8.7.1 General principles of transgenesis
Transgenic organisms contain foreign DNA that has been introduced using biotechnology. Foreign DNA (the transgene) is defined here as DNA from another species, or else recombinant DNA from the same species that has been manipulated in the laboratory then reintroduced. The terms transgenic organism and genetically modified organism (GMO) are generally synonymous. The process of creating transgenic organisms or cells to be come whole organisms with a permanent change to their germline has been called either transformation or transfection. (Unfortunately, both words have alternate meanings. Transformation also refers to the process of mammalian cell becoming cancerous, while transfection also refers to the process of introducing DNA into cells in culture, either bacterial or eukaryote, for a temporary use, not germ line changes.) Transgenic organisms are important research tools, and are often used when exploring a gene’s function. Transgenesis is also related to the medical practice of gene therapy, in which DNA is transferred into a patient’s cells to treat disease. Transgenic organisms are widespread in agriculture. Approximately 90% of canola, cotton, corn, soybean, and sugar beets grown in North America are transgenic. No other transgenic livestock or crops (except some squash, papaya, and alfalfa) are currently produced in North America.
To make a transgenic cell, DNA must first be transferred across the cell membrane, (and, if present, across the cell wall), without destroying the cell. In some cases, naked DNA (meaning plasmid or linear DNA that is not bound to any type of carrier) may be transferred into the cell by adding DNA to the medium and temporarily increasing the porosity of the membrane, for example by electroporation. When working with larger cells, naked DNA can also be microinjected into a cell using a specialized needle. Other methods use vectors to transport DNA across the membrane. Note that the word “vector” as used here refers to any type of carrier, and not just plasmid vectors. Vectors for transformation/transfection include vesicles made of lipids or other polymers that surround DNA; various types of particles that carry DNA on their surface; and infectious viruses and bacteria that naturally transfer their own DNA into a host cell, but which have been engineered to transfer any DNA molecule of interest. Usually the foreign DNA is a complete expression unit that includes its own cis-regulators (e.g. promoter) as well as the gene that is to be transcribed.
When the objective of an experiment is to produce a stable (i.e. heritable) transgenic eukaryote, the foreign DNA must be incorporated into the host’s chromosomes. For this to occur, the foreign DNA must enter the host’s nucleus, and recombine with one of the host’s chromatids. In some species, the foreign DNA is inserted at a random location in a chromatid, probably wherever strand breakage and non-homologous end joining happen to occur. In other species, the foreign DNA can be targeted to a particular locus, by flanking the foreign DNA with DNA that is homologous to the host’s DNA at that locus. The foreign DNA is then incorporated into the host’s chromosomes through homologous recombination.
Furthermore, to produce multicellular organisms in which all cells are transgenic and the transgene is stably inherited, the cell that was originally transformed must be either a gamete or must develop into tissues that produce gametes. Transgenic gametes can eventually be mated to produce homozygous, transgenic offspring. The presence of the transgene in the offspring is typically confirmed using PCR or Southern blotting, and the expression of the transgene can be measured using reverse-transcription PCR (RT-PCR), RNA blotting, and Western (protein blotting).
The rate of transcription of a transgene is highly dependent on the state of the chromatin into which it is inserted (i.e. position effects), as well as other factors. Therefore, researchers often generate several independently transformed/transfected lines with the same transgene, and then screen for the lines with the highest expression. It is also good practice to clone and sequence the transgenic locus from a newly generated transgenic organism, since errors (truncations, rearrangements, and other mutations) can be introduced during transformation/transfection.
8.7.2 Producing a transgenic plant
The most common method for producing transgenic plants is Agrobacterium-mediated transformation (Figure 8.17). Agrobacterium tumifaciens is a soil bacterium that, as part of its natural pathogenesis, injects its own tumor-inducing (Ti) plasmid into cells of a host plant. The natural Ti plasmid encodes growth-promoting genes that cause a gall (i.e. tumor) to form on the plant, which also provides an environment for the pathogen to proliferate. Molecular biologists have engineered the Ti plasmid by removing the tumor-inducing genes and adding restriction sites that make it convenient to insert any DNA of interest. This engineered version is called a T-DNA (transfer-DNA) plasmid; the bacterium transfers a linear fragment of this plasmid that includes the conserved “left-border (LB)”, and right-border (RB)” DNA sequences, and anything in between them (up to about 10 kb). The linear T-DNA fragment is transported into the nucleus, where it recombines with the host-DNA, probably wherever random breakages occur in the host’s chromosomes. In Arabidopsis and a few other species, flowers can simply be dipped in a suspension of Agrobacterium, and ~1% of the resulting seeds will be transformed. In most other plant species, cells are induced by hormones to form a mass of undifferentiated tissues called a callus. The Agrobacterium is applied to a callus and a few cells are transformed, which can then be induced by other hormones to regenerate whole plants (Figure 8.18). Some plant species are resistant (i.e. “recalcitrant”) to transformation by Agrobacterium. In these situations, other techniques must be used such as particle bombardment, whereby DNA is non-covalently attached to small metallic particles, which are accelerated by compressed air into callus tissue, from which complete transgenic plants can sometimes be regenerated. In all transformation methods, the presence of a selectable marker (e.g. a gene that confers antibiotic resistance or herbicide resistance) is useful for distinguishing transgenic cells from non-transgenic cells at an early stage of the transformation process.
Figure 8.17: Production of a transgenic plant using Agrobacterium-mediated transformation. The bacterium has been transformed with a T-DNA plasmid that contains the transgene and a selectable marker that confers resistance to a herbicide or antibiotic. A bacterial culture and plant tissue (e.g. a leaf punch) are co-cultured on growth medium in a Petri dish. Some of the plant cells will become infected by the bacterium, which will transfer the T-DNA into the plant cytoplasm. In some cases the transgene will become integrated into the chromosomal DNA of a plant cell. In the presence of certain combinations of hormones, the plant cells will dedifferentiate into a mass of cells called callus. The presence of a selective agent (e.g. herbicide or antibiotic) in the growth medium prevents untransformed cells from dividing. Therefore, each callus ideally consists only of transgenic plant cells. The resistant calli are transferred to media with other combinations of hormones that promote organogenesis, i.e. differentiation of callus cells into shoots and then roots. The regenerated transgenic plants are transferred to soil. Their seeds can be harvested and tested to ensure that the transgene is stably inherited. (Original-Deyholos-CC:AN)
Figure 8.18: Organogenesis of flax shoots from calli. (Original-J. McDill-CC:AN)
8.7.3 Producing a transgenic mouse
In a commonly used method for producing a transgenic mouse, stem cells are removed from a mouse embryo, and a transgenic DNA construct is transferred into the stem cells using electroporation, and some of this transgenic DNA enters the nucleus, where it may undergo homologous recombination (Figure 8.19). The transgenic DNA construct contains DNA homologous to either side of a locus that is to be targeted for replacement. If the objective of the experiment is simply to delete (“knock-out”) the targeted locus, the host’s DNA can simply be replaced by selectable marker, as shown. It is also possible to replace the host’s DNA at this locus with a different version of the same gene, or a completely different gene, depending on how the transgenic construct is made. Cells that have been transfected and express the selectable marker (i.e. resistance to the antibiotic neomycin resistance, neoR, in this example) are distinguished from unsuccessfully transfected cells by their ability to survive in the presence of the selective agent (e.g. an antibiotic). Transfected cells are then injected into early stage embryos, and then are transferred to a foster mother. The resulting pups are chimeras, meaning that only some of their cells are transgenic. Some of the chimeras will produce gametes that are transgenic, which when mated with a wild-type gamete, will produce mice that are hemizygous for the transgene. Unlike the chimeras, these hemizygotes carry the transgene in all of their cells. Through further breeding, mice that are homozygous for the transgene can be obtained.
Figure 8.19: Production of a transgenic mouse. Stem cells are removed from an embryo, and are transfected (using electroporation) with a transgenic construct that bears a neomycin resistance gene (neor) flanked by two segments of DNA homologous to a gene of interest. In the nucleus of a transgenic cell, some of the foreign DNA will recombine with the targeted gene, disrupting the targeted gene and introducing the selectable marker. Only cells in which neor has been incorporated will survive selection. These neomycin resistant cells are then transplanted into another embryo, which will grow into a chimera within a foster mother. (Wikipedia-Kiaergaard-CC:AN)
8.7.4 Human gene therapy
Many different strategies for human gene therapy are under development. In theory, either the germline or somatic cells may be targeted for transfection, but most research has focused on somatic cell transfection, because of risks and ethical issues associated with germline transformation. Gene therapy approaches may be further classified as either ex vivo or in vivo, with the former meaning that cells (e.g. stem cells) are transfected in isolation before being introduced to the body, where they replace defective cells. Ex vivo gene therapies for several blood disorders (e.g. immunodeficiencies, thalassemias) are undergoing clinical trials. For in vivo therapies, the transfection occurs within the patient. The objective may be either stable integration, or non-integrative transfection. As described above, stable transfection involves integration into the host genome. In the clinical context, stable integration may not be necessary, and carries with it higher risk of inducing mutations in either the transgene or host genome). In contrast, transient transfection does not involve integration into the host genome and the transgene may therefore be delivered to the cell as either RNA or DNA. Advantages of RNA delivery include that no promoter is needed to drive expression of the transgene. Besides mRNA transgenes, which could provide a functional version of a mutant protein, there is great interest in delivery of siRNA (small-inhibitory RNAs), which can be used to silence specific genes in the host cell’s genome.
Vectors for in vivo gene therapy must be capable of delivering DNA or RNA to a large proportion of the targeted cells, without inducing a significant immune response, or having any toxic effects. Ideally, the vectors should also have high specificity for the targeted cell type. Vectors based on viruses (e.g. lentiviruses) are being developed for in both in vivo and ex vivo gene therapies. Other, non-viral vectors (e.g. vesicles and nanoparticles) are also being developed for gene therapy as well.
Dr. Todd Nickle and Isabelle Barrette-Ng (Mount Royal University) The content on this page is licensed under CC SA 3.0 licensing guidelines.