Genetic modification of roosters’ germ cells using various methodological approaches

Gonad germ cells of male farm birds are considered as promising target cells for introducing recombinant DNA for the purpose of targeted genetic modification of their genome. Germ cell precursors (primordial germ cells and spermatogonia cells) are of most interest for modifications. The transplantation of these transformed cells and their successful colonization in the gonads of recipient individuals would allow a population of transformed mature germ cells - sperm - to be obtained, which can be used for female insemination in order to obtain transgenic offspring. The results of studies on the genetic modification of rooster germ cells by transfection of primordial germ cells in embryos and spermatogenous cells in the testes were presented in this work. The novelty of the research consists in the development and optimization of individual stages of local transformation in roosters’ spermatogenic cells in vivo to obtain a genetically modified sperm. Our aim was to evaluate the effectiveness of the genetic transformation in roosters’ germ cells using various methodological approaches. The study was carried out with poultry ( Gallus gallus domesticus ) of the Russian White breed. Primordial germ cells were isolated from 6-day-old embryos. The resulting culture of PGCs was transformed by electroporation using the Neon system (Thermo Fisher Scientific, USA). For transfection, the ZsGreen1-N1 plasmid (Addgene, USA) with the ZsGreen gene under the CMV promoter was used. Transformed cells in the amount of 400, 700 and 1000 were introduced into the dorsal aorta of 2.5-day-old embryos. The embryos of the control group were injected with DMEM growth medium in the dorsal aorta. To transform spermatogenic cells in vivo, a viral preparation was used, which was injected directly into the testes of roosters by multiple injection. The introduction of the viral drugs was carried out once at the age of 3 or 4 months and twice at the age of 3 and 4 months. The viral preparation at a concentration of 1½107 CFU/ml was introduced at the rate of 0.5 ml per testis. The lentiviral vector contained the ZsGreen reporter gene under the CMV promoter. Histological sections of the testes from experimental males were obtained and analyzed to assess the efficiency of colonization and development of donor primordial germ cells (PGCs) in the gonads of recipients, as well as to evaluate the effectiveness of spermatogenic cell transformation in vivo. As a control, we used histological sections of the testes from non-transgenic roosters, selected on the basis of analogues (age, breed). The fertilizing ability of the sperm from experimental roosters and the proportion of embryos with ZsGreen gene expression were evaluated. The transformation efficiency of target cells was determined by expression of the ZsGreen reporter gene using a Nikon Ni-U microscope (Nikon, Japan). The chicken embryonic cell culture obtained in the first stage of the experiment consisted of the several types of cells. The proportion of PGCs did not exceed 3 %. The percentage of PGCs in the cell suspension increased to 81 % after separating the different types of embryonic cells by adhesion. The cell culture transformation efficiency of PGCs was 12 %.The presence of fluorescent spermatogenic cells in the testes seminiferous tubules was established both with the introduction of transformed donor PGCs and with a lentiviral vector. With the introduction of donor PGCs at a concentration of 400, 700 and 1000 cells per embryo, the percentage of chickens with transformed germ cells was 16 %, 23 % and 26 %, respectively. With the twofold introduction of the viral drug into the testes at the age of 3 and 4 months, the highest transformation efficiency of spermatogenic testicular cells in vivo was established, which amounted to 10 %. With a single injection of the viral drug, this indicator was 2 times lower. The possibility of using the obtained individuals with transformed germ cells to obtain transgenic offspring is shown. The efficiency of obtaining transgenic embryos is 6-10 %.