Gene transfer to the embryo: Strategies for the delivery and expression ofproteins at 48 to 56 hours postfertilization

Background/Purpose: Although gene and protein transfer may potentiate the c ure of genetic disease, current strategies involving fetal gene therapy rem ain nonfocal and confounded by the lack of imaging techniques and in vivo m arkers for precise gene transfer. Methods: Fourteen white Leghorn chick eggs were incubated for 48 to 56 hour s postfertilization until they reached stages 11 to 16, about 3 mm in size. In 7 chick embryos, a glass needle was placed at the midbrain/hindbrain le vel and 1 x 10(7) pfu of an adenovirus containing the green fluorescent pro tein (GFP) reporter gene was injected into the lateral head. In another 7 c hicken embryos, colored agarose beads coated with Sonic hedgehog (Shh) prot ein were implanted at the level of the hindbrain under direct microscopy. T he eggs were then sealed, incubated at 37 degreesC for 24 hours, and reimag ed using fluorescent microscopy and confocal laser microscopy. Results: At 24 hours postinjection, all embryos were alive and were imaged in vivo. Fluorescent microscopic imaging showed green fluorescence in the r egion of the injection site in all the embryos. In embryos that underwent b ead placement, the beads were visualized under microscopy in the lateral hi ndbrain of all embryos, and the presence of the Shh protein was confirmed u sing fluorescein isothiocyanate (FITC)-conjugated secondary antibody. Conclusions: This study shows that embryonic 3-mm chick embryos survive ade noviral transduction or agarose bead implantation in a focal manner in vivo and that this delivery results in production of imageable levels of protei n. This may be used in mammalian systems, including humans, to introduce ge nes and proteins. J Pediatr Surg 36:1304-1307. Copyright (C) 2001 by W.B. S aunders Company.