Development of optimal technological approaches for obtaining PGCs in Pushkin breed chickens for further transformation by the CRISPR / Cas9 system

Abstract

The CRISPR / Cas9 genome editing system is a very convenient and promising method for creating highly productive chickens of meat direction with improved productive qualities. The biological feature of the amniotes structure creates certain difficulties for the an editing structure introduction into a unicellular zygote. The aim of the study is to optimize technological approaches for obtaining PGCs (Primordial Germ Cells) in Pushkin chickens from the Bioresource collection RRIFAGB “Genetic collection of rare and endangered chicken breeds” (Pushkin, St. Petersburg) for further transformation by the CRISPR / Cas9 system. All experiments on chickens were conducted with the 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). The optimal egg weight varied from 55 to 62 grams for incubation It has been revealed that embryos obtained from large eggs from 62 grams or more developed poorly or did not reach the stage of PGCs selection at all. The eggs were incubated with occasional shaking at 37–38 ° C and 65–70% relative humidity. It was better to select PGCs of Pushkin breed chickens on the 3rd day of incubation from the dorsal aorta of embryos (Hamburger Hamilton stage 14). Cell selection was performed using a microinjector (Narishige IM-11-2, Japan). The cells were cultured for 21 days at 37 ° C with 5% CO2. PGCs have been found to reproduce better in a serum-free medium (MEM Non-Essential Amino Acids Solution, Gibco, Thermo Fisher) of a specific chemical composition than in a medium containing serum (Opti-MEM with Chicken Serum, Gibco, Thermo Fisher). The expression of PGCs various genes characteristic was determined by real-time PCR using conservative primer sets.