Evaluation of combinatorial cis-regulatory elements for stable gene expression in chicken cells

Area of Science:

• Avian biotechnology
• Molecular biology
• Genetics

Background:

• Bird embryos offer unique biotechnological potential due to manipulability and simple egg proteins.
• However, transgene expression is often unstable, affected by factors influencing gene expression and leading to silencing.
• Previous studies have not comprehensively evaluated multiple cis-acting elements for sustained gene expression in chickens.

Purpose of the Study:

• To investigate optimal transcriptional regulatory elements for enhancing stable transgene expression in chicken cells.
• To analyze the efficacy of various cis-acting elements in sustaining transgene expression in chicken fibroblasts.

Main Methods:

• Generated eight constructs encoding enhanced green fluorescent protein (eGFP) with CMV or CAG promoters and regulatory elements (cMAR, cHS4, WPRE).
• Transformed immortalized chicken embryonic fibroblasts via electroporation and selected cells under G418.
• Analyzed mRNA levels (quantitative real-time PCR) and mean fluorescence intensity (flow cytometry).

Main Results:

• Construct size negatively correlated with copy number (R² = 0.701).
• CAG promoter showed 1.6-fold higher transcription than CMV (P = 0.027).
• Cis-acting elements (cMAR, cHS4) increased eGFP expression 2-3 fold and reduced gene silencing and expression variability (P < 0.0001).

Conclusions:

• An optimal combination of cis-acting elements and promoters/enhancers can sustain gene expression in chicken cells.
• These findings offer valuable insights for avian transgenesis and poultry gene function studies.