Expert of Antibody Production and Genetic Modification

Plasmid Construction - DNA microinjection

Animal transgenesis with DNA microinjection

Transgenic model organisms can be routinely generated by purified DNA microinjection. This technique is commonly used in animal transgenesis since 1980 's. [1]

During this method, the solution of purified and linear DNA is injected into the pronuclei of fertilized oocytes. The process of microinjection is implemented under invert microscope equipped with two micromanipulators connected to holder, and injection capillaries. The injected genetic elements can integrate into the genome during chromosome replication. This method belongs to additive transgenesis.

Fertilized oocytes are obtained from hormone treated females. After the embryo flushing, the collected one cell stage embryos are injected with the required DNA solution within few hours. Following pronuclear injection, surviving embryos are surgically transferred into the oviducts of pseudopregnant females.

The transgene integration efficiency is generally low, 1-25% of animals born from these implanted embryos may contain the injected construct. The overall efficiency can depend on the choice of animal species and strains, [2] and it is usually inversely proportionate to the size of the animal species used.

The integration of DNA into the genome is random and normally stable germ-line transmission can be observed. Transgenes often integrate as concatamers into the genome, so copy number of the gene of interest can be high [3]. The advantage of the method is that even large size of DNA fragments can be microinjected (minichromosomes).

As integration is random the expression of the transgene is unpredictable so establishment of more than one transgenic line is highly recommended.


[1]      J. W. Gordon, G. A. Scangos, D. J. Plotkin, J. A. Barbosa, and F. H. Ruddle, “Genetic transformation of mouse embryos by microinjection of purified DNA.,” Proc. Natl. Acad. Sci. U. S. A., vol. 77, no. 12, pp. 7380–4, 1980.
 
[2]      R. L. Brinster, H. Y. Chen, M. E. Trumbauer, M. K. Yagle, and R. D. Palmiter, “Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs.,” Proc. Natl. Acad. Sci. U. S. A., vol. 82, no. 13, pp. 4438–42, 1985.
 
[3]      R. L. Brinster, H. Y. Chen, M. Trumbauer, A. W. Senear, R. Warren, and R. D. Palmiter, “Somatic expression of herpes thymidine kinase in mice following injection of a fusion gene into eggs,” Cell, vol. 27, no. 1 PART 2, pp. 223–231, 1981.