The 91-kb murine PU.1 genomic clone directs high-level reporter gene expression in stably transfected cell lines.

(A) Northern blot analysis of Thy1 mRNA in stable cell-line transfectants harboring the murine 91-kb PU.1 P1 clone. The 91-kb P1 DNA was linearized and stable transfectants established in myeloid U937 cells. The blot was sequentially probed with murine Thy1 cDNA (top) and GAPDH (bottom). Numbers at the top of the blot indicate clone number (lanes 1-10) as well as the negative control U937 cells (lane 11) and positive control BW5147 T-cell line (lane 12). Lanes 13 to 18 represent a limiting exposure of the left-hand blot (lanes 7-12). The position of migration of 18S ribosomal RNA is shown on the left, and the arrow on the right indicates the position of endogenous Thy1.1 mRNA in BW5147 T cells. (B) Sequence of the cryptic splice resulting in loss of Thy1.1 protein expression. Flanking primers were used to derive cDNA from the region spanning PU.1 exon 4, the IRES, and Thy1.1 cDNA (Figure1A). Shown in the upper line is the sequence of the PU.1-Thy1.1-P1 plasmid, and below it is the actual sequence derived from mRNA from stably transfected U937 cells. Alignment of the 2 sequences demonstrated a gap in the cDNA in which the IRES and 5′ end of the Thy1.1 cDNA (including the start ATG) are missing. Also shown in bold and underlined are nucleotides presumptively serving as the signals for the cryptic splice donor (GT) and acceptor (AG).