Traditionally, the delivery of Cre recombinase has relied on viral vectors or plasmid DNA (pDNA); however, these methods often face challenges such as genomic integration risks, prolonged expression leading to off-target toxicity, and low transfection efficiency in non-dividing cells. CD BioGlyco offers a cutting-edge solution through our Cre mRNA synthesis service, providing high-purity, messenger RNA (mRNA) encoding Cre recombinase.
By delivering Cre as an mRNA transcript, researchers achieve rapid and transient protein expression directly in the cytoplasm. This "hit-and-run" approach ensures that the recombinase is only present long enough to perform the desired genetic modification, significantly enhancing safety and specificity. Whether you are developing complex animal models or engineering sophisticated cell lines, our specialized synthesis platform delivers tailored Cre mRNA optimized for maximum biological activity and minimal immunogenicity.
We utilize high-fidelity RNA polymerases and optimized DNA templates to produce full-length Cre mRNA. This process includes the fusion of a nuclear localization sequence (NLS) to the Cre coding region, ensuring the expressed protein is efficiently trafficked to the nucleus, where the genomic reside.
To bypass the cellular innate immune response, we offer the incorporation of modified nucleotides, such as N1-methylpseudouridine or 5-methoxyuridine (5moU). These modifications reduce the activation of Toll-like receptors (TLRs), thereby increasing the half-life and translation yield of the mRNA in sensitive primary cells and in vivo environments.
Our synthesis includes sophisticated co-transcriptional capping (Cap 1) and enzymatic polyadenylation. A stable 5' cap and a long poly(A) tail are critical for protecting the mRNA from exonuclease degradation and ensuring robust recognition by the ribosome.
As a vital component of our therapeutic nucleic acid development platform and, more specifically, our therapeutic oligonucleotide synthesis service, CD BioGlyco provides end-to-end solutions for Cre mRNA production. Our service is engineered to address the critical pain points of traditional gene-editing tools, such as delivery challenges and expression kinetics. We don't just provide standardized Cre mRNA; we deliver highly customized synthesis strategies tailored to your specific application, whether it involves complex in vivo tissue targeting or transfection into immune-sensitive primary cells.
We begin by optimizing the Cre recombinase sequence, incorporating NLS tags and species-specific codons. This step ensures that the mRNA is not only stable but also produces a highly functional protein upon delivery.
A high-quality plasmid DNA (pDNA) template containing a T7 promoter, the Cre gene, and optimized untranslated regions (UTRs) is constructed. The pDNA is then linearized and purified to serve as a precise template for transcription.
Using our optimized IVT system, the DNA template is transcribed into RNA. During this phase, modified nucleotides are incorporated if requested, ensuring the resulting transcript meets the client's specific immunogenicity requirements.
The mRNA undergoes 5' capping (typically Cap 1) and 3' polyadenylation. These modifications are essential for mimicking endogenous mature mRNA, enhancing its stability and translation efficiency within the target cells.
We employ advanced purification techniques, including tangential flow filtration (TFF) and high-performance liquid chromatography (HPLC), to remove double-stranded RNA (dsRNA), DNA templates, and enzymatic byproducts.
Every batch undergoes stringent QC testing, including capillary electrophoresis for integrity, Nanodrop for concentration, and limulus amoebocyte lysate (LAL) assays for endotoxin levels. We guarantee high-purity, bioactive mRNA ready for immediate use.
DoI: 10.3390/ijms22084050
Journal: International Journal of Molecular Sciences
IF: 4.9
Published: 2021
Results: This study investigates whether extracellular vesicles (EVs) transfer Cre mRNA between endothelial cells (EC) and adipose-derived stromal/stem cells (ASC) during vascular network formation, using an adapted Cre-loxP system. Functional controls confirmed the Cre construct and reporter system worked, but neither supernatant transfer from Cre+ cells nor direct coculture induced Cre-mediated recombination in reporter cells. PCR analysis showed Cre mRNA was expressed in Cre+ cells but undetectable in their EVs. TNF-α stimulation of human umbilical vein endothelial cell (HUVEC) did not promote Cre mRNA loading into EVs or recombination. EV characterization via non-template control (NTC) and FT-FC revealed successful enrichment and cell-type-specific release patterns, but no functional Cre mRNA transfer. The findings indicate the Cre-loxP system is unsuitable for studying EV-mediated communication in this in vitro coculture model, highlighting the need for optimized cargo loading strategies.
Fig.1 Scheme of the strategy applying the adapted Cre-loxP system for EV uptake evaluat. (Schneider, et al., 2021)
Conditional Gene Knockout/Knock-in
Cre mRNA is ideal for inducing spatial or temporal gene deletion in animal models or cell lines. Its transient nature prevents the long-term presence of the recombinase, reducing off-target genomic damage.
Cell Line Engineering
Researchers use Cre mRNA to excise selection markers or activate reporter genes in complex cellular systems. This approach is highly effective in hard-to-transfect primary cells where traditional DNA-based methods often fail.
Lineage Tracing and Fate Mapping
In developmental biology, the transient delivery of Cre allows for precise "pulse-labeling" of cell populations. This enables scientists to track cell progeny over time without the confounding effects of continuous Cre expression.
Therapeutic Proof-of-Concept
Cre mRNA serves as a powerful tool in gene therapy research, allowing for the validation of site-specific recombination strategies in disease models before transitioning to complex therapeutic candidates.
High Recombination Efficiency
By incorporating optimized NLS sequences and utilizing high-purity transcripts, CD BioGlyco's Cre mRNA achieves superior recombination rates even in non-dividing and difficult-to-transfect cell types.
Reduced Immunogenicity
Through the use of modified nucleotides like N1-methylpseudouridine, our mRNA minimizes the induction of the cellular innate immune response, allowing for higher dosing and better cell viability during experiments.
Precise Dose Control
The relationship between mRNA concentration and protein expression is highly linear. This allows researchers to fine-tune the amount of Cre recombinase produced, optimizing the balance between recombination efficiency and cellular health.
Rapid Development Cycles
Our synthesis process is significantly faster than generating viral lots or high-quality plasmids. This enables researchers to move from sequence design to experimental results in a fraction of the time.
"The Cre mRNA from CD BioGlyco showed exceptional efficiency in our primary neuronal cultures. Unlike the viral vectors we used previously, we saw zero cytopathic effects, and the recombination was complete within 24 hours."
– J.R., Senior Researcher
"We ordered a large batch of N1-methylpseudouridine-modified Cre mRNA for a series of mouse embryo injections. The purity was outstanding, and the results were highly reproducible across multiple litters."
– D.G., Principal Investigator
"The turnaround time was impressive. We received our custom-optimized Cre mRNA in less than four weeks, allowing us to stay ahead of our project milestones. The quality control provided gave us total confidence in the integrity of the product."
– A.P., Lead Scientist
CD BioGlyco is dedicated to empowering the scientific community with high-performance Cre mRNA synthesis services. By combining advanced IVT technology, precise chemical modifications, and rigorous purification, we provide the tools necessary for safe, efficient, and precise genetic recombination. Our expertise in the therapeutic oligonucleotide synthesis service ensures that your project benefits from the highest industrial standards and deep scientific insight. Please feel free to contact us to discuss your sequence design, scale requirements, and delivery strategies.
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