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

Abstract

Background/Purpose: Although gene and protein transfer may potentiate the cure of genetic disease, current strategies involving fetal gene therapy remain nonfocal and confounded by the lack of imaging techniques and in vivo markers for precise gene transfer. Methods: Fourteen white Leghorn chick eggs were incubated for 48 to 56 hours 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 level and 1 × 10^7 pfu of an adenovirus containing the green fluorescent protein (GFP) reporter gene was injected into the lateral head. In another 7 chicken embryos, colored agarose beads coated with Sonic hedgehog (Shh) protein were implanted at the level of the hindbrain under direct microscopy. The eggs were then sealed, incubated at 37°C for 24 hours, and reimaged 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 region of the injection site in all the embryos. In embryos that underwent bead placement, the beads were visualized under microscopy in the lateral hindbrain of all embryos, and the presence of the Shh protein was confirmed using fluorescein isothiocyanate (FITC)-conjugated secondary antibody. Conclusions: This study shows that embryonic 3-mm chick embryos survive adenoviral transduction or agarose bead implantation in a focal manner in vivo and that this delivery results in production of imageable levels of protein. This may be used in mammalian systems, including humans, to introduce genes and proteins.