401 USE OF FLOW CYTOMETRY TO EVALUATE THE CAPACITY OF BOAR SPERM TO BIND TO EXOGENOUS DNA OF DIFFERENT SIZES

Abstract

Sperm-mediated gene transfer (SMGT) is based on the ability of sperm cells to bind exogenous DNA and transfer it into the egg at fertilization (Lavitrano et al. 1989 Cell 57, 717–723). SMGT has been reported in several species, but different degrees of efficiency have been reported in different laboratories. In this study, in order to optimize and understand the mechanism of SMGT, the capacity of spermatozoa to bind 2 different sizes of exogenous DNA (previously marked with fluorescein using flow cytometry), the kinetics of DNA binding, and the effect of the male used were evaluated. Semen from 7 fertile boars was recovered and immediately diluted 1 : 10 in SFM (swine fertilize medium) at 37°C and later centrifuged (800g 10 min, 25°C), discarding the seminal plasma to avoid a detrimental effect on DNA binding to cells. Two groups were established depending on the size of DNA (Group A: 5.4 kb; Group B: 20 kb). Linearized plasmid DNA (5.4 kb and 20 kb), marked by random primed DNA labeling with fluorescein-12-dUTP (Roche Diagnostics, Mannheim, Germany) was added (1 × 10⁸ spermatozoa/mL + 5 µg of DNA/mL) in each group and incubated at 16°C. Initial spermatozoa contact with DNA was the starting point, initiating the measurements in the cytometer. Time measurements were taken at 0, 15, 30, 60, 90, and 120 min of incubation. Samples were analyzed on a Coulter Epics XL flow cytometer (Beckman Coulter, Inc., Fullerton, CA, USA). A total of 10 000 sperm cells were counted per sample and 3 measurements in each incubation time, with sample running rates of approximately 600 events/s. Fluorescence was measured using the FL1 sensor, with a 525-nm band-pass filter to detect fluorescein isothiocyanate. The results showed that no significant effect on the parameters studied was associated with the kind of DNA employed, and the main DNA binding to spermatozoa was achieved as soon as 15 min in both groups (Group A: 13.43 ± 0.74%; Group B: 13.97 ± 0.77%); thereafter, no further significant increase in the binding was detected (Group A: 14.27 ± 0.88%, 14.16 ± 1.05%, 13.41 ± 1.11%, 13.53 ± 1.26%; Group B: 13.97 ± 0.77%, 15.23 ± 0.93%, 15.19 ± 0.92%, 15.17 ± 1.05%, at 30, 60, 90, and 120 min, respectively). Nevertheless, a significant boar effect was detected. These data suggest that boar sperm is able to bind 20 kb with the same efficiency as 5 kbp and that flow cytometry is a practical tool to evaluate sperm DNA binding capacity in a specific and objective manner and to select the boar that produces better results.

This work was supported by BIOCARM 10BIO2005/01-6463.