Gene transfer to chicks using lentiviral vectors administered via the embryonic chorioallantoic membrane.

The lack of affordable techniques for gene transfer in birds has inhibited the advancement of molecular studies in avian species. Here we demonstrate a new approach for introducing genes into chicken somatic tissues by administration of a lentiviral vector, derived from the feline immunodeficiency v...

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Autores principales:	Gideon Hen, Sara Yosefi, Dmitry Shinder, Adi Or, Sivan Mygdal, Reba Condiotti, Eithan Galun, Amir Bor, Dalit Sela-Donenfeld, Miriam Friedman-Einat
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2012
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/437e5b61e79640ba94be725054b41893
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spelling	oai:doaj.org-article:437e5b61e79640ba94be725054b418932021-11-18T07:19:04ZGene transfer to chicks using lentiviral vectors administered via the embryonic chorioallantoic membrane.1932-620310.1371/journal.pone.0036531https://doaj.org/article/437e5b61e79640ba94be725054b418932012-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22606269/?tool=EBIhttps://doaj.org/toc/1932-6203The lack of affordable techniques for gene transfer in birds has inhibited the advancement of molecular studies in avian species. Here we demonstrate a new approach for introducing genes into chicken somatic tissues by administration of a lentiviral vector, derived from the feline immunodeficiency virus (FIV), into the chorioallantoic membrane (CAM) of chick embryos on embryonic day 11. The FIV-derived vectors carried yellow fluorescent protein (YFP) or recombinant alpha-melanocyte-stimulating hormone (α-MSH) genes, driven by the cytomegalovirus (CMV) promoter. Transgene expression, detected in chicks 2 days after hatch by quantitative real-time PCR, was mostly observed in the liver and spleen. Lower expression levels were also detected in the brain, kidney, heart and breast muscle. Immunofluorescence and flow cytometry analyses confirmed transgene expression in chick tissues at the protein level, demonstrating a transduction efficiency of ∼0.46% of liver cells. Integration of the viral vector into the chicken genome was demonstrated using genomic repetitive (CR1)-PCR amplification. Viability and stability of the transduced cells was confirmed using terminal deoxynucleotidyl transferase (dUTP) nick end labeling (TUNEL) assay, immunostaining with anti-proliferating cell nuclear antigen (anti-PCNA), and detection of transgene expression 51 days post transduction. Our approach led to only 9% drop in hatching efficiency compared to non-injected embryos, and all of the hatched chicks expressed the transgenes. We suggest that the transduction efficiency of FIV vectors combined with the accessibility of the CAM vasculature as a delivery route comprise a new powerful and practical approach for gene delivery into somatic tissues of chickens. Most relevant is the efficient transduction of the liver, which specializes in the production and secretion of proteins, thereby providing an optimal target for prolonged study of secreted hormones and peptides.Gideon HenSara YosefiDmitry ShinderAdi OrSivan MygdalReba CondiottiEithan GalunAmir BorDalit Sela-DonenfeldMiriam Friedman-EinatPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 7, Iss 5, p e36531 (2012)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Gideon Hen Sara Yosefi Dmitry Shinder Adi Or Sivan Mygdal Reba Condiotti Eithan Galun Amir Bor Dalit Sela-Donenfeld Miriam Friedman-Einat Gene transfer to chicks using lentiviral vectors administered via the embryonic chorioallantoic membrane.
description	The lack of affordable techniques for gene transfer in birds has inhibited the advancement of molecular studies in avian species. Here we demonstrate a new approach for introducing genes into chicken somatic tissues by administration of a lentiviral vector, derived from the feline immunodeficiency virus (FIV), into the chorioallantoic membrane (CAM) of chick embryos on embryonic day 11. The FIV-derived vectors carried yellow fluorescent protein (YFP) or recombinant alpha-melanocyte-stimulating hormone (α-MSH) genes, driven by the cytomegalovirus (CMV) promoter. Transgene expression, detected in chicks 2 days after hatch by quantitative real-time PCR, was mostly observed in the liver and spleen. Lower expression levels were also detected in the brain, kidney, heart and breast muscle. Immunofluorescence and flow cytometry analyses confirmed transgene expression in chick tissues at the protein level, demonstrating a transduction efficiency of ∼0.46% of liver cells. Integration of the viral vector into the chicken genome was demonstrated using genomic repetitive (CR1)-PCR amplification. Viability and stability of the transduced cells was confirmed using terminal deoxynucleotidyl transferase (dUTP) nick end labeling (TUNEL) assay, immunostaining with anti-proliferating cell nuclear antigen (anti-PCNA), and detection of transgene expression 51 days post transduction. Our approach led to only 9% drop in hatching efficiency compared to non-injected embryos, and all of the hatched chicks expressed the transgenes. We suggest that the transduction efficiency of FIV vectors combined with the accessibility of the CAM vasculature as a delivery route comprise a new powerful and practical approach for gene delivery into somatic tissues of chickens. Most relevant is the efficient transduction of the liver, which specializes in the production and secretion of proteins, thereby providing an optimal target for prolonged study of secreted hormones and peptides.
format	article
author	Gideon Hen Sara Yosefi Dmitry Shinder Adi Or Sivan Mygdal Reba Condiotti Eithan Galun Amir Bor Dalit Sela-Donenfeld Miriam Friedman-Einat
author_facet	Gideon Hen Sara Yosefi Dmitry Shinder Adi Or Sivan Mygdal Reba Condiotti Eithan Galun Amir Bor Dalit Sela-Donenfeld Miriam Friedman-Einat
author_sort	Gideon Hen
title	Gene transfer to chicks using lentiviral vectors administered via the embryonic chorioallantoic membrane.
title_short	Gene transfer to chicks using lentiviral vectors administered via the embryonic chorioallantoic membrane.
title_full	Gene transfer to chicks using lentiviral vectors administered via the embryonic chorioallantoic membrane.
title_fullStr	Gene transfer to chicks using lentiviral vectors administered via the embryonic chorioallantoic membrane.
title_full_unstemmed	Gene transfer to chicks using lentiviral vectors administered via the embryonic chorioallantoic membrane.
title_sort	gene transfer to chicks using lentiviral vectors administered via the embryonic chorioallantoic membrane.
publisher	Public Library of Science (PLoS)
publishDate	2012
url	https://doaj.org/article/437e5b61e79640ba94be725054b41893
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Gene transfer to chicks using lentiviral vectors administered via the embryonic chorioallantoic membrane.

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