| As is known to all,pig is an important agricultural economic animal and biomedical animal.It is of great significance to carry out efficient and accurate gene editing and establish a gene modification model of pig.As far as agricultural breeding is concerned,genetic modification of pig models can effectively improve the growth rate of individual animals,improve production conditions,enhance disease resistance and promote the breeding of new varieties.The pig is an ideal animal model for biomedical research.Compared with rodents,pigs are closer to humans in terms of anatomy,physiology,biochemical characteristics,nutrient metabolism,and immune system.Compared with primates,pigs have a shorter reproductive cycle,produce more offspring,and have fewer ethical constraints.In recent years,nuclease gene editing technologies,including ZFN,Talen and CRISPR/Cas9 gene editing tools,have emerged.Due to their high efficiency,they provide powerful tools for animal gene editing,especially for large animals such as pigs,rabbits and monkeys.However,due to the imperfection of the underlying technical systems such as superovulation,in vitro embryo construction and in vitro embryo manipulation,large animal gene editing has not been widely used.In order to solve the underlying technical problems of pig application in agriculture and regenerative medicine,this study took miniature pigs as experimental animal model,took the typical immunodeficiency gene FOXN1 as the target gene,combined with the simultaneous estrus,superovulation and CRISPR/Cas technology,and constructed a pig model with FOXN1 gene knockout.The results showed that the estrus rate of sows was the highest when pregnant horse serum gonadotropin PMSG was injected.According to the body weight of the experimental individuals,PMSg at concentrations of 0 IU/kg,10 IU/kg,16 IU/kg,22 IU/kg,28 IU/kg and 34 IU/kg were injected along the gradient,and it was found that the effect of 22 IU/kg treatment was better than that of other treatment groups.Based on gene sequencing,T cell count and thymus volume assessment,it was confirmed that the single-allele FOXN1 gene knockout pig model was successfully constructed.The gene knockout pig technology established in this study can effectively improve the efficiency of embryo production and has important reference value for the establishment of a fast and stable experimental platform for gene knockout pig model animals. |
