BAC transgenic expression efficiency: bicistronic versus ATG-fusion strategies

Area of Science:

• * Molecular Biology
• * Genetics
• * Genomics

Background:

• * Bacterial artificial chromosomes (BACs) are vital tools for generating transgenic organisms.
• * Transgene expression can be achieved through bicistronic methods or ATG-fusion to the target gene's start codon.
• * Optimizing transgene expression levels is critical for various research applications.

Purpose of the Study:

• * To compare the efficiency of bicistronic versus ATG-fusion methods for BAC transgene expression.
• * To evaluate green fluorescent protein (GFP) expression levels in mice engineered with different BAC constructs.
• * To identify the underlying mechanisms responsible for differences in transgene expression.

Main Methods:

• * Generation of mice expressing GFP from the nucleostemin locus using bicistronic (NSiGFP) or ATG-fusion (NSmGFP) BAC constructs.
• * Creation of multiple transgenic lines with varying copy numbers of the NSiGFP transgene (1, 2, and 4 copies) and NSmGFP transgene (1 copy).
• * Quantitative analysis of GFP protein and nucleostemin RNA transcript levels in different transgenic mouse lines.

Main Results:

• * Only the 4-copy NSiGFP line achieved GFP protein expression levels comparable to the NSmGFP lines.
• * Analysis of RNA transcripts ruled out Internal Ribosome Entry Site (IRES) inefficiency as a cause for lower GFP expression in bicistronic constructs.
• * Evidence suggests premature termination of the bicistronic message limits GFP expression.

Conclusions:

• * The ATG-fusion method demonstrates superior efficiency for achieving high transgene expression compared to the bicistronic approach in BAC transgenesis.
• * Premature transcript termination is identified as a key factor limiting expression in bicistronic BAC transgenes.
• * Findings offer crucial guidance for designing BAC transgenesis strategies where precise transgene expression levels are paramount.