Detection of Enhancer Activity of the Spermatogenic Gene
The enhancer activity of a spermatid-specific gene using a luciferase reporting system was studied to examine the possible application of an intratubular injection and in vivo EP as a new transient expression assay for spermatogenic specific genes. The Prm-1 gene encodes a sperm-specific chromosomal protein that replaces histone proteins during spermiogenesis. The well-defined enhancer element Prm-1 was monitored as a typical indicator of sper-matogenic stage-specific expression. buy flovent inhaler
The four luciferase reporting plasmids were separately injected into adult mice (8 wk old), and the luciferase activities were determined 18 h after EP. The results are summarized in Table 1. By setting the luciferase activity of the promoterless vector (Basic-luc) injection at 1.0, the injections of the SV40-enhancer vector (SV40 E/P-luc) and the enhancerless vector (SV40P-luc) resulted in 44.3-fold and 5.2-fold increases in the luciferase activity, respectively, meaning that the SV40 enhancer itself showed about 8.5-fold enhancing activity. Transfection of PrmE/SV40P-luc driven by the Prm-1 enhancer resulted in a 10.0-fold increase, equivalent to about 1.9-fold compared with that of the enhancerless vector (SV40P-luc) and 22% of the enhancing activity of the SV40-enhancer vector.
TABLE 1. Transient expression assay of luciferase reporting transgenes (means ± SD).
|Corrected valuec||Relative fold (X)d|
|SV40 E/P-luc||677.6 ± 194.0||44.3|
|Prm E/SV40 P-luc||153.7 ± 54.8||10.0|
|SV40 P-luc||79.8 ± 11.0||5.2|
|Basic-luc||15.3 ± 2.4||1.0|
a Each plasmid construct was coinjected with pRL-CMV (internal control). b Results from three independent experiments.
c The ratio of luciferase activity divided by internal control activity is indicated as the corrected value (X10~3).
d Values are calculated based on a value of Basic-luc set at 1.0.