title: Gene transfer to the embryo: strategies for the delivery and expression of proteins at 48 to 56 hours postfertilization.
abstract: OBJECTIVE
  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 x 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 degrees 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.
authors.affiliation: Division of Pediatric Surgery, UCLA School of Medicine, California Institute of Technology, Los Angeles, CA 90095, USA.
authors.forename: A
authors.initials: A
authors.lastname: Thakur
authors.forename: R
authors.initials: R
authors.lastname: Lansford
authors.forename: V
authors.initials: V
authors.lastname: Thakur
authors.forename: J N
authors.initials: JN
authors.lastname: Narone
authors.forename: J B
authors.initials: JB
authors.lastname: Atkinson
authors.forename: T
authors.initials: T
authors.lastname: Buchmiller-Crair
authors.forename: S E
authors.initials: SE
authors.lastname: Fraser
mesh_terms: D000818:Animals; D002642:Chick Embryo; D005314:Embryonic and Fetal Development; D005260:Female; D015870:Gene Expression; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000032:analysis*; D018613:Microscopy, Confocal; D011247:Pregnancy; D011256:Pregnancy Outcome; D011270:Pregnancy, Animal; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins