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

Thakur, Anjani and Lansford, Rusty and Thakur, Vijaya and Narone, Jogendra N. and Atkinson, James B. and Buchmiller-Crair, Terry and Fraser, Scott E. (2001) Gene transfer to the embryo: Strategies for the delivery and expression of proteins at 48 to 56 hours postfertilization. Journal of Pediatric Surgery, 36 (8). pp. 1304-1307. ISSN 0022-3468. https://resolver.caltech.edu/CaltechAUTHORS:20160112-082307734

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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.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1053/jpsu.2001.25804DOIArticle
http://www.sciencedirect.com/science/article/pii/S0022346801013422PublisherArticle
ORCID:
AuthorORCID
Lansford, Rusty0000-0002-2159-3699
Fraser, Scott E.0000-0002-5377-0223
Additional Information:© 2001 W.B. Saunders Company. Published by Elsevier Inc. Presented at the 2000 Annual Meeting of the Section on Surgery of the American Academy of Pediatrics, Chicago, Illinois, October 28-November 1, 2000. Recipient of Poster Prize.
Subject Keywords:Fetal gene therapy, green fluorescent protein, sonic hedgehog
Issue or Number:8
Record Number:CaltechAUTHORS:20160112-082307734
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20160112-082307734
Official Citation:Anjani Thakur, Rusty Lansford, Vijaya Thakur, Jogendra N. Narone, James B. Atkinson, Terry Buchmiller-Crair, Scott E. Fraser, Gene transfer to the embryo: Strategies for the delivery and expression of proteins at 48 to 56 hours postfertilization, Journal of Pediatric Surgery, Volume 36, Issue 8, August 2001, Pages 1304-1307, ISSN 0022-3468, http://dx.doi.org/10.1053/jpsu.2001.25804. (http://www.sciencedirect.com/science/article/pii/S0022346801013422)
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:63566
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:12 Jan 2016 22:20
Last Modified:03 Oct 2019 09:29

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