Receptor-mediated transport of foreign DNA into preimplantation mammalian embryos.
Mouse and rabbit preimplantation embryos with intact zona pellucida were incubated for 3 hr with DNA-carrying constructs containing insulin as an internalizable ligand: (insulin-polylysine)-DNA and (insulin-polylysine)-DNA-(streptavidin-polylysine)-(biotinylated adenovirus). Video-intensified microscopy demonstrated that the constructs penetrated the zona pellucida and accumulated in the blastomere perinuclear space. The percentage of blastocysts formed was about 70% after incubation of zygotes and two-cell embryos with the constructs. Foreign DNA was detected after 51 hr in 80% of rabbit embryos and after 96 hr in 73% of mouse embryos. Inclusion of various adenoviruses into the construct improved foreign DNA preservation in early embryos. Blot hybridization revealed genome-integrated foreign DNA in 12- and 15-day mouse embryos and in a newborn. Thus, the ligand-mediated mechanism can be employed for introducing foreign genetic material into early mammalian embryos; insulin provides for delivery inside the cell and to the nucleus, while adenoviruses ensure release from endosomes.
Ivanova
M M
MM
Laboratory of Genetic Engineering and Molecular Diagnostics of Microorganisms, Moscow, Russia.
Rosenkranz
A A
AA
Smirnova
O A
OA
Nikitin
V A
VA
Sobolev
A S
AS
Landa
V
V
Naroditsky
B S
BS
Ernst
L K
LK
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D015139:Blotting, Southern; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism*; D004622:Embryo, Mammalian / Q000378:metabolism; D047108:Embryonic Development / Q000235:genetics*; D004705:Endocytosis; D005260:Female; D018014:Gene Transfer Techniques; D007328:Insulin / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D018715:Microscopy, Video; D008957:Models, Genetic; D010957:Plasmids; D011247:Pregnancy; D011817:Rabbits; D014162:Transfection
D007328:Insulin; D004247:DNA