BAC transgenic expression efficiency: bicistronic versus ATG-fusion strategies

Bacterial artificial chromosome (BAC)-based transgene can be expressed bicistronically with the target gene or fused to its translation start codon. To compare the transgene expression efficiencies of these two methods, mice were created that expressed green fluorescent protein (GFP) from the genomic locus of nucleostemin using the bicistronic (NSiGFP) or the ATG-fusion approach (NSmGFP). Three lines with 1, 2, and 4 copies of the NSiGFP transgene, and two lines with 1 copy of the NSmGFP transgene were generated. Of the three NSiGFP lines, only the 4-copy line can match the NSmGFP lines in their GFP protein expression levels. Analyses of the GFP and nucleostemin RNA transcript levels exclude IRES inefficiency and suggest premature termination of the bicistronic message as the cause for low GFP expression in the NSiGFP mice. This work provides important information for designing BAC transgenics when the transgene expression level is crucial.