DNA uptake in swine sperm: effect of plasmid topology and methyl-beta-cyclodextrin-mediated cholesterol depletion.
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.
Department of Biomedical Sciences, University of Teramo, Teramo, Italy. soddi@unite.it
Sergio
S
Oddi
Nicola
N
Bernabò
Monia
M
Di Tommaso
Clotilde Beatrice
CB
Angelucci
Elisa
E
Bisicchia
Mauro
M
Mattioli
Mauro
M
Maccarrone
D000818:Animals; D001692:Biological Transport; D002784:Cholesterol / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004270:DNA, Circular / Q000378:metabolism*; D005306:Fertilization / Q000235:genetics; D018014:Gene Transfer Techniques; D008297:Male; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D013094:Spermatozoa / Q000166:cytology / Q000378:metabolism; D034421:Sus scrofa; D047392:beta-Cyclodextrins / Q000494:pharmacology*
D004270:DNA, Circular; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D004247:DNA; D002784:Cholesterol