GENE-TRANSFER EFFICIENCY DURING GESTATION AND THE INFLUENCE OF CO-TRANSFER OF NON-MANIPULATED EMBRYOS ON PRODUCTION OF TRANSGENIC MICE

Litter size of DNA microinjected zygotes is lower than for non-manipul ated zygotes. The rate of embryonic and fetal survival in early, mid a nd late gestation was determined to assess whether DNA integration was responsible for embryonic losses. Also, the effect of including non-m icroinjected embryos with injected embryos on pregnancy rate and trans genic pup production was determined. In Experiment I, one-cell embryos from immature CD-1 mice were microinjected with a whey acidic protein promoter-human protein C gene construct. One hour after microinjectio n embryos were transferred to pseudopregnant recipients (45 transfers of 30 embryos each). Fifteen recipients were sacrificed on day 4, 12 a nd Is of gestation and the embryos/fetuses analysed for the transgene. The percentage of embryos or fetuses that were positive for the trans gene was not significantly different at any day. However, the number o f viable embryos at day 4 was significantly greater than fetuses on da ys 12 or 18. In addition, a high degree of mosaicism was observed in d ay 18 fetuses and placentae recovered. In Experiment 2, one-cell embry os from CD-1 mice were microinjected and co-transferred with non-manip ulated embryos (C57BL/6). Pregnancy rate and the total number of pups born were improved by addition of non-injected embryos. However, the n umber of transgenic mice produced was similar whether non-injected emb ryos were included or not. There were 32.2% (15/46) transgenic pups wh en 0 non-injected embryos were transferred compared with 15.1% (13/86) transgenic pups when 4 or 8 non-injected embryos were added to the tr ansfers. In summary, a high degree of embryonic and fetal mortality oc curs among microinjected embryos. Furthermore, since the percentage of transgenesis did not change throughout pregnancy, DNA integration doe s not appear to account for all of the embryonic losses. Other factor( s) related to the microinjection procedure may be involved in the embr yonic and fetal failure of microinjected embryos. Addition of non-inje cted embryos, although it increased pregnancy rate and the number of p ups born from microinjected embryos, actually decreased the number of transgenic pups obtained per pregnancy.