Gene delivery to the neurulating embryo during culture.
Modulating expression of specific genes during embryogenesis will help elucidate their role in development. Transient overexpression of specific genes can be accomplished by adding additional copies, or else antisense transcripts can be used to block expression. Manipulation of gene expression requires an efficient, nontoxic gene delivery system. We compared a plasmid and a replication-defective adenovirus (Ad5) as methods of delivering genes to the embryo during the neurulation stage of development. Both vectors utilized a construct containing the bacterial beta-galactosidase reporter gene under the control of the human cytomegalovirus early gene promoter and the SV40 polyadenylation signal. Vectors were delivered by intraamniotic microinjection to embryos prepared for whole-embryo culture. Plasmid transfection experiments were done with and without polycationic lipid (lipofectamine, 20 or 125 micrograms/microliter) enhancement at 0.1 and 0.01 microgram per embryo. Twenty-six hours after transfection with plasmid only, embryos appeared normal, but had very weak gene expression which was detected only after extended periods of staining. In contrast, adenovirus gene delivery was successful. While high concentrations of virus (6 x 10(8) particles/ microliter) elicited significant malformations, lower concentrations (1.5 x 10(8) particles/microliter) produced no malformations and intense gene expression. Time-course studies revealed staining at 6 hr postinjection, and intense staining at 26 hr. Staining appeared primarily in the neurectoderm and cells derived from the neurectoderm. This pattern of gene expression was confirmed using a green fluorescent protein-expressing adenovirus. Rapid induction of gene expression with no toxicity is critical to the utility of this technique within the whole-embryo culture system. Clearly, Ad5 transduction provides a more useful tool than plasmid vectors.
Hartig
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Division of Reproductive Toxicology, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711, USA. hartig.phillip@epamail.epa.gov
Hunter
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D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D046508:Culture Techniques; D004622:Embryo, Mammalian / Q000378:metabolism*; D005260:Female; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D008297:Male; D051379:Mice; D008845:Microinjections; D008856:Microscopy, Fluorescence; D011247:Pregnancy