Functional study of a Novel Acrosome-specific Gene (AEP1/VAD1.3) using Conditional Gene Knock Out Approach

Abstract

Objective: AEP1/VAD1.3, a novel acrosome gene, was first identified from a retinol treated Vitamin A deficiency (VAD) rat model with synchronized spermatogenesis. AEP1 was expressed specifically in the testes from Day 25 when spermatids form. AEP1 immunoreactivity was localized at the acrosome region of the rat, human, monkey and porcine spermatids and spermatozoa, suggesting that AEP1 may play a significant role on acrosome formation. We hypothesized that AEP1 deficiency may cause acrosomal defect in mouse sperm leading to infertility. Methods: To elucidate the role of AEP1 during spermatogenesis, a Cre-LoxP conditional gene knock out approach will be used to generate AEP1 deficient mice for functional studies. Results: Conditional target vector was constructed by inserting the LoxP sites flanking the exons 2 and 3 of the AEP1 gene. A neo cassette flanked by Frt sites and a splice-receptor conjugated EGFP gene for in vitro selection of recombinant embryonic stem (ES) cells and Loxed AEP1 allele in sperm, respectively, were inserted downstream of the exons 3. In vitro recombinant occurred when the targeting vector was transformed into E. coli harboring Cre or Flp recombinases targeting for LoxP and Frt sites of the targeting vector, respectively. The conditional targeting construct was used to electrophorate 129/SvEv embryonic stem (ES) cells to generate conditional KO mice. Conclusion: The conditional knockout targeting vector for AEP1 gene was generated and characterized. Generation of AEP1 conditional knockout animal may allow us to understand the role of AEP1 on acrosome formation and infertility in human. (This work was supported in part by a RGC grant HKU7537/05M to KFL.)