2A self-cleaving peptide-based multi-gene expression system in the silkworm Bombyx mori

Area of Science:

• * Functional genomics
• * Transgenic silkworm technology

Background:

• * Silkworm research is advancing into the functional genomics era.
• * Simultaneous expression of multiple genes is crucial for complex biological functions and material production.

Purpose of the Study:

• * To develop and validate a multi-gene expression system (MGES) for transgenic silkworms.
• * To optimize the efficiency of 2A self-cleaving peptides for gene expression and cleavage.

Main Methods:

• * Engineered a multi-gene expression system utilizing 2A self-cleaving peptides with glycine-serine-glycine (GSG) spacers.
• * Tested and compared the cleavage efficiency of six 2A variants, identifying the porcine teschovirus-1 2A (P2A-GSG) as optimal.
• * Validated P2A-GSG in transgenic silkworms for expressing fusion proteins and secreting functional silk materials.

Main Results:

• * The glycine-serine-glycine spacer significantly enhanced 2A cleavage efficiency.
• * Porcine teschovirus-1 2A with GSG (P2A-GSG) showed the highest cleavage efficiency across tested insect cell lines.
• * P2A-GSG successfully mediated simultaneous expression and secretion of fusion proteins (e.g., human serum albumin, silkworm vitellogenin receptor fragment, DsRed, EGFP) in silkworm silk glands and cocoons.

Conclusions:

• * The developed MGES, particularly using P2A-GSG, is an efficient tool for simultaneous gene expression in silkworms.
• * This system facilitates gene function research and the creation of novel functional silk materials for medicine and cosmetics.