Expression of human immunodeficiency virus long terminal repeat-coupled genes in early cleaving embryos.
Early studies suggested that exogenous retroviral genes are not expressed by mouse embryos infected at early cleavage stages. The general view has therefore been that, since human eggs are non-dividing cells, they could not be infected with HIV before fertilization, nor would HIV be expressed if infection occurred during early cleavage stages following fertilization. In contrast with this view, more recent work has shown that genes coupled to the long terminal repeat (LTR) promotor elements in Rous Sarcoma Virus are readily expressed in early cleaving mouse embryos; in addition, pHIV-LTR, a plasmid construct with HIV-LTR coupled to the reporter gene, lacZ (which encodes beta-galactosidase), is expressed when transfected into a human embryonic carcinoma cell line. To determine if HIV LTR coupled genes would be expressed in early cleaving embryos, one nucleus of mouse zygotes were microinjected with approximately 5000 copies of the pHIV-LTR plasmid. Following microinjection, zygotes were cultured for 48 h to the four cell stage and stained for expression of beta-galactosidase. A plasmid construct containing lacZ without HIV LTR was microinjected as control. A total of 25 of the 111 mouse zygotes microinjected with pHIV-LTR stained positively for beta-galactosidase activity. Blastomeres within individual embryos were not uniformly stained, suggesting a mosaic pattern of distribution of the microinjected plasmids among the cleaving blastomeres. None of the 22 mouse zygotes microinjected with control plasmid stained positively. A total of six polypronuclear human eggs were obtained as discarded by-products of a human IVF program. All four human eggs microinjected with approximately 6000 copies of HIV-LTR reporter gene construct and cultured for 48 h stained highly positive for beta-galactasidase. Staining intensity varied among blastomeres, suggesting a mosaic pattern similar to the mouse embryos. The two human eggs microinjected with control plasmid were negative for beta-galactosidase activity. These results suggest that HIV-LTR is active in both early cleaving mouse embryos and in early cleaving polypronuclear human eggs. These findings indicate that human eggs infected at the time of fertilization with HIV could express viral proteins during early embryonic development.
Kiessling
A A
AA
Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, Boston, MA 02115, USA. akiessl@bidmc.harvard.edu
D000818:Animals; D002970:Cleavage Stage, Ovum / Q000821:virology*; D005314:Embryonic and Fetal Development; D005260:Female; D015870:Gene Expression; D005814:Genes, Viral; D016325:HIV Long Terminal Repeat; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008297:Male; D051379:Mice; D015053:Zygote; D001616:beta-Galactosidase / Q000235:genetics
D001616:beta-Galactosidase