| Abstract: | A circular recombinant plasmid composed of a 12.5 kb fragment of human DNA including the entire insulin gene and the 4.3 kb bacterial plasmid pBR322 was microinjected into fertilized C57BL/6 mouse eggs. 753 eggs were injected with 30000 gene copies in a volume of 1-2 pl; 379 eggs survived micromanipulation and were subsequently cultured to the blastocyst stage. From 282 embryos that were transferred into the uteri of pseudopregnant ICR/Swiss foster females, 60 fetuses and corresponding placentas could be recovered at day 16-19 of pregnancy. High molecular weight DNA was extracted from these tissues and was screened with radioactively labelled hybridization probes for the presence of the injected DNA sequences. By restriction endonuclease analysis in conjunction with Southern blot hybridization, we found that in two normally developed fetuses at day 18, the fetal and placental tissues contained the human insulin gene including the flanking regions and bacterial plasmid sequences. Our results indicate that the injected DNA integrated into the mouse genome within its pBR322 region and properly replicated with the host DNA during development. The intensities of the hybridization bands suggest that at least one copy of foreign plasmid DNA was present per cell in the two fetuses and their placentas. |
