There could be multiple reasons. Perhaps the best wat to start testing it would be to use another gene (a positive control), which was shown previously to be well expressed in mice.

• If this is successful, then IV injections, and all the donwstream steps are OK. You will need then to focus on your specific mRNA, checking first its in vivo stability, then continuing with other steps.

• If it (the control) is not successful, perhaps there is already a problem with IV injections.

Hm...

1. Cellular Regulation and Homeostasis

• Gene Regulation: In vivo systems have tight regulatory mechanisms (transcriptional, translational, and post-translational) that control protein production to maintain cellular balance and prevent toxicity.
• Feedback Inhibition: Cells often downregulate gene expression if excess protein production disturbs cellular homeostasis.

2. Proteolytic Degradation

• Proteases: In vivo systems have active protease enzymes that degrade misfolded or excess proteins to prevent aggregation or toxicity.
• Protein Stability: Proteins expressed in vivo may lack stabilizing modifications, making them more prone to degradation.

3. Limited Transcription and Translation Rates

• Promoter Strength: Natural promoters used in vivo are weaker than synthetic or inducible promoters often used in vitro systems.
• Codon Optimization: Codon usage may not be optimized for host cells, reducing translation efficiency.
• Ribosome Availability: Competition for ribosomes and other translational machinery slows protein synthesis.

4. Energy and Resource Constraints

• Cells in vivo must balance protein production with other metabolic demands, including growth, replication, and repair.
• Limited availability of amino acids, ATP, and cofactors may restrict protein synthesis rates.

5. Post-Translational Modifications

• In vivo systems often require complex post-translational modifications (e.g., glycosylation, phosphorylation) for protein function, which can slow down production.
• Incorrect folding or lack of chaperone assistance may trigger degradation rather than accumulation.

6. Immune System Constraints

• In multicellular organisms, excessive production of foreign or misfolded proteins may trigger immune responses that limit expression and induce degradation pathways.

7. Gene Delivery and Integration Challenges

• In vivo systems often require gene delivery methods (e.g., viral vectors or plasmids) that may result in low transfection efficiency compared to highly optimized in vitro systems.
• Gene silencing mechanisms (e.g., RNA interference) may further reduce expression levels.

While in vitro systems optimize expression conditions—providing abundant nutrients, high plasmid copy numbers, and inducible promoters—in vivo systems prioritize regulation, stability, and cellular balance, leading to lower protein yields but better control and biological relevance.