301 EVALUATION OF THE SPERM-MEDIATED GENE TRANSFER (SMGT) TECHNIQUE BY IN VITRO FERTILIZATION IN PIGS USING RecA PROTEIN

Abstract

Sperm-mediated gene transfer (SMGT) is a transgenesis technique used in pigs mainly byAI (Lavitrano ML et al. 2002 Proc. Natl. Acad. Sci. USA 99, 14 230–14 235), and by intracytoplasmic spermi injection (ICSI) (Garcia-Vazquez FA et al. 2006 Reprod. Domest. Anim. 41, 338), but up to now, it has not been reported by IVF (Bolling LC et al. 2003 Transgenics 4, 77–86). The aim of this study was to evaluate the efficiency of SMGT by IVF in pigs and the use of recombinase RecA to avoid possible exogenous DNA degradation by endonucleases. We designed a study with 3 experimental groups: (1) sperm incubated without exogenous DNA (control group), (2) sperm incubated with exogenous DNA (DNA group), and (3) sperm incubated with the complex RecA:DNA (RecA group). Ejaculates from mature boars were recovered and the seminal plasma was discarded to avoid detrimental effects on DNA binding. The spermatozoa were incubated with DNA or RecA-DNA and used as vectors for transferring linealized plasmid DNA [5.7 kb enhanced green fluorescent protein (EGFP)] into in vitro-matured porcine oocytes by IVF. Spermatozoa and oocytes were coincubated for 2 h in TALP medium; then, the fertilized oocytes were transferred into the culture drops with NCSU-23 medium. The binding of the DNA to the spermatozoa was confirmed by the use of enzymatic fluorescein-12-dUTP-labeled DNA and measured by flow cytometry. The total number of oocytes used was 584 (n = 59; n = 382; n = 143 for the 3 experimental groups, respectively). Embryos were examined for cleavage rate at 48 h after fertilization, and for embryo development at 144 h. Expression of EGFP in embryos was examined using a fluorescence inverted microscope. The results in our experiment showed that the coincubation of sperm with exogenous DNA induced a lower cleavage rate than when the RecA:DNA complex was used (DNA: 25.13 ± 2.22 v. RecA: 41.26 ± 4.13%, P < 0.05), and both no different from the control group (38.98 ± 6.40). On the other hand, the production of blastocysts was similar in the 3 groups (Control: 21.74 ± 8.79 v. DNA: 21.87 ± 4.24 v. RecA: 15.25 ± 4.72%) as well as the quality of the obtained embryos. The average number of cells per blastocyst was similar in the 3 groups (36.40 ± 9.28 v. 37.26 ± 3.32 v. 28.45 ± 3.34, respectively). None of the produced embryos was detected for expressing protein EGFP. In conclusion, under our experimental conditions, IVF is not an effective technique for SMGT, whereas using ICSI-SMGT under the same conditions (DNA and DNA:RecA groups), we obtained a high percentage of transgenic embryos (Garcia-Vazquez FA et al. 2006 Reprod. Domest. Anim. 41, 338). Three main causes are hypothesized to be probably related to this conclusion: (i) the penetration of the oocytes is achieved only by the not DNA-bound viable spermatozoa in a competitive system, (ii) the DNA was only bound to altered membrane or dead spermatozoa, and (iii) the exogenous DNA is only present on sperm surface and in the process of fusion with oocyte membrane, the DNA is not internalized.