mRNA has become an emerging class of gene therapy because of its negligible cytotoxicity and immunogenicity. So, there is an increasing demand for efficient and robust production of mRNA molecules in both pharmaceutics and research field. In vitro transcription (IVT) is an elegant way to obtain highly pure and uniform mRNA oligonucleotides of lengths ranging from about 15 to several thousand nucleotides. It is transcribed from a double-stranded DNA template by RNA polymerase (T7, T3 or SP6 RNA polymerase). Creative Biolabs is a leading service provider in custom mRNA IVT synthesis for basic research and clinical applications. We provide best-fit in vitro transcription vectors that are simple and efficient systems for RNA synthesis, applicable for a variety of research purposes.
IVT has received increasing attention as a powerful alternative genetic material by exogenous delivery for protein expression. Compared with conventional pDNA expression, IVT mRNA-based protein expression has several advantages: 1) mRNA can directly encode the protein through a single translation step; 2) mRNA has extremely high protein expression comparable to viral systems; 3) rapid onset time for the expression of target proteins; 4) mRNA is a non-viral system that relieves some safety concerns; 5) plasmid-based template by IVT meets the synthetic demands for long RNA (up to thousands of bases).
Transcription of RNA is started with the appropriate choice of IVT vector that contains promoter sequence (T3, T7 or SP6) for generating high yields of transcript, appropriate restriction sites for cloning of the gene of interest (GOI), 5' UTR, 3' UTR, and/or a polyA tail. Typically, T7 is a standard and well-described polymerase used to transcribe a linearized DNA template into RNA. Therefore, it is often necessary to optimize the structural elements in the IVT vectors for stable expression of target proteins.
Effective in vitro transcription starts with a high-quality template. We have constructed a range of novel IVT vectors containing functional T3, T7 or SP6 RNA polymerase promoters and different restriction sites to meet different demands. Our vectors are simple and efficient systems for mRNA IVT synthesis, biochemical studies, protein expression, and other applications requiring mRNAs of defined sequences. Usually, we utilize a T7 promoter upstream of your transgene, facilitating the highly efficient production of mRNA by T7 bacteriophage RNA polymerase (T7 RNAP).
Fig.1 In vitro Transcription Vector.
Usually, a fully dsDNA template often increases transcription yield. We provide different types of DNA templates for in vitro transcription, including the DNA plasmid, PCR-product, synthetic short oligo and cDNA. The DNA templates can be designed and synthesized according to the sequence of interest.
Fig.2 DNA templates for IVT.
7-methylguanosine capped mRNA transcripts (5' end) are essential for RNA stability, efficient translation, nuclear transport, and splicing. RNA capping can be carried out either co-transcriptionally by using cap analogs (ARCA and anti-reverse cap analog), or post-transcriptionally by using capping enzymes.
Poly(A) tails can be added after transcription using poly(A) polymerase and ATP. Alternatively, poly(A) stretches in transcription templates can be encoded in plasmid templates or added during PCR-based generation of transcription templates.
As one of the leading biotechnology companies, Creative Biolabs is dedicated to providing ideally suited IVT vectors and professional IVT mRNA synthesis services. If you are interested in our products, please feel free to contact us.
