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 spec ific genes can be accomplished by adding additional copies, or else an tisense transcripts can be used to block expression. Manipulation of g ene expression requires an efficient, nontoxic gene delivery system. W e compared a plasmid and a replication-defective adenovirus (Ad5) as m ethods of delivering genes to the embryo during the neurulation stage of development. Both vectors utilized a construct containing the bacte rial beta-galactosidase reporter gene under the control of the human c ytomegalovirus early gene promoter and the SV40 polyadenylation signal . Vectors were delivered by intraamniotic microinjection to embryos pr epared for whole-embryo culture. Plasmid transfection experiments were done with and without polycationic lipid (lipofectamine, 20 or 125 mu g/mu l) enhancement at 0.1 and 0.01 mu g 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 peri ods of staining. In contrast, adenovirus gene delivery was successful. While high concentrations of virus (6 x 10(8) particles/mu l) elicite d significant malformations, lower concentrations (1.5 x 10(8) particl es/mu l) produced no malformations and intense gene expression. Time-c ourse studies revealed staining at 6 hr postinjection, and intense sta ining at 26 hr. Staining appeared primarily in the neurectoderm and ce lls derived from the neurectoderm. This pattern of gene expression was confirmed using a green fluorescent protein-expressing adenovirus. Ra pid induction of gene expression with no toxicity is critical to the u tility of this technique within the whole-embryo culture system. Clear ly, Ad5 transduction provides a more useful tool than plasmid vectors. (C) 1998 Wiley-Liss, Inc.(+)