FIG. 4.

Imprinting analyses of transgenes RAA-21 and RAA-14. (A) Methylation analyses of the Air promoter. A Southern blot is shown of genomic spleen DNA digested with EcoRV (− lanes) or EcoRV and MluI (+ lanes) and hybridized with probe MS detecting both endogenous (wt) and transgenic (tg) Air fragments (sizes in kilobases are indicated). (B) Methylation analyses of the Aprt promoter. A Southern blot is shown of spleen DNA cut with NcoI (− lanes) or NcoI and SmaI (+ lanes) and hybridized with probe SE, which detects both endogenous (wt) and transgenic (tg) Aprt fragments (sizes in kilobases are indicated). (C) Expression of the Aprt and Air promoters by Northern blot analyses of adult cardiac tissue total RNA. Probe PS consists of Aprt exon 1 and 2 (top) and detects both endogenous and transgenic Aprt RNA and transgenic Air RNA due to the reverse orientation of the Air promoter (see Fig. 1). The signal from transgenic Aprt is not visible at this exposure with this short probe. Probe DE consists of Aprt exon 3 to 5 (middle) and detects endogenous and transgenic Aprt RNA as indistinguishable bands. Transgenic Aprt expression from line RAA-14 was analyzed in an Aprt^−/− background. Gapdh was used as a loading control (bottom). Lanes: p and m, paternal and maternal transmission of the transgene, respectively; wt, nontransgenic sample.