RESEARCH ARTICLE Molecular Reproduction & Development (2012) DNA Uptake in Swine Sperm: Effect of Plasmid Topology and Methyl-Beta-Cyclodextrin-Mediated Cholesterol Depletion SERGIO ODDI, 1,2 * NICOLA BERNABO ` , 1 MONIA DI TOMMASO, 1 CLOTILDE BEATRICE ANGELUCCI, 1 ELISA BISICCHIA, 1,2 MAURO MATTIOLI, 1 AND MAURO MACCARRONE 1,2 1 Department of Biomedical Sciences, University of Teramo, Teramo, Italy 2 European Center for Brain Research (CERC)/IRCCS Santa Lucia Foundation, Rome, Italy SUMMARY Sperm-mediated gene transfer (SMGT), the ability of sperm cells to spontaneously incorporate exogenous DNA and to deliver it to oocytes during fertilization, has been proposed as an easy and efficient method for producing transgenic animals. SMGT is still undergoing development and optimization to improve the uptake efficiency of foreign DNA by sperm cells, which is a preliminary, yet critical, step for successful SMGT. Towards this aim, we developed a quantitative, real-time PCR-based assay to assess the absolute number of exogenous plasmids internalized into the spermato- zoon. Using this technique, we found that the circular form of the DNA is more efficiently taken up than the linearized form. We also found that DNA internalization into the nucleus of porcine sperm cells is better under specific methyl-b-cyclodextrin (MCD)-treated conditions, where the plasma membrane properties were altered without significantly compromising sperm physiology. These results provide the first evidence that membrane cholesterol depletion by MCD might represent a novel strategy for enhancing the ability of sperm to take up heterologous DNA. Mol. Reprod. Dev. 2012. ß 2012 Wiley Periodicals, Inc. Received 5 June 2012; Accepted 8 October 2012 * Corresponding author: Department of Biomedical Sciences University of Teramo Piazza Aldo Moro 45 Teramo 64100, Italy. E-mail: soddi@unite.it Grant sponsor: Fondazione TERCAS; Grant number: 2009–2012 Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/mrd.22124 INTRODUCTION Mature sperm can spontaneously take up exogenous DNA molecules and transfer them to oocytes during fertili- zation (Brackett et al., 1971; Arezzo, 1989; Lavitrano et al., 1989; Atkinson et al., 1991; Castro et al., 1991; Magnano et al., 1998). This capability has been exploited over the course of the last two decades to develop an alternative strategy for animal transgenesis, called sperm-mediated gene transfer (SMGT) (Nakanishi and Iritani, 1993; Lavi- trano et al., 1997a, 2006; Smith and Spadafora, 2005). SMGT is simpler, less expensive, and more user-friendly than other techniques such as DNA microinjection, nuclear transfer, and retroviral infection (Kues and Niemann, 2004; Smith and Spadafora, 2005; Niemann and Kues, 2007; Robl et al., 2007; Bacci et al., 2009). Indeed, protocols for SMGT have been successfully applied to a variety of animal species, including fish (Patil and Khoo, 1996), chicken (Nakanishi and Iritani, 1993), mouse (Maione et al., 1998), and swine (Lavitrano et al., 1997a). Despite its success, the efficacy and reproducibility of SMGT technology has remained low due to a number of unresolved issues, including the unknown molecular mechanism(s) underlying gene transfer. Further, param- eters that improve DNA uptake and integration in sperm cells without interfering with their motility and fertilizing capacity remain elusive. Thus in the present study, we sought to quantify the effect of some relevant experimental parameters on the ability of boar (Sus scrofa) sperm to Abbreviations: MCD, methyl-b-cyclodextrin; qRT-PCR, quantitative real- time PCR; SMGT, sperm-mediated gene transfer; sZP, solubilized zona pellucida. ß 2012 WILEY PERIODICALS, INC.