Although the terms genetically modified, genetically engineered, and transgenic organism apply to any life form whose genome has been deliberately altered, it is often used in reference to plants and animals that possess genes that were not acquired through traditional breeding. The basic techniques covered in this chapter have given scientists a limited ability to custom-build an organism that expresses a certain trait, in a sense accelerating the pace of evolution. Because of the universal nature of the genetic code, however, scientists can take nature one step further and combine genes from more than a single species, creating plants or animals that would never have existed otherwise. Examples of genetically modified organisms include plants with added nutritional value, animals that excrete clot-dissolving proteins in their milk, and hu mans who have gained the ability to produce an enzyme crucial to the immune response.
Transgenic Plants
Engineering plants to produce new traits often revolves around Agrobacterium tumefaciens, a plant pathogen particularly adept at transferring small amounts of DNA to plant cells. These bacteria live in soil and can invade injured plant tissues. Inside the wounded tissue, the bacteria adhere to plant cells and introduce a large plasmid termed Ti (tumor-inducing). This plasmid inserts into the genome of the infected plant cell and transforms it (figure 1). Over time this causes development of a tumor called crown gall disease, a mass of undifferentiated tissue on the stem.
Fig1. Bioengineering of plants. (a). Most techniques employ a natural tumor-producing bacterium called Agrobacterium tumefaciens (b–e). The example here shows the insertion of a gene to create a herbicide-resistant plant (f) Crown gall at the base of a rose bush. (f): Nigel Cattlin/Alamy Stock Photo
The primary factor that induces this transforma tion is a discrete region of the Ti plasmid called T-DNA. This series of genes is integrated into a plant cell’s chromosome, where it directs the synthesis of nutrients to feed the bacteria and hormones to stimulate plant growth. Even after the bacteria in a tumor are dead, the plasmid genes remain in the cell nucleus and the tumor continues to grow.
This plasmid is an effective vector for inserting foreign genes into plant genomes. The procedure in volves removing the Ti plasmid, inserting a specially selected gene such as herbicide resistance into it, and returning the plasmid to Agrobacterium. Infection of the plant by the recombinant bacteria automatically transfers the plasmid with the foreign gene into the plant cells. Transformed cells can be grown into mature plants that express the donor gene and transmit it to their offspring. The insertion of genes with a Ti plasmid works primarily to engineer important plants such as potatoes, tomatoes, cotton, and grapes.
For plants that cannot be transformed with Agrobacterium, seed embryos have been successfully implanted with additional genes using gene guns—small shotguns that shoot the genes into plant embryos with tiny gold bullets.
Although more than 1,200 genetically modified plants have been approved for field testing, only 11 are currently available, including apples that don’t easily brown, potatoes that don’t bruise, and rice engineered to contain genes that supply a precursor to vitamin A. However, the most common genes engineered into plants provide resistance to insects and tolerance to herbicides. The principal concern about plants with these last two traits is the potential for genetic recombination with nearby wild plants, leading to the creation of “superweeds” that are resistant to herbicides and disease. This has already happened in parts of Canada, where canola plants have transferred their genes to wild mustard. Because this could alter the ecology of natural environments, the U.S. Department of Agriculture is increasing the regulation of all releases of transgenic plants. See table 1 for examples of plants that have been engineered.
Table1. Examples of Genetically Modified Plants
Transgenic Animals: Engineering Embryos
Animals such as mice, pigs, sheep, cattle, fish, and poultry are prominent candidates for genetic engineering. Thousands of animal strains now exist for the purposes of agriculture, industry, and research into genetic diseases.
The most effective way to insert genes into animals is to use a virus to transfect the fertilized egg or early embryo (germ-line engineering). Foreign genes can also be delivered into a fertilized egg by pulsing the egg with high voltage or by injection (figure 2). The success rate does not have to be high, because once created, a transgenic animal will pass the genes on to its offspring.
Fig2. Microinjection of DNA into a fertilized egg. After being removed from the female, the egg is held with a pipette while a small needle is used to inject the transgene. Several injected embryos will be placed within the oviduct of a female mouse. After birth, a small piece of the tail of each new mouse is snipped off and examined to ensure the presence of the gene of interest. Zephyr/Science Source
The dominant animals in the genetic engineer’s laboratory are transgenic forms of mice. In one pioneering experiment, fertilized mouse embryos were injected with a gene from a jellyfish that pro duces a green fluorescent protein. The cells of these mice fluoresce in ultraviolet radiation. One serious use has been to study the developmental stages in mice. This strategy has led to the design of animal models to study human genetic diseases and to use these natural systems to test new genetic therapies before they are used in humans. Animals such as sheep or goats can also be engineered to become “factories” capable of manufacturing proteins and excreting them in their milk or semen, a process often referred to (when done to produce medically useful proteins) as “pharming.”
One benefit of transgenic animals is that they will automatically transcribe and translate eukaryotic genes and accurately carry out posttranslational modification of the protein. By ensuring that the protein product is produced in the milk or semen of animals, recovery and purification are made that much simpler. For example, alpha-1 antitrypsin (AAT), produced in sheep, is used to treat an inherited form of emphysema. A single animal produces AAT at concentrations of 35 grams per liter of milk. A few hundred sheep could easily meet the needs of the 20,000 people a year who suffer from this debilitating disease. Dolly the sheep was the first animal ever to be cloned precisely because she produced this protein. See table 2 to examine some other examples and applications of transgenic animals.
Table2. Selected Applications for Transgenic Animals
An inventive newer technology known as CRISPR—an acronym of clustered regularly interspaced short palindromic repeats— is rapidly becoming the chosen method for manipulating DNA. CRISPR and a related protein, CAS-9 (CRISPR-associated protein 9), were discovered in bacterial cells, where they work together to excise viral DNA from the genome of the cell.
The CRISPR DNA, along with adjacent viral DNA sequences, are transcribed to produce a CRISPR RNA. Hybridization between the CRISPR RNA and complementary sequences in the viral DNA create a “guide” for the Cas-9 enzyme. The Cas-9 enzyme is a re striction endonuclease and will cut any DNA to which it is guided, which in nature is the location in the genome where viral DNA has chosen to integrate. The CRISPR/Cas-9 system acts like a prokaryotic immune system, allowing bacterial cells to excise integrated viral DNA. And, like the immune system, a memory is created as the CRISPR RNA remains in the cell, ready to respond to any future attack by the same virus.
Where CRISPR/Cas-9 has been most useful (at least to hu mans) has been in the laboratory. By synthesizing a specific sequence of nucleotides, a guide RNA can be made that will hybridize to any location in the genome (figure 3). In this way, CRISPR/ Cas-9 can be used to edit DNA with a specificity never before possible. CRISPR/Cas-9 has already been used to study the effect of a gene by creating “knockout” strains of bacteria and mice, allowing scientists to see how organisms missing that specific gene function. Potential uses also include introducing genes into mosquitoes, making them sterile, thereby limiting their ability to spread diseases like malaria and Zika virus (see this chapter’s Case Study). Further in the future, scientists may be able to use CRISPR/Cas-9 to genetically engineer pigs to produce human organs for transplant or insert a CCR-5 mutation into white blood cells to prevent HIV infection.
