Considering that the population of round spermatid cells that specifically transcribe the Prm-1 gene is about 30% of all testicular cells, the relatively lower activity of the Prm-1 enhancer appeared to be mainly due to the difference in the ratio of cells expressing each transgene. Moreover, when immature mouse testes (18-20 days after birth), which rarely have haploid cells, were used for the same experiments, the Prm-1 enhancer vector showed a small enhancing activity of about 0.9-fold compared with that of the SV40-enhancer vector. These results indicate that the transcriptional effect of the spermatogenic stage-specific Prm-1 enhancer is transiently detectable by this EP transfection method. buy yasmin online
Figure 2A shows the p-gal staining of testes prepared two days after intratubular injection of Prm-lacZ plasmid. Although p-gal-positive tubules were interspersed throughout the testis, sections of the positive tubules showed that the p-gal-positive cells were detected only in the innermost layer of the seminiferous tubules, and the cells were in the elongated-spermatid stage (Fig. 2, B and C). Neither Sertoli cells nor spermatogenic germ cells of other stages exhibited any p-gal staining. This expression pattern of the transfected Prm-lacZ was identical with the expression of the endogenous Prm-1 gene.
Long-Lasting Expression of the Transgene
One interesting aspect of this in vivo gene transfer technique is the possibility of tracing the developmental behavior of the transfected cells by detecting transgene expression as a long-lasting marker. When the testes at 4 wk after transfection of CMV-lacZ were examined with p-gal staining, a small number of p-gal-expressing cells were found to be scattered throughout the entire testis.
FIG. 2. Spermatid-specific expression of the Prm-lacZ transgene. Nuclear-targeted p-gal is driven by the mouse Protamine-1 enhancer. A) Testis at 48 h after in vivo gene transfer. Several parts of the seminiferous tubules are stained. Bar = 500 ^m. B) Cross section (8 ^m) of the p-gal-positive tubules. The p-gal expression is concentrated on the lumen side of the seminiferous tubule. Bar = 100 ^m. C) A higher magnification of B. Nuclear localized p-gal expression is detected in the nuclei of the numerous elongating spermatids. Bar = 20 ^m.