A Low Frequency of Chromosomal Integration of Cloned DNA after Microinjection into the Cytoplasm of Fertilized Eggs of Chickens.

Abstract

To evaluate the efficiency of transgenesis in chickens by the microinjection method, we have investigated the expression and integration of DNA which was microinjected into the cytoplasm of fertilized chicken eggs by histochemical staining, PCR and Southern analyses. We found that DNA injected into the cytoplasm was incorporated into the nucleus at an early cleavage stage and transcribed into mRNA. However, the DNA incorporated into the nucleus was rapidly lost during early development without integration into chromosomes. After 3 days of incubation, expression of the injected DNA was still observed in 38% of embryos, but the population of positive cells was very low in most of the embryos. PCR analysis indicated that a low amount of DNA was still retained in 35% of embryos after 7 days of incubation. However, in Southern analysis, injected DNA was detected in only one out of 76 embryos (1.3%). This embryo showed a single positive band after EcoRI digestion. The copy number was estimated to be about 0.2 copy per haploid genome from the intensity of the band, suggesting that chromosomal integration may have occurred after several cell divisions even if the detected DNA was integrated one. We obtained no case in which the injected DNA was integrated at the single cell stage, even though DNA was injected into the fertilized egg before pronucleus fusion. These results suggested that the DNA injected into the cytoplasm of the fertilized eggs may be incorporated into the cleavage nucleus at an early stage of development, but is rarely integrated into the chromosomal DNA. We concluded that the cytoplasmic injection method used in this study is a reliable method for expressing injected DNA transiently in early chicken embryos, but not an efficient method for generating transgenic chickens.