Noninvasive fluorescent screening of microinjected bovine embryos to predict transgene integration

Most transgenic domestic animals are generated by direct microinjection of DNA fragments into zygote pronuclei. It has generally been assumed that the majority of integration events should occur prior to the first round of chromosomal DNA replication. The aim of this study was to investigate the expression of GFP in bovine preimplantation embryos by using a gfp reporter gene consisting of chicken beta-actin promoter, the CMV-IE enhancer, gfp cDNA (EGFP) (732 bp) and rabbit beta-globin polyadenylation sequences. In five experiments 302 bovine zygotes were injected while 75 served as a control. The fluorescence intensity was detected at 72 and 168 h following fertilization in bovine embryos injected with 3 ng/microl in experiments 1-3, and injected with 5 ng/microl in experiments 4-5. Eight embryos were considered as expressing green fluorescence protein; 2 of them were 100% fluorescent after microinjection of a higher dose of the DNA; one was 75%, two--50%, and three 25% transgenic. The mosaicism was assumed to be at 75%. The results indicated that the fluorescence could be analyzed at any time of bovine embryo development. It was therefore concluded, that chicken beta-actin promoter together with the CMV-IE enhancer would confer a strong expression of the gfp reporter gene in preimplantation bovine embryos. Therefore, using GFP that could be simply detected in live bovine (transgenic) embryos would be very promising in establishing transgenic lines of domestic animals producing in their fluids human therapeutic proteins.