BY4741 (his3Δ1, leu2Δ0, ura3Δ0, met15Δ0) either transformed with one or all four plasmids, respectively, for either the four non-fluorescent or fluorescent vectors, were cultured in 25 mL of YPD media for 48 hr then plated at 1 100,000 dilution on YPD solid media.
Fluorescence signals were corrected for background autofluorescence measured in cells transfected with an empty non-fluorescent vector.
Enhanced cyan fluorescent protein vector (ECFP-C1) was obtained from Clontech (CA, USA).
Bonano et al. constructed another fluorescent reporter vector by using the genomic fragment around exon 8, and they visualized the regulation of exon 8 inclusion in the mouse [32].
A fluorescent Tax vector was constructed that allows the identification of Tax-expressing HeLa/Fucci2 cells.
The PCR product was digested and ligated into pEGFP (plasmid enhanced green fluorescent protein) vector (Clontech, Mountain View, CA, USA) to produce RANK-c fused to green fluorescent protein (GFP).
The enhanced fluorescent protein vectors (pECFP-C1 and pEYFP-C1) were purchased from Clontech.
To compare dual and single transfectants, an additional 1 µg control non-fluorescent empty vecontrol non-fluorescentch singlemptynsfection reaction.
Newman et al. generated fluorescent reporter vectors with the minimum genomic region that can reflect endogenous splicing regulation in AT-3 and DT-3 cells.
Although the experiments shown were performed using H9 cells (WiCell WA09), similar outcomes were obtained with the PGK-H2B fluorescent protein vectors, Rex- and αMHC-driven fluorescent and selectable markers using HUES13 (Harvard) and human induced pluripotent stem cells (hIPSCs) derived in our laboratory.
