Introducing Recombinant Molecules into Eukaryotic Hosts

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Electroporation is one laboratory technique used to introduce DNA into eukaryotic cells.

Source: OpenStax Microbiology

OpenStax Microbiology

The use of bacterial hosts for genetic engineering laid the foundation for recombinant DNA technology; however, researchers have also had great interest in genetically engineering eukaryotic cells, particularly those of plants and animals. The introduction of recombinant DNA molecules into eukaryotic hosts is called transfection. Genetically engineered plants, called transgenic plants, are of significant interest for agricultural and pharmaceutical purposes. The first transgenic plant sold commercially was the Flavr Savr delayed-ripening tomato, which came to market in 1994. Genetically engineered livestock have also been successfully produced, resulting, for example, in pigs with increased nutritional value and goats that secrete pharmaceutical products in their milk.

Electroporation

Compared to bacterial cells, eukaryotic cells tend to be less amenable as hosts for recombinant DNA molecules. Because eukaryotes are typically neither competent to take up foreign DNA nor able to maintain plasmids, transfection of eukaryotic hosts is far more challenging and requires more intrusive techniques for success. One method used for transfecting cells in cell culture is called electroporation. A brief electric pulse induces the formation of transient pores in the phospholipid bilayers of cells through which the gene can be introduced. At the same time, the electric pulse generates a short-lived positive charge on one side of the cell’s interior and a negative charge on the opposite side; the charge difference draws negatively charged DNA molecules into the cell.

Microinjection

An alternative method of transfection is called microinjection. Because eukaryotic cells are typically larger than those of prokaryotes, DNA fragments can sometimes be directly injected into the cytoplasm using a glass micropipette.

Microinjection is another technique for introducing DNA into eukaryotic cells. A microinjection needle containing recombinant DNA is able to penetrate both the cell membrane and nuclear envelope.

Source: OpenStax Microbiology

Gene Guns

Transfecting plant cells can be even more difficult than animal cells because of their thick cell walls. One approach involves treating plant cells with enzymes to remove their cell walls, producing protoplasts. Then, a gene gun is used to shoot gold or tungsten particles coated with recombinant DNA molecules into the plant protoplasts at high speeds. Recipient protoplast cells can then recover and be used to generate new transgenic plants.

Heavy-metal particles coated with recombinant DNA are shot into plant protoplasts using a gene gun. The resulting transformed cells are allowed to recover and can be used to generate recombinant plants. (a) A schematic of a gene gun. (b) A photograph of a gene gun. (credit a, b: modification of work by JA O’Brien, SC Lummis)

Shuttle Vectors

Another method of transfecting plants involves shuttle vectors, plasmids that can move between bacterial and eukaryotic cells. The tumor-inducing (Ti ) plasmids originating from the bacterium Agrobacterium tumefaciens are commonly used as shuttle vectors for incorporating genes into plants. In nature, the Ti plasmids of A. tumefaciens cause plants to develop tumors when they are transferred from bacterial cells to plant cells. Researchers have been able to manipulate these naturally occurring plasmids to remove their tumor-causing genes and insert desirable DNA fragments. The resulting recombinant Ti plasmids can be transferred into the plant genome through the natural transfer of Ti plasmids from the bacterium to the plant host. Once inside the plant host cell, the gene of interest recombines into the plant cell’s genome.

The Ti plasmid of Agrobacterium tumefaciens is a useful shuttle vector for the uptake of genes of interest into plant cells. The gene of interest is cloned into the Ti plasmid, which is then introduced into plant cells. The gene of interest then recombines into the plant cell’s genome, allowing for the production of transgenic plants.

Viral Vectors

Viral vectors can also be used to transfect eukaryotic cells. In fact, this method is often used in gene therapy to introduce healthy genes into human patients suffering from diseases that result from genetic mutations. Viral genes can be deleted and replaced with the gene to be delivered to the patient; the virus then infects the host cell and delivers the foreign DNA into the genome of the targeted cell. Adenoviruses are often used for this purpose because they can be grown to high titer and can infect both nondividing and dividing host cells. However, use of viral vectors for gene therapy can pose some risks for patients.

Source:

Parker, N., Schneegurt, M., Thi Tu, A.-H., Forster, B. M., & Lister, P. (n.d.). Microbiology. Houston, Texas: OpenStax. Access for free at: https://openstax.org/details/books/microbiology


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