A method for uptake quantification of multiple fluorescent DNAs in boar semen as an alternative to radiolabeling.
Sperm-mediated gene transfer (SMGT) is a simple and efficient method for producing multitransgenic organisms. Until now, the exogenous DNA uptake efficiencies have been quantified, performing coincubation of spermatozoa with (3)H-DNA. This method has significant limitations; from a researcher's point of view, radioactivity-based experiments are hazardous and require specialistic skills, and in technical analysis, the signal does not allow the simultaneous discrimination of two or more types of labeled constructs. Considering these remarkable points, the present work aims to develop a method for differential uptake quantification of various transgenes alternative to the use radioactive material. The main approach relies on fluorescent-specific peaks for each construct, and their diminution during the sperm--DNA-coincubation phase. The obtained results were confirmed by real-time PCR analysis and fluorescence microscopy imaging. This method becomes of primary importance when the SMGT technique has to be applied on various constructs, as it allows preliminary conclusions to be drawn about multiple transgenesis events and to approach further research about eventual sperm membrane preferences in sequences or structures for constructs.
De Cecco
Marco
M
Department of Veterinary Morphophysiology and Animal Production-DIMORFIPA, University of Bologna, Bologna, Italy. marco.dececco2@unibo.it
Zannoni
Augusta
A
Bernardini
Chiara
C
Lavitrano
Marialuisa
M
Bacci
Maria Laura
ML
Forni
Monica
M
D000818:Animals; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism*; D018014:Gene Transfer Techniques; D007553:Isotope Labeling / Q000379:methods*; D008297:Male; D008856:Microscopy, Fluorescence; D008858:Microscopy, Phase-Contrast; D010957:Plasmids / Q000235:genetics; D011868:Radioisotopes; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012661:Semen / Q000378:metabolism*; D013552:Swine / Q000378:metabolism*
D005456:Fluorescent Dyes; D011868:Radioisotopes; D004247:DNA