Case Study

• Poly(A) Tail Length Impacts Protein Expression

  Hereby, we demonstrate the positive correlation of poly(A) length and protein expression by adding different lengths of adenine ribonucleotides to the pUC57-eGFP plasmid. When the Poly(A) tail length increased from 50 to 100 adenine ribonucleotides, there was a twofold surge in GFP expression.

  

  Figure 1. Correlation between Poly(A) tail length and protein expression. eGFP encoding mRNA with various poly(A) tail length was generated. 293T cells were transfected with 0.1 µg of the mRNA using the Lipofectamine MessengerMAX reagent (Invitrogen). GFP expression was observed 48 hours after transfection. The results highlight the pivotal role of the Poly(A) tail length in fine-tuning protein expression according to specific needs and preferences.

• GenScript Poly(A) Strains Offer Superior Perseverance of Poly(A) Sequence

  Choosing the right bacterial strain for the propagation of plasmids with a Poly(A) tail is crucial to maintain its integrity. Different bacterial strains have distinct levels of exonuclease activity, which can profoundly affect the stability of the Poly(A) tail in the plasmid DNA. Recognizing this challenge, GenScript introduced the GenScript Poly(A) Strains V2 and V3. These strains are tailor-made to effectively preserve and propagate plasmids embedded with poly(A) sequences.

  GenScript Poly(A) Strain Provides Superior Poly(A) Stability

  

  

  Figure 1. GenScript Poly(A) Strains underwent rigorous testing to evaluate their capability in preserving the fidelity and stability of Poly(A) Plasmids. We examined six plasmids, each with a Poly(A) tail ranging between 100-120 adenines. These plasmids were propagated in both GenScript Poly(A) Strains and the NEB Stable strain. For each plasmid, 8 clones were selected to assess the integrity of the Poly(A) sequence in both the 1st and 3rd generations. The results are presented in a graph that displays the percentage of clones that met the QC standards set by GenScript's Poly(A) Guarantee Service.

• GenScript Poly(A) Strain Capable of Refining Poly(A) Sequences

  GenScript Poly(A) Strain Capable of Refining Poly(A) Sequences

  

  Figure 1. An mRNA template plasmid possessing a Poly(A) tail of 110 adenines was introduced into the GenScript Poly(A) Strain V3 for growth and replication. Subsequent Sanger Sequencing was employed to validate the poly(A) sequences. The resulting chromatogram from the sequencing revealed that after growth in the GenScript Poly(A) Strain V3 , the "noise" (baseline) peaks were significantly diminished, highlighting the capacity of the GenScript Poly(A) Strain V3 to enhance the clarity of plasmids with elongated poly(A) sequences.

Resources

Application Note:
Overcoming Traditional Limitations: GenScript’s Advanced mRNA Applied Vector for Superior Cloning and Efficient Plasmid Production

In response to the growing demand for mRNA-based therapeutics, we have engineered the GS-mK and GS-CMV vectors to address the limitations of traditional vectors such as pUC57 and pVAX1.

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Application Note:
GenScript Poly(A) Strain: Overcoming Challenges in Pertaining Long Poly(A) Tail Sequence Integrity

To address the challenges associated with maintaining and prorogating plasmids containing poly(A) sequences, GenScript have developed a suite of exclusive Poly(A) strains.

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Poster:
Enhancing Poly(A) Tail Stability and Plasmid Yield: GenScript's Innovative Vector and Strain Solutions for mRNA Production

Correlation Between Poly(A) Tail Length and Protein Expression. Methods to Enhance Poly(A) Tail Stability During Plasmid Propagation

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