Genetic transformation

In molecular biology transformation is the genetic alteration of a cell resulting from the uptake, incorporation and expression of exogenous genetic material (DNA) that is taken up through the cell wall(s). Transformation occurs most commonly in bacteria and in some species occurs naturally. Transformation can also be effected by artificial means. Bacteria that are capable of being transformed, whether naturally or artificially, are called competent. Genetic material can also be transferred between cells by conjugation or transduction. Conjugation involves the direct contact of two different bacterial cells with the DNA being transferred from one cell to the other. In transduction, viruses called bacteriophages inject the foreign DNA into their host. Introduction of foreign DNA into eukaryotic cells is usually called "transfection". Transformation is also used to describe the insertion of new genetic material into nonbacterial cells including animal and plant cells.

History

Transformation was first demonstrated in 1928 by Frederick Griffith, an English bacteriologist searching for a vaccine against bacterial pneumonia. Griffith discovered that a harmless strain of Streptococcus pneumoniae could be made virulent after being exposed to heat-killed virulent strains. Griffith hypothesized that some "transforming factor" from the heat-killed strain was responsible for making the harmless strain virulent. In 1944 this "transforming factor" was identified as being genetic by Oswald Avery, Colin MacLeod, and Maclyn McCarty. They isolated DNA from a virulent strain of S. pneumoniae and using just this DNA were able to make a harmless strain virulent. They called this uptake and incorporation of DNA by bacteria "transformation." See Avery-MacLeod-McCarty experiment.

The results of Avery et al.'s experiments were at first sceptically received by the scientific community and it was not until the development of genetic markers and the discovery of other methods of genetic transfer (conjugation in 1947 and transduction in 1953) by Joshua Lederberg that Avery's experiments were accepted. Transformation did not become routine procedure in laboratories until 1972 when Stanley Cohen, Annie Chang and Leslie Hsu successfully transformed Escherichia coli by treating the bacteria with calcium chloride. This created an efficient and convenient procedure for transforming bacteria and opened the way for molecular cloning in biotechnology and research.

Transformation using electroporation was developed in the late 1980s thus increasing the efficiency and number of bacterial strains that could be transformed. Transformation of animal and plant cells was also investigated with the first transgenic mouse being created by injecting a gene for a rat growth hormone into a mouse embryo in 1982. In 1907 a bacterium that caused plant tumors, Agrobacterium tumefaciens, was discovered and in the early 1970s the tumor inducing agent was found to be a DNA plasmid called the Ti plasmid. By removing the genes in the plasmid that caused the cancer and adding in novel genes researchers were able to infect plants with A. tumefaciens and let the bacteria insert their chosen DNA into the genomes of the plants. Not all plant cells are susceptible to infection by A. tumefaciens so other methods were developed including electroporation and micro-injection. Particle bombardment was made possible with the invention of the Biolistic Particle Delivery System (gene gun) by John Sanford in 1990.

Mechanisms

Bacteria

Bacteria transformation may be referred to as a stable genetic change brought about by the uptake of naked DNA (DNA without associated cells or proteins) and competence refers to the state of being able to take up exogenous DNA from the environment. Two forms of competence exist: natural and artificial.

Natural competence

Main article: Competence (biology)

About 1% of bacterial species are capable of naturally taking up DNA under laboratory conditions; many more are able to take it up in their natural environments. Such bacteria carry sets of genes that provide the protein machinery to bring DNA across the cell membrane(s).

Artificial competence

Artificial competence is induced by laboratory procedures and involves making the cell passively permeable to DNA by exposing it to conditions that do not normally occur in nature.

Calcium chloride transformation is a method of promoting competence. Chilling cells in the presence of divalent cations such as Ca²⁺ (in CaCl₂) prepares the cell membrane to become permeable to plasmid DNA. The cells are incubated on ice with the DNA and then briefly heat shocked (e.g., 42°C for 30–120 seconds) thus allowing the DNA to enter the cells. This method works very well for circular plasmid DNA. An excellent preparation of competent cells will give ~10⁸ colonies per microgram of plasmid. A poor preparation will be about 10⁴/μg or less. Good, non-commercial preparations should give 10⁵ to 10⁶ transformants per microgram of plasmid. The method, however, usually does not work well for linear DNA, such as fragments of chromosomal DNA, probably because the cell's native exonuclease enzymes rapidly degrade linear DNA. Interestingly, cells that are naturally competent are usually transformed more efficiently with linear DNA than with plasmid DNA.

Electroporation is another method of promoting competence. In the method the cells are briefly shocked with an electric field of 10-20 kV/cm that creates holes in the cell membrane through which the plasmid DNA enters. This method is amenable to the uptake of large plasmid DNA. After the electric shock the holes are rapidly closed by the cell's membrane-repair mechanisms.

The efficiency with which a competent culture can take up exogenous DNA and express its genes is known as Transformation efficiency.

Plasmid transformation

In order to be stably maintained in the cell a plasmid DNA molecule must contain an origin of replication, which allows it to be replicated in the cell independent of the replication of the cell's own chromosome. Because transformation usually produces a mixture of relatively few transformed cells and an abundance of non-transformed cells a method is needed to identify the cells that have acquired the plasmid. The method usually consists of using a plasmid that contains a gene that gives the bacterial cells resistance to an antibiotic that they are naturally sensitive to. The mixture of cells are then plated on media that contains the antibiotic thus only the transformed cells are able to grow. Cells that did not take up the plasmid are killed in the media.

Another selection method called blue-white screen uses a plasmid that contains an antibiotic resistance gene and the lacZ gene. The lacZ gene codes for the lacZ-α subunit of the enzyme β-galactosidase, a homo-tetramer with each monomer composed of one lacZ-α subunit and one lacZ-ω subunit. The method also requires an E. coli strain that possesses in its genome the code for only the lacZ-ω subunit and not the lacZ-α subunit. One of the first steps in any transformation is the production of a recombined plasmid obtained by the successful ligation of the gene of interest into its corresponding vector, which in this method results in the disruption of lacZ because the gene of interest is inserted within the lacZ code. A cell that takes up a recombined plasmid would thus not be able to express the lacZ-α subunit and would, in turn, not be able to produce a functional β-galactosidase. Conversely, a cell that has taken up non-recombined plasmid (perhaps one formed by the ligation of the vector's own two ends) will express the lacZ-α subunit and thus produce a functional β-galactosidase. A cell that does not take up any plasmid is not conferred with antibiotic resistance and will die upon plating. Consequently, the blue-white screen method allows for the ready detection of not just transformed cells, but, most importantly, cells that have been transformed by a successfully recombined plasmid. Selection occurs as a result of the action of β-galactosidase on its substrate X-gal, which is included in the media along with the appropriate antibiotic. X-gal is a colorless, modified galactose sugar whose hydrolysis by β-galactosidase produces galactose and the pre-chromophore 5-bromo-4-chloro-3-hydroxyindole. The latter is subsequently oxidized to 5,5'-dibromo-4,4'-dichloro-indigo, an insoluble, blue product that is readily seen by the naked eye. Colonies of cells that have been transformed by a successfully recombined plasmid will thus appear white whereas those that have been transformed by non-recombined plasmid will appear blue.

Plants

A number of mechanisms are available to transfer DNA into plant cells:

• Agrobacterium mediated transformation is the easiest and most simple plant transformation. Plant tissue (often leaves) is cut into small pieces, e.g. 10x10mm, and soaked for 10 minutes in a fluid containing suspended Agrobacterium. Some cells along the cut will be transformed by the bacterium, that inserts its DNA into the cell. Placed on selectable rooting and shooting media, the plants will regrow. Some plants species can be transformed just by dipping the flowers into suspension of Agrobacterium and then planting the seeds in a selective medium. Unfortunately, many plants are not transformable by this method.
• Particle bombardment: Particles of gold or tungsten are coated with DNA and then shot into young plant cells or plant embryos. Some genetic material will stay in the cells and transform them. This method also allows transformation of plant plastids. The transformation efficiency is lower than in agribacterial mediated transformation, but most plants can be transformed with this method.
• Electroporation: make transient holes in cell membranes using electric shock; this allows DNA to enter as described above for Bacteria.
• Viral transformation (transduction): Package the desired genetic material into a suitable plant virus and allow this modified virus to infect the plant. If the genetic material is DNA, it can recombine with the chromosomes to produce transformant cells. However genomes of most plant viruses consist of single stranded RNA which replicates in the cytoplasm of infected cell. For such genomes this method is a form of transfection and not a real transformation, since the inserted genes never reach the nucleus of the cell and do not integrate into the host genome. The progeny of the infected plants is virus free and also free of the inserted gene.

Animals

Introduction of DNA into animal cells is usually called transfection, and is discussed in the corresponding article.

External links

• Bacterial Transformation (a Flash Animation)