Selection of culture media for chicken PGC long‐term cultivation

Abstract

Primordial germ cells (PGCs) are precursors of eggs and spermatozoa and serve as an effective means for the transgenic chickens production, including using CRISPR / Cas9 technology. The aim study is selection of PGCs culture media suitable for long‐term cultivation. All experiments on chickens were conducted with ethical approval of the Russian Research Institute of Farm Animal Genetics and Breeding ‐ Branch of the L.K. Ernst Federal Research Center for Animal Husbandry (Protocol Number: 2020‐3). For the work, eggs of the Pushkin breed chickens (n = 21) were selected from the Bioresource collection RRIFAGB (Pushkin, St. Petersburg). PGCs were selected from the embryos dorsal aorta for 3‐4 days of incubation (stage Hamburger‐Hamilton 14) using a microinjector (Narishige IM‐11‐2, Japan). Cells were cultured for 21 days at 37 ° C with 5% CO2. Three variants of the culture medium were used. The first basic medium was Opti‐MEM (Reduced Serum Medium, GlutaMAX Supplement) (Gibco, Thermo Fisher) with the addition of sodium pyruvate 1M, nucleosides 100X ‐ 2.5 ml, Chicken Serum (Gibco, Thermo Fisher) ‐ 2%, 2‐mercaptoethanol (NF, VWR) ‐ 3.9 μl. On day 21, the concentration of cells in the first culture reached 1,2х103, and some of the wells with cells were overgrown. The second base medium was similar to the first base medium, but with the addition of antimycotic antibiotic (Thermo Fisher) up to 1X. The medium with the use of the antibiotic‐antimycotic was less susceptible to overgrowth, on day 21, the concentration of cells in the culture reached 3.5x104. The third basic medium was KnockOut DMEM / F‐12, without L‐glutamine (Gibco, ThermoFisher) with the addition sodium pyruvate 1M, nucleosides 100X ‐ 2.5 ml, Human Activin A Recombinant Protein (Gibco, Thermo Fisher) ‐ 25 ng / μl, Human FGF‐basic (FGF‐2 / bFGF) Recombinant Protein (Gibco, Thermo Fisher) ‐ 10 ng / μl, Chicken Serum (Gibco, Thermo Fisher) ‐ 2%, 2‐mercaptoethanol (NF, VWR) ‐ 3.9 μl), antimycotic antibiotic (Thermo Fisher) up to 1X. The third medium with the addition of Activin A and FGF‐2 / bFGF supported the cell culture better, overgrowth was practically not observed, cells were actively proliferating, оn day 21, the concentration of cells in the culture reached 2.7x105. The PGCs presence was confirmed by expression high level (p≤0.01) specific marker genes PGCs such as CXCR4 (chemokine receptor CXC type 4) and PIWIL1 (Piwi‐like protein 1) in cultured cells. In a control tissue small intestine sample of an adult chicken (330 days old), the above genes expression was completely absent.