329 FLOW CYTOMETRIC ANALYSIS OF SPERMATOZOA FROM REPORTER TRANSGENIC BOARS DERIVED BY PRECISION GENETIC ENGINEERING

Abstract

Recently, we produced 2 founder boars with a non-autonomous Sleeping Beauty (SB) system carrying 3 monomeric integrations of a Venus transposon cassette and showing transgene segregation during meiosis (Garrels et al. 2011 PLoS One 6, e27563). It was possible to show transmission of the reporter protein to fertilized oocytes by confocal microscopy. The aim of this study was to assess the suitability of different fluorophore reporters for in vivo labelling of pig spermatozoa. Therefore, we used Venus transposon fibroblasts from a F1 boar, which carry a single integration of the transposon cassette and used these fibroblasts for a Cre-mediated cassette exchange against an mCherry reporter. These cells were used for somatic cell nuclear transfer (SCNT) to derive a syngene clone cohort of boars, which differ only in the fluorophore reporter cDNAs (either Venus or mCherry). Importantly, this methodology did not request any antibiotic selection cassette and allows precise genetic modifications in a livestock species where no authentic embryonic stem cells are available (Garrels et al. 2012 Trends in Biotechnology 30, 386–393). A total of 8 male piglets carrying the Venus transposon, and 4 male piglets carrying the mCherry reporter were born. Three Venus boars and 2 mCherry boars were raised to sexual maturity, and ejaculated sperm was obtained with the help of a phantom. A detailed flow cytometric analysis revealed that the spermatozoa samples were specifically Venus or mCherry positive [Gallios, Beckmann Coulter, Krefeld, Germany; solid-state laser (488 nm; 22 mW), filter for green fluorescence (525 BP); filter for red fluorescence: (620/30)], respectively. In direct comparative measurements, the spermatozoa samples from transgenic boars (Venus and Cherry) and wildtype controls could be discriminated. Interestingly, spermatozoa were uniformly Venus- or mCherry-positive and gave a distinct fluorescence peak in flow-cytometric measurements. The monomeric transgenes were transmitted through the germ line according to Mendelian rules with the expected ratio of 50% transgenic and 50% nontransgenic piglets. Fluorescence microscopic analysis and Western blotting confirmed the uniform presence of Venus and mCherry in boar spermatozoa, respectively. This is the first characterisation of spermatozoa from a pig cohort carrying a targeted cassette exchange. This large animal model may help to elucidate the function of paternally transmitted components to fertilized oocytes.