GSE161135
Antisense RNA technology induces the loss of function of steroidogenic factor 1 leading to abortion of tilapia
As a precise and efficient genetic engineering method, gene editing technology for animals and plants will gradually move towards large-scale applications, and will have a disruptive impact on the breeding of related traits. By using antisense RNA interference technology to interfere with the mRNA expression and protein translation of the SF-1 gene, we have successfully cultivated tilapia with defective gonadal development. The results of the study showed that the weights of male and female fish in the experimental group were significantly higher than those of the control group. The gonadal weight and gonadal index of the experimental group were significantly lower than those of the control group and the negative control group. HE staining showed that the number of spermatogonia in the testis tissue of the experimental group was significantly reduced, the cavity in the seminiferous tubules was significantly enlarged and increased, the number of sperm cells was significantly decreased, and the number of sperm was relatively rare. However, a large number of stage V oocytes accumulated in the ovarian tissue of female fish in the experimental group, and many of them were gray-white. The mRNA and protein expression levels of SF-1 gene in the gonadal tissues were extremely significantly lower than those of the control and negative controls. The combined analysis of transcriptome and proteome showed that Steroid hormone biosynthesis, Arachidonic acid metabolism and Cell adhesion molecules are involved in the regulation of the gonads of male fish after SF-1 gene silencing. However, ECM-receptor interaction, Retinol metabolism, Nicotinate and nicotinamide metabolism and cytochrome P450 may be involved in the regulation of female fish SF-1 gene silencing. SF-1 silencing may cause developmental defects in the external reproductive organs of male and female fishes and inhibit the development of gonadal tissues through different regulatory pathways. This provides data support for the further functional analysis of SF-1 on fish and the treatment of abnormal human SF-1 expression. Overall design: The use of the present disclosure preferably comprises the following steps: 1) cloning the Anti-SF1-1-I, Anti-SF1-1-II, Anti-SF1-2-I and Anti-SF1-2-II into expression vectors to obtain recombinant vectors; 2) using the recombination vectors as templates, and performing PCR amplification with specific primers to obtain four amplified products; 3) after mixing the four amplified products with a cationic liposome and a spermatozoa preservative fluid respectively, equilibrating at 15-30oC for 20-40 min to obtain transfection reagents; 4) mixing the transfection reagents with the eggs for transfection to obtain transfected eggs; 5) mixing the transfected eggs with sperms for fertilization and hatching to obtain abortive tilapia fries.