Transgenesis biotechnological procedures influence on domestic duck embryos survival

Abstract

Due to its high reproductive potential, short interval between generations and embryonic development outside the mother's body, the bird provides unique opportunities for its use in fundamental and applied biological research. The creation of a transgenic bird is complicated by the structure of its opaque egg cell with a large yolk and a unique reproductive system of this class. Direct microinjection of DNA into an oocyte, which is often used in mammals, is practically impossible for birds, since fertilization occurs in the infudibulum of the reproductive tract and can be polyspermic. Therefore, manipulations with the zygote turned out to be difficult for their use in creating a transgenic bird. Over the past decades, some alternative strategies have been developed for producing transgenic poultry using bizarre animals created by transferring blastodermal cells. However, to date, the efficiency of creating transgenic poultry in many cases remains very low, and the technique of using ducks to create transgenic poultry is practically not developed. Busulfan is used to suppress cell proliferation. Injection of busulfan into the pidembryonic cavity is one of the methods that increases the number of donor cells when creating chimeras. However, until now, methods of creating hermentative ducks chimeras face difficulties associated with the structure of the shell of waterfowl. Therefore, the aim of the work was to establish the effect of factors influencing the survival of transgenic embryos when using various methods of introducing a DNA construct into the duck genome. The objects of the study were ducks (Anas platyrhynchos) of the Shan partridge duck and Shaoxing breeds kept at the duck farm of Zhuji Guowei Poultry Development Co., Ltd, China. The studies were carried out in the poultry genetics laboratory of the Zhejiang Academy of Agricultural Sciences and on the duck farm of Zhejiang Generation Biological Science and Technology Co., Ltd. (Zhejiang Province, PRC). For the analysis of survival, we used embryos obtained by using various methods of introducing the DNA (insertion of the EGFP gene, mediated by homologous repair (HDR)) 1) direct injection of the DNA construct into the sub-embryonic cavity; 2) transfection of DNA with sperm; 3) injection of transfected donor blastomeres into recipient embryos after exposure to busulfan or ultraviolet radiation. A total more than 1100 eggs were examined. As a result of the direct injection of a transgenic DNA construction ( sub-embryonic cavity of 300 embryos, 35.7% of embryos did not develop after injection, 36% stopped developing at the time of the first ovoscopy (day 9 of incubation), 8% died within 10-15 days, 17, 3% - 16-25 days. In total, after direct injections, 9 live ducklings were received (the survival rate was 3%), of which 4 were transgenic. After insemination of ducks transfected with sperm, 292 eggs were laid for incubation. After the first ovoscopy, 51.4% of the eggs were unfertilized; 0.7% of embryos stopped developing at the time of the first ovoscopy (9 day of incubation), 1.0% died within 10-15 days, 17.8% - 16-25 days, 6.2% suffocated during hatching. In total, after using the transfected sperm, 67 live ducklings were obtained (the survival rate of embryos from fertilized eggs was 47.2%). Among 31 adult animals, 19 were transgenic. To sterilize recipient cells for the use of busulfan at a concentration of 300 ng per egg, followed by injection of blastodermal transfected donor cells, 200 embryos were examined, among which 61.0% of embryos developed after injection, 17.0% stopped in development at the time of the first ovoscopy (day 9 of incubation ), 12.5% of those died in the period of 10-15 days, 9.0% - 16-25 days. In total, after injections of busulfan at a concentration of 300 ng per egg, 1 live duckling was obtained (the survival rate was 0.5%). Using busulfan at a concentration of 150 ng per egg, 100 embryos were examined, among which 68.0% of embryos developed after injection, 11.0% stopped developing at the time of the first ovoscopy (day 9 of incubation), 5% died within 10-15 days, 14.0% - 16-25 days. In total, after injections of busulfan at a concentration of 150 ng per egg, 2 live ducklings were obtained (the survival rate was 0.5%). Using busulfan at a concentration of 75 ng per egg, 100 embryos were examined, among which 12.0% of embryos developed after injection, 27.0% stopped developing at the time of the first ovoscopy (9 day of incubation), 14.0% died in the period 10-15 days, 42.0% - 16-25 days. In total, after injections of busulfan at a concentration of 75 ng per egg, 5 live ducklings were obtained (the survival rate was 5%). Ultraviolet irradiation of 200 embryos for 1 hour followed by injection of blastodermal transfected donor cells resulted in death after injection of 20%, stopped developing 27.5% (9 days of incubation), 7.5% died within 10-15 days , 35.0% - 16-25 days. A total of 20 live ducklings were obtained using ultraviolet radiation (survival rate was 10%). Among 13 adult animals gave offspring, 7 were transgenic chimeras. The use of ultraviolet light has reduced the impact of egg infection due to the structure of waterfowl shells. Thus, the safest for the survival of embryos was the method of insemination of ducks with transfected sperm, using which 47.2% of embryos survived.