Efficiency of busulfan use for eliminating primordial germ cells in the gonads of chicken embryos

The use of primordial germ cells to produce genetically modified and chimeric poultry is one of the promising areas of biotechnology. It is also considered as an alternative to traditional methods of selection and genome modification. The production of transgenic and chimeric animals using this technology provides the introduction of donor primordial germ cells into the dorsal aorta of recipient embryos during the migration of this cell type from the blood to the gonads. In the case of the colonization by donor germ cells of the recipient embryos gonads, further differentiation of donor cells to mature germ cells (both male and female) is possible. One of the main factors that determine the effectiveness of conducted manipulations is eliminating own primordial germ cells in a recipient embryos. In this regard, it remains urgent to develop effective methodical techniques for removing this type of cells. The aim of our study was to optimize methodological approaches for eliminating primordial germ cells in the gonads of chicken embryos. Busulfan was used as an alkylating agent in concentrations from 10 to 250 μg/embryo. Dimethyl sulfoxide (DMSO) was used to dilute busulfan, as well as its combination with DMEM medium in various ratios. The busulfan solution was injected into the chicken embryos prior to incubation or after 24 hour incubation. The efficiency of elimination of primordial germ cells in embryonic gonads was assessed on day 7of incubation based on histological studies using immunohistochemistry on the expression of the SSEA-1 gene (stage-specific embryonic antigen-1). It has been established that the method of preparation and introduction of busulfan into embryos, as well as the dose of this drug determine the effectiveness of elimination primordial germ cells. Reduction of the negative effect on the development of chicken embryos when using DMSO for the dilution of busulfan was shown when the DMEM medium was included in the solution formulation at a concentration of 10 %. The high efficiency of eliminating primordial germ cells was observed when busulfan was used at a concentration of 100 μg per embryo. In this case, the number of germ cells in the gonads declined by 92 % compared to the control. The use of busulfan at a higher concentration while maintaining the effectiveness of this drug was characterized by an increase in embryonic mortality. At the same time, higher effectiveness of eliminating primordial germ cells was achieved with the introduction of busulfan after 24-hour incubation of embryos: the number of germ cells in the gonads of 7-day-old chicken embryos was 12.5 % less than in the case of injection of this drug before incubation.