DNA uptake in swine sperm: Effect of plasmid topology and methyl-beta-cyclodextrin-mediated cholesterol depletion

Sergio Oddi, Nicola Bernabò, Monia Di Tommaso, Clotilde Beatrice Angelucci, Elisa Bisicchia, Mauro Mattioli, Mauro Maccarrone

Research output: Contribution to journalArticle

Abstract

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 spermatozoon. 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-β-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.

Original languageEnglish
Pages (from-to)853-860
Number of pages8
JournalMolecular Reproduction and Development
Volume79
Issue number12
DOIs
Publication statusPublished - Dec 2012

Keywords

  • Cholesterol depletion
  • DNA incorporation
  • DNA topology
  • Swine spermatozoa

ASJC Scopus subject areas

  • Genetics
  • Developmental Biology
  • Cell Biology

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