Prediction of transgene integration by noninvasive bioluminescent screening of microinjected bovine embryos

Transgenesis in domestic species, as a research tool and in biotechnologica l applications, has been limited by the expense of producing transgenic off spring by standard microinjection techniques. A major factor is the ineffic iency of maintaining large numbers of recipient females, when a high percen tage of these carry nontransgenic fetuses. There are two approaches to redu ce this cost, the fusion of transfected fetal fibroblasts with enucleated o ocytes, and the screening of microinjected embryos for transgene integratio n in blastocysts, prior to transfer. Here, we develop a luminescent screeni ng system to select transgenic bovine embryos. A transgene with scaffold at tachment regions flanking the murine HSP70.1 promoter linked to firefly luc iferase cDNA, was microinjected into pronuclei of in vitro produced zygotes . At the blastocyst stage, the transgene was induced by heat shock (45 degr ees C, 15 min) and 4-6 h later, luciferase expression was analyzed by photo n counting imaging. Screened blastocysts were transferred to recipients and day 50 fetuses or calves were analyzed by PCR and Southern blot for transg ene integration. When nonluminescent blastocysts were transferred, transgen e integration was never observed. Of 13 fetuses derived from luminescent bl astocysts, 3 contained integrated transgenes that were functional in all ti ssues examined. Image analysis of the signal emitted by positive blastocyst s revealed that 9 nontransgenic fetuses were obtained from blastocysts that exhibited a localized luminescent signal. On the other hand, 3 of 4 fetuse s derived from blastocysts that emitted light over more than 70% of their s urface were transgenic. Thus, by selecting luminescent blastocysts on the b asis of both signal intensity and distribution, the number of recipient fem ales required to produce transgenic offspring can be greatly reduced. Using this technique it should also be possible to improve the efficiency of tra nsgenesis by microinjection through studies in which vector design and inte gration conditions are examined at the blastocyst stage.