Abstract
At present, the most reliable method for creating genetically modified chickens is the modification of the DNA sequence of primordial germ cells (PGCs). However, during embryogenesis, only a small number of chicken PGCs can be obtained. Therefore, in vitro PGC culturing is necessary to obtain sufficient cells for further genetic engineering. Previously reported PGC culturing methods lack versatility. We report here a new protocol for stable and efficient culturing of chicken PGCs using small-molecule inhibitors. The growth rate of PGCs was investigated following the addition of three small-molecule inhibitors, including blebbistatin, into the culture medium. Chicken PGC survival and proliferation rates increased after the addition of small-molecule inhibitors, compared with the untreated control. Blebbistatin was shown to be the most effective inducer of PGC growth. Long-term culturing of PGCs with blebbistatin maintained the morphology of typical PGCs, and these cells expressed marker proteins such as chicken vasa homolog (CVH) and NANOG. Additionally, PGCs transfected with a fluorescent protein gene were shown to migrate into the gonads of the recipient embryo, and progeny derived from PGCs cultured by this method were efficiently obtained. These results demonstrate that small-molecule inhibitors represent a useful tool for stable and efficient chicken PGC culturing.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F (2013) Multiplex genome engineering using CRISPR/Cas systems. Science 339:819–823
Dimitrov L, Pedersen D, Ching KH, Yi H, Collarini EJ, Izquierdo S, van de Lavoir MC, Leighton PA (2016) Germline gene editing in chickens by efficient CRISPR-mediated homologous recombination in primordial germ cells. PLoS ONE.https://doi.org/10.1371/journal.pone.0154303
Ge C, Yu M, Petitte JN, Zhang C (2009) Epidermal growth factor-induced proliferation of chicken primordial germ cells: involvement of calcium/protein kinase C and NFKB1. Biol Reprod 80:528–536
Hamburger V, Hamilton HL (1951) A series of normal stages in the development of the chick embryo. J Morphol 88:49–92
Kovács M, Tóth J, Hetényi C, Málnási-Csizmadia A, Sellers JR (2004) Mechanism of blebbistatin inhibition of myosin II. J Biol Chem 279:35557–35563
Leighton PA, van de Lavoir MC, Diamond JH, Xia C, Etches RJ (2008) Genetic modification of primordial germ cells by gene trapping, gene targeting, and phiC31 integrase. Mol Reprod Dev 75:1163–1175
Macdonald J, Taylor L, Sherman A, Kawakami K, Takahashi Y, Sang HM, McGrew MJ (2012) Efficient genetic modification and germ-line transmission of primordial germ cells using piggyBac and Tol2 transposons. Proc Natl Acad Sci USA 109:E1466–E1472
Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM (2013) RNA-guided human genome engineering via Cas9. Science 399:823–826
Miller JC, Tan S, Qiao G, Barlow KA, Wang J, Xia DF, Meng X, Paschon DE, Leung E, Hinkley SJ, Dulay GP, Hua KL, Ankoudinova I, Cost GJ, Urnov FD, Zhang HS, Holmes MC, Zhang L, Gregory PD, Rebar EJ (2011) A TALE nuclease architecture for efficient genome editing. Nat Biotechnol 29:143–148
Naito M, Harumi T, Kuwana T (2010) Long term in vitro culture of chicken primordial germ cells isolated from embryonic blood and incorporation into germline of recipient embryo. J Poult Sci 47:57–64
Nakano M, Arisawa K, Yokoyama S, Nishimoto M, Yamashita Y, Sakashita M, Ezaki R, Matsuda H, Furusawa S, Horiuchi H (2011) Characteristics of novel chicken embryonic stem cells established using chicken leukemia inhibitory factor. J Poult Sci 48:64–72
Nemudryi AA, Valetdinova KR, Medvedev SP, Zakian SM (2014) TALEN and CRISPR/Cas genome editing systems: tools of discovery. Acta Naturae 6:19–40
Nishijima K, Iijima S (2013) Transgenic chickens. Dev Growth Differ 55:207–216
Ohgushi M, Matsumura M, Eiraku M, Murakami K, Aramaki T, Nishiyama A, Muguruma K, Nakano T, Suga H, Ueno M, Ishizaki T, Suemori H, Narumiya S, Niwa H, Sasai Y (2010) Molecular pathway and cell state responsible for dissociation-induced apoptosis in human pluripotent stem cells. Cell Stem Cell 7:225–239
Oishi I, Yoshii K, Miyahara D, Kagami H, Tagami T (2016) Targeted mutagenesis in chicken using CRISPR/Cas9 system. Sci Rep. https://doi.org/10.1038/srep23980
Park TS, Han JY (2012) PiggyBac transposition into primordial germ cells is an efficient tool for transgenesis in chickens. Proc Natl Acad Sci USA 109:9337–9341
Park TS, Lee HJ, Kim KH, Kim JS, Han JY (2014) Targeted gene knockout in chickens mediated by TALENs. Proc Natl Acad Sci USA 111:12716–12721
Perry MM (1988) A complete culture system for the chick embryo. Nature 331:70–72
Pesce M, Farrace MG, Piacentini M, Dolci S, De Felici M (1993) Stem cell factor and leukemia inhibitory factor promote primordial germ cell survival by suppressing programmed cell death (apoptosis). Development 118:1089–1094
Schusser B, Collarini EJ, Yi H, Izquierdo SM, Fesler J, Pedersen D, Klasing KC, Kaspers B, Harriman WD, van de Lavoir MC, Etches RJ, Leighton PA (2013) Immunoglobulin knockout chickens via efficient homologous recombination in primordial germ cells. Proc Natl Acad Sci USA 110:20170–20175
Stankiewicz TR, Linseman DA (2014) Rho family GTPases: key players in neuronal development, neuronal survival, and neurodegeneration. Front Cell Neurosci. https://doi.org/10.3389/fncel.2014.00314
Taylor L, Carlson DF, Nandi S, Sherman A, Fahrenkrug SC, McGrew MJ (2017) Efficient TALEN-mediated gene targeting of chicken primordial germ cells. Development 144:928–934
Tres LL, Rosselot C, Kierszenbaum AL (2004) Primordial germ cells: what does it take to be alive? Mol Reprod Dev 68:1–4
Tsunekawa N, Naito M, Sakai Y, Nishida T, Noce T (2000) Isolation of chicken vasa homolog gene and tracing the origin of primordial germ cells. Development 127:2741–2750
van de Lavoir MC, Diamond JH, Leighton PA, Mather-Love C, Heyer BS, Bradshaw R, Kerchner A, Hooi LT, Gessaro TM, Swanberg SE, Delany ME, Etches RJ (2006) Germline transmission of genetically modified primordial germ cells. Nature 441:766–769
Vick L, Li Y, Simkiss K (1993) Transgenic birds from transformed primordial germ cells. Proc Biol Sci 251:179–182
Whyte J, Glover JD, Woodcock M, Brzeszczynska J, Taylor L, Sherman A, Kaiser P, McGrew MJ (2015) FGF, insulin, and SMAD signaling cooperate for avian primordial germ cell self-renewal. Stem Cell Rep 5:1171–1182
Wickman GR, Julian L, Mardilovich K, Schumacher S, Munro J, Rath N, Zander SA, Mleczak A, Sumpton D, Morrice N, Bienvenut WV, Olson MF (2013) Blebs produced by actin-myosin contraction during apoptosis release damage-associated molecular pattern proteins before secondary necrosis occurs. Cell Death Differ 10:1293–1305
Acknowledgements
We would like to thank Editage (http://www.editage.jp) for English language editing. This work was supported by the Japan Society for the Promotion of Science KAKENHI Grant Numbers 19H03107, and 19K22286.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Ethical approval
The experimental methods were approved by the animal use and care committee at the institution of Hiroshima University.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Ezaki, R., Hirose, F., Furusawa, S. et al. An improved protocol for stable and efficient culturing of chicken primordial germ cells using small-molecule inhibitors. Cytotechnology 72, 397–405 (2020). https://doi.org/10.1007/s10616-020-00385-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10616-020-00385-9