Cultivation and characterization of primordial germ cells from blue layer hybrids (Araucana crossbreeds) and generation of germline chimeric chickens

Abstract The chicken (Gallus gallus) is one of the most common and widespread domestic species, with an estimated total population of 25 billion birds worldwide. The vast majority of chickens in agriculture originate from hybrid breeding programs and is concentrated on few commercially used high per...

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Autores principales:	Stefanie Altgilbers, Sabine Klein, Claudia Dierks, Steffen Weigend, Wilfried A. Kues
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/f0989119e0d04542ba759247cf8881fd
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spelling	oai:doaj.org-article:f0989119e0d04542ba759247cf8881fd2021-12-02T16:07:04ZCultivation and characterization of primordial germ cells from blue layer hybrids (Araucana crossbreeds) and generation of germline chimeric chickens10.1038/s41598-021-91490-y2045-2322https://doaj.org/article/f0989119e0d04542ba759247cf8881fd2021-06-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-91490-yhttps://doaj.org/toc/2045-2322Abstract The chicken (Gallus gallus) is one of the most common and widespread domestic species, with an estimated total population of 25 billion birds worldwide. The vast majority of chickens in agriculture originate from hybrid breeding programs and is concentrated on few commercially used high performance lines, whereas numerous local and indigenous breeds are at risk to become extinct. To preserve the genomic resources of rare and endangered chicken breeds innovative methods are necessary. Here, we established a solid workflow for the derivation and biobanking of chicken primordial germ cells (PGCs) from blue layer hybrids. To achieve this, embryos of a cross of heterozygous blue egg layers were sampled to obtain blood derived and gonadal male as well as female PGCs of different genotypes (homozygous, heterozygous and nullizygous blue-allele bearing). The total efficiency of established PGC lines was 45% (47/104) within an average of 49 days until they reached sufficient numbers of cells for cryopreservation. The stem-cell character of the cultivated PGCs was confirmed by SSEA-1 immunostaining, and RT-PCR amplification of the pluripotency- and PGC-specific genes cPOUV, cNANOG, cDAZL and CVH. The Sleeping Beauty transposon system allowed to generate a stable integration of a Venus fluorophore reporter into the chicken genome. Finally, we demonstrated that, after re-transfer into chicken embryos, Venus-positive PGCs migrated and colonized the forming gonads. Semen samples of 13 raised cell chimeric roosters were analyzed by flow cytometry for the efficiency of germline colonization by the transferred PGCs carrying the Venus reporter and their proper differentiation into vital spermatids. Thus, we provide a proof-of-concept study for the potential use of PGCs for the cryobanking of rare breeds or rare alleles.Stefanie AltgilbersSabine KleinClaudia DierksSteffen WeigendWilfried A. KuesNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-12 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Stefanie Altgilbers Sabine Klein Claudia Dierks Steffen Weigend Wilfried A. Kues Cultivation and characterization of primordial germ cells from blue layer hybrids (Araucana crossbreeds) and generation of germline chimeric chickens
description	Abstract The chicken (Gallus gallus) is one of the most common and widespread domestic species, with an estimated total population of 25 billion birds worldwide. The vast majority of chickens in agriculture originate from hybrid breeding programs and is concentrated on few commercially used high performance lines, whereas numerous local and indigenous breeds are at risk to become extinct. To preserve the genomic resources of rare and endangered chicken breeds innovative methods are necessary. Here, we established a solid workflow for the derivation and biobanking of chicken primordial germ cells (PGCs) from blue layer hybrids. To achieve this, embryos of a cross of heterozygous blue egg layers were sampled to obtain blood derived and gonadal male as well as female PGCs of different genotypes (homozygous, heterozygous and nullizygous blue-allele bearing). The total efficiency of established PGC lines was 45% (47/104) within an average of 49 days until they reached sufficient numbers of cells for cryopreservation. The stem-cell character of the cultivated PGCs was confirmed by SSEA-1 immunostaining, and RT-PCR amplification of the pluripotency- and PGC-specific genes cPOUV, cNANOG, cDAZL and CVH. The Sleeping Beauty transposon system allowed to generate a stable integration of a Venus fluorophore reporter into the chicken genome. Finally, we demonstrated that, after re-transfer into chicken embryos, Venus-positive PGCs migrated and colonized the forming gonads. Semen samples of 13 raised cell chimeric roosters were analyzed by flow cytometry for the efficiency of germline colonization by the transferred PGCs carrying the Venus reporter and their proper differentiation into vital spermatids. Thus, we provide a proof-of-concept study for the potential use of PGCs for the cryobanking of rare breeds or rare alleles.
format	article
author	Stefanie Altgilbers Sabine Klein Claudia Dierks Steffen Weigend Wilfried A. Kues
author_facet	Stefanie Altgilbers Sabine Klein Claudia Dierks Steffen Weigend Wilfried A. Kues
author_sort	Stefanie Altgilbers
title	Cultivation and characterization of primordial germ cells from blue layer hybrids (Araucana crossbreeds) and generation of germline chimeric chickens
title_short	Cultivation and characterization of primordial germ cells from blue layer hybrids (Araucana crossbreeds) and generation of germline chimeric chickens
title_full	Cultivation and characterization of primordial germ cells from blue layer hybrids (Araucana crossbreeds) and generation of germline chimeric chickens
title_fullStr	Cultivation and characterization of primordial germ cells from blue layer hybrids (Araucana crossbreeds) and generation of germline chimeric chickens
title_full_unstemmed	Cultivation and characterization of primordial germ cells from blue layer hybrids (Araucana crossbreeds) and generation of germline chimeric chickens
title_sort	cultivation and characterization of primordial germ cells from blue layer hybrids (araucana crossbreeds) and generation of germline chimeric chickens
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/f0989119e0d04542ba759247cf8881fd
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Cultivation and characterization of primordial germ cells from blue layer hybrids (Araucana crossbreeds) and generation of germline chimeric chickens

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