Derivation and characterization of primordial germ cells from Guangxi yellow-feather chickens

Wang, L.; Chen, M. J.; Chen, D. Y.; Peng, S. F.; Zhou, Xueliang; Liao, Yuying; Yang, Xiaogan; Xu, Huiyan; Lu, Sen; Zhang, M.; Ke-huan, LU; Lu, Yangqing

doi:10.3382/ps/pew387

Cited by 12 publications

(7 citation statements)

References 24 publications

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“…We isolated the PGCs from the chicken embryos, which were characterized with several markers and cytochemistry, and the results are similar to the ndings of earlier studies [9][10][11][12][13][14][15][16][17] . The chromosomes revealed both macro and micro-chromosomes in PGCs, which was i n agreement with the earlier reports 18 .…”

Section: Discussionsupporting

confidence: 78%

Production of Transgenic Chimeric Chicken from Cryopreserved Primordial Germ Cells and its Validation by Developing shRNA Transgenic Chicken Chimera

Divya

Shukla

Chatterjee

et al. 2021

Preprint

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Primordial germ cells (PGCs) are precursors of gametes in birds. For ex-situ conservation and production of transgenic birds, there is a limitation for preservation of oocytes in birds as compared to other mammalian species. To overcome those limitations, PGCs have been used as candidate cells, which have been cryopreserved and manipulated and used as to produce transgenic birds. In this study, cryo-preserved PGCs were used to produce transgenic birds. The protocol for production of transgenic birds with cryo-preserved PGCs was developed and the success rate for production of transgenics 16.7% in the protocols established in the study. The same gene transfer protocol through PGCs was validated by transferring shRNA molecule of SREBP-1 gene to the host genome to produce transgenic chimeric birds and the success rate for production of transgenic chimeric chicken was 40%. Finally, it is concluded that a standard protocol for ex-situ conservation of birds through PGCs and production of transgenic birds from cryo-preserved PGCs and knock down birds from PGCs were developed. It may be suggested that these protocols for resurrecting live birds from cryo-preserved PGCs may be applied as model for protecting the endangered birds from their extinction.

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Section: Discussionsupporting

confidence: 78%

Production of Transgenic Chimeric Chicken from Cryopreserved Primordial Germ Cells and its Validation by Developing shRNA Transgenic Chicken Chimera

Divya

Shukla

Chatterjee

et al. 2021

Preprint

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“…In previous studies, purifying PGCs removed debris and improved lipofection efficiency. [ 12,13 ] This study showed that removing sodium heparin from the PGC medium improved lipofection efficiency. This method also works well for CRISPR/Cas9‐mediated knock‐outs and knock‐ins.…”

Section: Discussionmentioning

confidence: 99%

“…It is a simple and superior gene transfer technique for the transient expression of transgenes; however, lipofection for avian PGCs is inefficient and a hurdle in a genome-edited chicken generation. [12,13] Therefore, lipofection requires improvement for efficient genome editing in chicken PGCs. PGC purification is reported to improve lipofection efficiency, suggesting the presence of debris and toxic substances in the culture medium.…”

Section: Introductionmentioning

confidence: 99%

Lipofection with Lipofectamine™ 2000 in a heparin‐free growth medium results in high transfection efficiency in chicken primordial germ cells

Watanabe

Ochi

Kajihara

et al. 2023

Biotechnology Journal

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Primordial germ cells (PGCs) that can differentiate into gametes are used to produce genome‐edited chickens. However, the transfection efficiency into PGCs is low in chickens; therefore, the yield efficiency of PGCs modified via genome editing is problematic. In this study, we improved transfection efficiency and achieved highly efficient genome editing in chicken PGCs. For transfection, we used lipofection, which is convenient for gene transfer. Chicken PGC cultures require adding heparin to support growth; however, heparin significantly reduces lipofection efficiency (p < 0.01). Heparin‐induced lipofection efficiency was restored by adding protamine. Based on these results, we optimized gene transfer into chicken PGCs. Lipofectamine™ 2000 and our PGC medium was the most efficient transfection reagent and medium, respectively. Finally, based on established conditions, we compared the gene knock‐out efficiencies of ovomucoid, a major egg allergen, and gene knock‐in efficiencies at the ACTB locus. These results indicate that optimized lipofection is useful for CRISPR/Cas9‐mediated knock‐out and knock‐in. Our findings may contribute to the generation of genome‐edited chickens and stimulate research in various applications involving them.This article is protected by copyright. All rights reserved

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“…DF-1 cells were cultured in DMEM/F12 medium containing 10% fetal bovine serum (FBS). The PGCs used were derived from a domestic chicken species (three-yellow chicken) and stored in our lab [5,36]. Chicken PGCs were cultured in a cKO medium composed of KO-DMEM, supplemented with 7.5% fetal bovine serum (FBS; Hyclone, USA), 20% BRL conditioned medium, 2.5% chicken serum (Sigma, St. Louis, MO, USA), 2 mM glutamine, 1 mM pyruvate, 1× nucleosides, 1× nonessential amino acids, 0.1 mM β-mercaptoethanol and 4 ng/mL human recombinant FGF.…”

Section: Cell Culture and Transfectionmentioning

confidence: 99%

HMEJ-mediated efficient site-specific gene integration in chicken cells

Xie

Sun

Mo³

et al. 2019

J Biol Eng

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Background: The production of transgenic chicken cells holds great promise for several diverse areas, including developmental biology and biomedical research. To this end, site-specific gene integration has been an attractive strategy for generating transgenic chicken cell lines and has been successfully adopted for inserting desired genes and regulating specific gene expression patterns. However, optimization of this method is essential for improving the efficiency of genome modification in this species. Results: Here we compare gene knock-in methods based on homology-independent targeted integration (HITI), homology-directed repair (HDR) and homology mediated end joining (HMEJ) coupled with a clustered regularly interspaced short palindromic repeat associated protein 9 (CRISPR/Cas9) gene editing system in chicken DF-1 cells and primordial germ cells (PGCs). HMEJ was found to be a robust and efficient method for gene knock-in in chicken PGCs. Using this method, we successfully labeled the germ cell specific gene DAZL and the pluripotency-related gene Pou5f3 in chicken PGCs through the insertion of a fluorescent protein in the frame at the 3′ end of the gene, allowing us to track cell migration in the embryonic gonad. HMEJ strategy was also successfully used in Ovalbumin, which accounts for more than 60% of proteins in chicken eggs, suggested its good promise for the mass production of protein with pharmaceutical importance using the chicken oviduct system. Conclusions: Taken together, these results demonstrate that HMEJ efficiently mediates site-specific gene integration in chicken PGCs, which holds great potential for the biopharmaceutical engineering of chicken cells.

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Derivation and characterization of primordial germ cells from Guangxi yellow-feather chickens

Cited by 12 publications

References 24 publications

Production of Transgenic Chimeric Chicken from Cryopreserved Primordial Germ Cells and its Validation by Developing shRNA Transgenic Chicken Chimera

Production of Transgenic Chimeric Chicken from Cryopreserved Primordial Germ Cells and its Validation by Developing shRNA Transgenic Chicken Chimera

Lipofection with Lipofectamine™ 2000 in a heparin‐free growth medium results in high transfection efficiency in chicken primordial germ cells

HMEJ-mediated efficient site-specific gene integration in chicken cells

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