Efficient and Reproducible Multigene Expression after Single-Step Transfection Using Improved BAC Transgenesis and...

Area of Science:

• Mammalian cell engineering
• Gene expression regulation
• Biotechnology

Background:

• Stable expression of single transgenes in mammalian cells is difficult.
• Simultaneous stable expression of multiple transgenes at reproducible levels is even more challenging.
• Previous BAC TG-EMBED approach achieved copy-number-dependent, chromosome-position-independent expression.

Purpose of the Study:

• To extend the BAC TG-EMBED approach for improved multi-gene stable expression.
• To develop a toolkit for fine-tuning relative transgene expression levels.
• To create a more efficient method for assembling multiple transgenes into BACs.

Main Methods:

• Developed a toolkit of endogenous promoters for tunable transgene expression (0.01- to 5-fold range).
• Demonstrated expression stability of reporter genes in various BAC genomic regions.
• Introduced BAC-MAGIC, a novel, time-efficient BAC assembly scheme for multiple transgenes.

Main Results:

• Simultaneously labeled three nuclear compartments with reproducible relative intensity in 94% of stable clones.
• Achieved simultaneous expression of 4 transgenes using BAC-MAGIC and a DHFR BAC scaffold.
• Showed minimal variation in expression level and long-term stability regardless of BAC scaffold activity.

Conclusions:

• The extended BAC TG-EMBED toolkit offers precise control over relative expression levels.
• BAC-MAGIC significantly improves the efficiency of multi-gene BAC construction.
• The combined approach provides a versatile platform for stable, simultaneous multi-gene expression.