Exogenous Gene Expression
Different approaches have been developed to over-express or ectopically express a protein in cells: peptide or full length recombinant protein transfer, viral gene transfer, non-viral DNA transfer and non-viral mRNA transfer. Non-viral mRNA transfer has been around for a long time, but it is not widely used because the expression is transient and generating large amount of mRNA is considered difficult. More recently, the benefits of transient expression, particularly without leaving a footprint in the host genome, have become recognized in many areas of study.
mRNA-mediated ectopic gene expression made a big splash recently through its use for iPSC reprogramming. iPSC factor mRNA enhances iPSC induction efficiency and completely avoids viral integration. This has tremendous implications for clinical use of iPSCs as compared to current methods, because any method involving DNA (e.g. non-integrating lentivirus or minicircle plasmids) presents the risk of random integration by partial recombination. In addition, because mRNA transfection does not require nuclear entry, it is generally easy to do and is not cell cycle-dependent.
RNA can be produced by in vitro transcription (IVT), a simple reaction requiring only a DNA template (double-stranded, or even single-stranded DNA with a double-stranded promoter region), RNA polymerase (from T7, SP6, or T3 phage), NTPs, and a reaction buffer that provides appropriate salt and pH. Using a high quality template; a single 20-50 µl reaction can routinely produce 40-50 µg of mRNA as a single band on an agarose gel from 1 µg of DNA template. At such high concentrations, IVT mRNAs are not nearly as sensitive to RNase-mediated degradation as low-abundance samples.
mRNA transfection has already been developed for use in several applications. Enzymes such as transposase and Cre recombinase have been commonly delivered in the form of mRNA. Other well-known examples of mRNA transfection include loading special cancer antigens or HIV antigens to dendritic cells (DCs) in vitro for personal immunotherapy. PSA antigen expressing DCs transfected by mRNA has moved on to Phase I Clinical Trials for this purpose.
These linear DNA templates are ready to use directly in IVT reactions. Within each template, the coding sequence for a protein is flanked by 5’ and 3’ untranslated regions designed to maximize the translational efficiency and cytoplasmic stability of the mRNA and validated in the iPSC application. The templates drive incorporation of 120-nt polyA tails into RNA transcripts, eliminating the need to enzymatically polyadenylate IVT products before use.
PCR templates to express functional proteins exogenously. Luciferase allows for the expression of the Luc gene for bioluminescence. Cre template allows for expression of recombinant Cre for the functional excision of DNA flanked by LoxP sites. To request additional functional enzymes (LacZ, Transposase, etc... please email email@example.com with your request).
PCR templates for fluorescent proteins offered exclusively by Allele Biotechnology. mWasabi is a truly monomeric green fluorescent protein (GFP) that is twofold brighter than eGFP. LanYFP is a tetrameric YFP derived from the lancelet species of fish, and is 10 times brighter than eGFP. Our entire line of fluorescent proteins will be offered in template form soon.
PCR templates that allow for exogenous expression or various transdifferentiation factors. We are aiming to carry templates for all known cell types (Neural, cardiomyocyte, adipocyte, etc) - to check our current offering click the link below. Factors that are linked to a product page are currently in stock, if you would like a factor that we don't have, please email firstname.lastname@example.org to request it. You may request factors that aren't on our list.
The NexGen iPSC Induction System supports footprint-free iPSC induction using mRNA transfection, a new reprogramming technology that’s faster and more efficient than conventional virus-based approaches. The kit includes in vitro transcription templates for large-scale synthesis of mRNA encoding six human reprogramming factors (Klf4, Myc, Oct4, Sox2, Lin28, and Nanog) and a fluorescent reporter.