EGFP and HiExpress™ Luciferase IVT mRNA
VectorBuilder offers off-the-shelf and ready-to-use EGFP and luciferase mRNA for in vitro and in vivo experiments. Our mRNA quality and efficacy have been fully validated for high levels of reporter expression in both cell culture and animal models, thus, it can be used to assess the efficiency of mRNA delivery and expression or used as control for your mRNA experiment. For custom mRNA production and delivery, check out VectorBuilder’s mRNA Gene Delivery Solutions.
- mRNA produced with Cap 1, validated 5’ & 3’ UTRs, a 110 nt polyA tail, and can be incorporated with modified nucleotides, such as N1-Methylpseudouridine (m1Ψ)
- Codon optimized HiExpressTM Firefly Luciferase mRNA for achieving robust expression
- Comprehensive quality control including sequence validation, integrity, purity, and expression validation
Shipping and storage
Our mRNA products are shipped on dry ice in a 1 mM sodium citrate buffer (pH 6.4). mRNA can be stored at -80°C for up to 12 months. Avoid freeze-thaw cycles.
Workflow of mRNA production process and QC
Figure 1. Typical workflow of RNA synthesis by in vitro transcription.
Our mRNA production workflow starts with designing and synthesis of the template DNA sequence with the consideration of preferred codons, GC content, and thermodynamic stability of RNA secondary structures, followed by its cloning into an in vitro transcription vector. The plasmid DNA is then purified, validated, and linearized before being subsequently subjected to the in vitro transcription reaction which results in the generation of the desired transcript. Modified nucleotides can be incorporated into the in vitro transcription reaction to improve in vivo translation and decrease immunogenicity. Highly efficient capping (>95%) can be achieved either using co-transcriptional or enzymatic approaches. The mRNA is then purified by mRNA-capture beads followed by additional quality control.
Figure 2. EGFP IVT mRNA expression in HeLa cells. The EGFP mRNA was generated with or without modified nucleotide, N1-Methylpseudouridine (m1Ψ). HeLa cells grown on a 12-well plate were transfected with 1 ug of mRNA per well. (A) EGFP expression at 24 h, 48 h, and 72 h post-transfection quantified by flow cytometry. Mean fluorescence intensities (MFI) are represented by colored bars and percentages of EGFP positive cells are represented by circles, squares, and triangles. Error bars indicate standard deviations (SD). (B) Representative images of HeLa cells transfected with EGFP mRNA at 48 h post-transfection.
Figure 3. HiExpressTM Firefly Luciferase IVT mRNA expression in vitro and in vivo. (A) Expression of HiExpressTM Firefly Luciferase mRNA and other luciferase mRNA in HEK293T cells. Cells grown on a 12-well plate were transfected with 0.5 ug of mRNA per well and luciferase activity was measured at 6 h, 24 h, 48 h, and 72 h post transfection. (B) Luciferase activity measured in adult C57BL/6 mice injected intramuscularly with 30 ug of LNP encapsulated mRNA at 6 h, 24 h, and 48 h post injection. FLuc WT indicates wild-type firefly luciferase. FLuc WT (co) indicates codon-optimized wild-type firefly luciferase. FLuc2 indicates Luc2 firefly luciferase.
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What are the differences between mRNA caps and capping methods?
Cap 0 refers to N7-methylguanosine (m7G) that is added to the 5’ end of eukaryotic mRNAs via a 5’ to 5’ triphosphate linkage. This modification is added via a series of enzymatic reactions that occur co-transcriptionally and functions to regulate nuclear export, transcript stability, and promotes translation of the mRNA through recognition by eukaryotic translation initiation factor (eIF4E). Cap 1 refers to the addition of a methyl group to the 2’O on the first nucleotide (m7GpppNm) of the transcribed mRNA sequence in addition to the m7G cap. In mammalian cells, cap 1 structure is an important marker for mRNA to be recognized as self and not targeted by innate immunity. Adding cap 1 structure to synthesized mRNA has been demonstrated to enhance mRNA expression in vivo and reduced its immunogenicity.
Capping for in vitro transcribed RNA can occur either co-transcriptionally with cap analogs or post-transcriptionally via enzymatic reactions. The efficiency of our capping method has been well validated using LC-MS.
Why should I consider incorporating modified nucleotides in mRNA and which ones can be included?
Cells contain cytosolic and endosomal RNA receptors that activate the immune response upon recognition of foreign RNA. Modified nucleotides are commonly found in endogenous cellular RNA. Incorporating certain modified nucleotides in mRNA reduces its immunogenicity, alters secondary structure, and increases translation efficiency and half-life in a sequence-dependent manner. We provide the commonly used N1-Methylpseudouridine (m1Ψ). N1-Methylpseudouridine (m1Ψ) is a naturally occurring nucleotide that was first identified in tRNAs, however, its use in coding mRNAs has only recently been appreciated. This methylated derivative of uridine can replace the canonical nucleotide uridine in mRNA IVT and translation without altering traditional Watson-Crick base pairing. A major advantage of its use in mRNA therapeutics is its ability to alter recognition by RNA immune receptors thus mitigating unwanted immune effects and enhancing transcript stability and translation.