Assessment of the proportion of transgene-bearing sperm by fluorescence insitu hybridization: A novel approach for the detection of germline mosaicism in transgenic male founders

Genetic mosaicism is frequent among transgenic animals produced by pronucle ar microinjection. A successful method for the screening of founder animals for germline mosaicism prior to mating would greatly reduce the costs asso ciated with the propagation of the transgenic lines, and improve the effici ency of transgenic livestock production. With this aim, we have devised a s imple method to detect integrated transgenes in individual spermatozoa usin g fluorescence in situ hybridization (FISH). The experiments reported here were undertaken to investigate the efficiency of this FISH-based approach t o accurately evaluate the proportion of transgene-bearing sperm and to be a pplied for the detection of potential germ line mosaics. Sperm samples from mice homozygous and hemizygous for a beta -lactoglobulin transgene were an alyzed in a first set of experiments. A high hybridization efficiency was a chieved, and the proportions of transgene-positive sperm cells in both homo zygous (94.8-98.2%) and hemizygous (49.8-51.9%) animals were close to the e xpected frequencies (100 and 50%, respectively). To evaluate the sensitivit y of the assay more directly, simulated mosaic samples with 5, 10, 15, 20 a nd 40% of transgene-bearing spermatozoa were then prepared and analyzed by FISH. Significant differences in the frequency of transgene-positive sperm were observed between all mosaic samples, indicating that even small deviat ions (5%) from the expected 50% transgene transmission rate in a founder an imal could be reliably detected with our assay. Therefore, the method propo sed represents a novel approach for the identification of germline mosaic f ounder males in livestock transgenic projects and a much more economic and faster alternative to breeding. (C) 2001 Wiley-Liss, Inc.