Development and Reporter Gene Expression in Transgenic Mouse Embryos After Positive Selection in Culture.

Abstract

A transgene in which polyoma enhancer was fused between the 5' upstream region of PGK-RU5/lacZ and that of PGK/neo was microinjected into pronuclei of mouse zygotes. These embryos were cultured in the presence (G-418 treatment) or absence (control treatment) of 150 μg/ml G-418 and those surviving were subsequently examined for the pattern of lacZ expression. The rate of development to the blastocyst stage after culture for 3.5 days was significantly reduced by the G-418 treatment (32.8%, 59/180 vs 17.6%, 40/227, P<0.05). The overall proportion of embryos expressing lacZ gene did not change significantly with treatment (72.8%, 131/180 vs 66.1%, 150/227). However, 54.2% (32/59) and 85.0% (34/40) of the blastocyst stage embryos obtained from the control and G-418 treatment groups showed signs of lacZ activity, respectively (P<0.05). These direct comparisons between the developmental potential and lacZ expression of embryos revealed that the transgene employed in this study can retain both neo and lacZ functions. Blastocysts were classified by blue deposits in the cytoplasm according to pattern and intensity. Almost all lacZ positive blastocysts were mosaic being composed of unstained and stained regions, and weakly or spotty stained and strongly stained regions within whole embryos. In both groups, all of the blastocysts in which the intensity of the staining cytoplasm was scored as weak or spotty, were categorized as having staining in less than 1/3 the area of whole embryo (56.2%, 18/32 vs 41.2%, 14/34). In the remaining blastocysts which had intense staining, the number of blastocysts scored as having staining in more than 1/3 the total area tended to increase on G-418 treatment (25.0%, 8/32 vs 38.2%, 13/34), but there was no clear relationship between the pattern of X-gal staining and the treatment. These results suggest that mouse embryos without exogenous DNA after microinjection can be eliminated during the preimplantation period.