FIG. 4.

Expression and mRNP association of transgene mRNA from mouse line 6a. (A) Confocal fluorescent and bright-field microscopy of whole live seminiferous tubules. Due to the high expression of GFP protein in this line, very low excitation energy was used (about 3% of that used for line 1a in Fig. 2). Arrows are as in Fig. 2. (B) Relative levels of nascent GFP transcripts in nuclei from lines 1c and 6a. RNase protection assays were performed as described above on the indicated amounts of RNA isolated from whole testis (total) or purified nuclei (nuclear), using the internal GFP probe (upper two autoradiograms) or the GAPD-s probe (below). At the left are given the mouse line used and the identity of each protected fragment. The schematic at the bottom shows that GAPD-s pre-mRNA retaining intron 1 hybridizes to a 147-base region of the probe (dark hatched line below the RNAs); mRNA with exon 1 spliced onto exon 2 hybridizes to a 177-base region of the probe. To compare relative levels of spliced and unspliced transcripts, the radioactivity of each band was determined by liquid scintillation counting and was corrected for differences in the number of radiolabeled UTP residues in each protected fragment (31 and 49 for nonspliced and spliced, respectively). By assuming equal hybridization efficiency, we calculate that 43% of the GAPD-s transcripts in testis nuclei have not yet removed intron 1. (C) Velocity sedimentation analysis of GFP transgene mRNA and endogenous Prm1 mRNA. Assays were as in Fig. 3 except that for detecting GFP mRNA, the internal probe was used.