Abstract: The present application provides an antibiotic resistance gene-free miniplasmid, comprising a nucleotide sequence encoding an antitoxin protein, and a replicon. A backbone of the antibiotic resistance gene-free miniplasmid of the present application is smaller and controlled within 1,000 bp, redundant useless fragments are reduced, and meanwhile, the miniplasmid does not comprise a resistance expression cassette, thereby improving the utilization rate of a sequence of interest in the plasmid and reducing the production burden. The miniplasmid has higher efficiency and safety in the plasmid DNA-mediated non-viral vector delivery, gene therapy, vaccine immunity, virus production, antibody production, and other genetic engineering.

Abstract: A dorsal aortic injection method of chicken embryos for improving transgenic efficiency, comprising the following steps: injecting adeno-associated virus particles into the dorsal aorta of each 2.5-day-old chicken embryo outside eggshell, individually transferring the injected chicken embryos into their recipient eggshells (coming from double yolk eggs), and proceeding to hatch. The disclosure realizes the outside-eggshell injection of the chicken embryo dorsal aorta through the modified dorsal aortic injection method, improves the accuracy of injection, and also realizes the direct observation of the development process of the chicken embryos.