable for both Central Nervous System (CNS) gene therapy and basic neurobiological studies, while adeno-associated virus (AAV) delivery systems enable long-term, stable gene expression with relatively little cytotoxicity. Lentiviral vectors have been reportedly used successfully for the delivery of short hairpin RNA (shRNA), a precursor of siRNA, into primary neurons to induce RNAi. The advantage of lentiviral system is the ability to transfect non-dividing cells.

In Vivo Transfection Reagents from Altogen Biosystems
RNAi has been used for in vivo target validation studies using animal models. The major challenge in performing RNAi studies in vivo is the effective, directed delivery of functional siRNA, shRNA, and miRNA molecules into specific tissues. ALTOGEN® In Vivo Transfection Reagents could be conjugated with siRNA and administered intratumorally (i.t) or systemically via intravenous (i.v) tail vein injection in order to provide directed gene silencing in specific tissues, including liver, pancreas, kidney, and tumors. Selective knockdown could be seen as early as 24 hours after injection.

Transfection Resource

siRNA Transfection || Transfection Methods || Stable Transfection || RNAi || siRNA Library Screening || RNAi Therapeutics || Cell Transfection || In Vivo Transfection Reagents || Cell Line Specific Transfection Reagents

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In Vivo Transfection Reagents || Fibroblast Cells Transfection Reagent || Astrocyte Cells Transfection Reagent || HEK-293 Transfection Reagent || MEF Transfection Reagent || HepG2 Transfection Reagent || CHO Reagent || MDA-MB Transfection Reagent || Cos7 Transfection Reagent || A549 Transfection Reagent || NIH3T3 Reagent || HUVEC Transfection Reagent || LNCaP Transfection Reagent || MCF-7 Reagent || PC-12 Reagent

Altogen Research Services

Altogen Custom Services provide specialized biotechnology and pharmaceutical services, including - generation of stable cell lines, RNA Interference (RNAi) services, assay development, screening and transfection services. Generation of stably-expressing cancer cell lines and primary cells can be very expensive and time-consuming. Altogen Services offers generation of stable cell lines by transforming the cell line of choice to stably express gene of interest or shRNA-encoding construct. Cloning and shRNA expression services are available.