There are four primary methods used in biology to produce transgenic organisms: transgene engineering, knock-in engineering using embryonic stem (ES) cells, knock-in engineering using CRISPR, and viral gene delivery. Here, I will go over the first two of these methods. Transgene engineering was the first time foreign genetic material was incorporated into an animal’s genome. Palmiter and Brinster added the gene for human growth hormone (HGH) to zygotic cells during the stage at which the genomes of each parent cell are fusing in order to get random incorporation of the gene. In order to get expression of this gene they also packaged a promoter with the transgene which would ensure its expression in transformed mice. The next step in genetic engineering came with knock-in engineering in ES cells developed by Capecchi, Martin, and Smithies. This method involves using a new kind of transgene that lacks a promoter but has gained a neomycin resistance gene and homologous arms. A promoter is unnecessary as the arms of homology will target the transgene to a specific place in the genome after a promoter that is already active. The transgenes are then added to cultured ES cells. The cells that take up the transgene and undergo a double-stranded (DS) break that matches the transgene’s arms of homology get transformed. Then, the antibiotic neomycin is added to the cultured cells to select for the cells that were transformed. These transformed cells are injected into a blastocyst where they can be expressed.
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