The Establishment Of PRP Overexpression Transgenic MIC

Objective:Our preliminary experiments indicated that PPR is expressed in the testis of male mice and is implicated in the regulation of testosterone production in vitro. Howerver, its roles in male reproductive functions in vivo need to be further investigated. Thus, in this study, we aimed to establish PRP transgenic mice, which had a Leydig cell-specific overexpression of PRP, to facilitate studies into the effects of PRP on testicular function in mice,Methods:1. Construction of vectorTo generate transgenic mice with a Leydig cell-specific overexpression of PRP, a fragment of the LHR promoter was used. The transgenic construct contained the following components:the upstream promoter region of the LHR, FLAG tags, the complete open reading frame of PRP, and and the SV40 poly(A) sequence. Subsequently, the resultant fragment was cloned into an 18-T simple vector for sequencing.2. Generation of mice with PRP overexpressionThe transgenic constructs were micro injected into the pronuclei of fertilized oocytes from C57 mice. To identify the founder transgenic mice, tail biopsies were collected for genomic DNA isolation. The resulting genomic DNA samples were screened by PCR. The amplified products were resolved using electrophoresis in 1.5% agarose gels.3. Detection of the FLAG-tagged PRP protein in testisWesternblotting and histopathological assay were employed to investigate the expression of PRP in testis.Results:Three males that were positive for the mouse PRP transgene were selected for further breeding. The 4-month-old mice, which were originally derived from the 3 male founder mice, showed an upregulation of PRP protein expression in their testes, but the control litter mate did not. There were no apparent differences in the levels of testicular PRP expression between the transgenic lines. Since the transgenic construct contains a FLAG tag, we also tested for the expression of the FLAG epitope in the testes, and the results showed that while there was no expression of the FLAG epitope in the control mice, its protein was detected in the testes from the transgenic animals at a molecular weight that was appropriate for PRP. Histological analysis also immunolocalized the FLAG epitope in the Leydig cells, which further substantiated the expression of the transgenic construct within the testes. Additionally, ELISA assay showed that PRP overexpression attenuated the testosterone production in mice.Conclusions:In conclusion, we have successfully established transgenic mice with PRP overexpression, whcih provided useful tools for study the roles of PRP in male reproduction in vivo.