328 ANALYSIS OF FLUOROPHORE-EXPRESSING SPERMATOZOA FROM TRANSGENIC BOARS PRODUCED BY SLEEPING BEAUTY TRANSPOSITION

Abstract

The pig is an important model for biomedical research. Recently, we described a method for producing transgenic pigs using a nonautonomous Sleeping Beauty (SB) transposon1 (Garrels et al. 2010 Reprod. Domest. Anim. 45, 65 abst.). Briefly, in vivo developed porcine zygotes were co-injected with a CAGGS-Venus transposon and hyperactive SB100. A total of 141 in vivo developed zygotes were injected and transferred to synchronized foster sows. Subsequent analysis revealed specific transposase-mediated integration of 1 to 5 copies of the Venus transposon in fetuses and piglets. This method results in highly efficient SB-mediated transgene transposition into the porcine genome: 57% of the fetuses examined and 42% of piglets were transgenic, representing 6.4% of the treated zygotes. The piglets showed persistent expression of the Venus reporter. Here, we present cellular analysis of 2 founder boars. Expression of the Venus reporter was observed in skin, cultured fibroblasts, leukocytes, and spermatozoa of both animals. However, flow cytometric measurement of leukocytes and cultured ear fibroblasts revealed that these boars carried both a Venus-fluorescence-positive population and a Venus-fluorescence-negative cell population. PCR analysis revealed that the Venus-fluorescence-negative cells were genotypically negative, indicating transgene mosaicism. Interestingly, all spermatozoa tested were Venus-positive and gave a distinct fluorescence peak in repeated flow-cytometric measurements (n = 6). Fluorescence microscopy revealed localisation of the Venus fluorophore in the sperm tail, in the midpiece, and in the equatorial segment of the sperm head. Motility of the transgenic sperm as measured by computer-assisted sperm analysis (Hamilton-Thorne, Beverly, MA, USA) indicated no decrease in percentage of motile sperm and the movement patterns. Sorting Hoechst 33342–stained transgenic sperm into X- and Y-chromosome bearing populations did not reveal any differences in Venus fluorescence between these 2 groups. To test the fertility of the transgenic sperm, 6 wild-type sows were artificially inseminated. Four pregnancies were established, 2 of these sows were sacrificed on Day 29 of gestation and a total of 9 Venus-positive normally developed fetuses and 2 degenerated fetuses were recovered. The other 2 pregnancies are ongoing at the time of writing. This is the first characterisation of spermatozoa from transposon transgenic pigs. The results show that Venus transposon–bearing transgenic spermatozoa are fertile and demonstrate germline transmission to the F₁ offspring. Sleeping Beauty-mediated transposition is thus a promising approach for genetic modification of the pig genome.