Exogenous DNA

In 1928, bacteriologist Fredrick Griffith observed exogenous DNA alongside bacterial transformation in the species Streptococcus pneumoniae.^[7][3] In further tests, physician Oswald Avery was able to isolate and confirm that the DNA used in the experiment originated from outside the cell and integrated itself into the cell's genome. Repeated experiments proved exogenous DNA integration was possible in other species of bacteria, prompting studies to extend to mammal cells.^[3] The technology for the injection of exogenous DNA into organisms was discovered by Lin in 1966. He was able to use a fine glass needle to insert laboratory-produced DNA into mouse zygotes without breaking their nuclei. In 1976, the first successful delivery of exogenous DNA into mice was performed by Jaenisch using the Moloney leukemia virus.^[5]

Transformation

The integration of exogenous DNA with the genome of a cell is called transformation (transfection in animal cells).^[3][8] Transformation is a naturally occurring process in bacteria. To successfully take up exogenous DNA, bacteria need to be in a state of competence. Some bacteria are naturally competent, but usually only for a brief time at a certain stage of their growth cycle.^[9] Bacteria can also be made competent through a variety of chemical treatments. These treatments typically involve making the targeted cell membrane more permeable towards accepting exogenous DNA, one such example being exposing the bacteria to a calcium ion solution, or a mixture of polyethylene glycol and dimethylsulfoxide.^[10] Another treatment method is the utilization of electricity (electroporation or electro transformation) to create holes in the cell membrane for the DNA to enter. Finally, liposome-mediated transformation can be used. The cell surface and the incoming DNA are both negatively charged, so the DNA is coated with lipids. By shielding the DNA and possibly merging with the membrane lipids, these liposomes can facilitate the entry of DNA.^[8]

Transformation of bacteria, plant cells and animal cells has important research and commercial functions. Targeted introduction of exogenous DNA is used to identify genes because the introduced DNA can cause a mutation or alter the expression of the targeted gene, providing a unique identifying signal. This technology, known as insertion mutagenesis, often employs retroviruses as the vectors of DNA delivery. Such insertion mutagenesis has been often used to identify many oncogenes in specific locations in tumor cells.^[11]

Transfection

Transfection is the process of introducing exogenous DNA into eukaryotic cells.^[12] It is a more specific term for animal cells, as the process of carcinogenesis in these cells is also included in the definition of transformation. Typically, transfection describes the changes in a cell's genome due to the introduction of foreign DNA.^[4] There are several ways of conducting artificial transfection. Chemical methods involve using chemicals as carriers to introduce DNA, such as calcium phosphate precipitation, DEAE-dextran complexation and lipid-mediated DNA transfer.^[13] Physical methods use techniques such as electroporation, microinjection, and cell squeezing to increase the permeability of the cell membrane for accepting DNA.^[14] Viral methods (or transduction) use recombinant, lab manipulated viruses as vectors to alter embryos and sperm cells.^[8]

Transgenesis

The use of exogenous DNA to transform cells has spawned the discipline of transgenesis: the use of recombinant DNA techniques to introduce new characters into organisms, mainly through transgenes.^[15] A transgene is an introduced DNA segment that be used to encode a gene in its host animal.^[16] Biologists uses transgenesis as a tool to breed genetically modified, or transgenic animals that provide a wide range of uses. These include the study of developmental genetics, disease processes and gene regulation.^[17] For example, transgenic farm animals can produce human pharmaceuticals alongside increased milk or meat production. Tissues and organs from transgenic animals can also be used in transfusions and transplants with a lesser chance of immune rejection.^[18]

Sperm cells

Using transgenesis to genetically modify animals has spawned a new division of using exogenous DNA to modify sperm cells. Epididymal sperm cells were shown to react to exogenous nucleic acids, allowing for DNA to reversibly bind to the spermatozoa through ionic interactions.^[19] The ability of sperm cells to locate and internalize exogenous DNA was then used to transfer foreign genes into an oocyte during fertilization to create transgenic animals.^[6] However, a low efficiency rate hinders this technique due to the low uptake of exogenous DNA by sperm cells compounded with the low fertilization rate of the oocyte.^[20]

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Transformation (genetics)

Transformation (genetics)

In molecular biology and genetics, transformation is the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material from its surroundings through the cell membrane(s). For transformation to take place, the recipient bacterium must be in a state of competence, which might occur in nature as a time-limited response to environmental conditions such as starvation and cell density, and may also be induced in a laboratory.

Transfection

Transfection

Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. It may also refer to other methods and cell types, although other terms are often preferred: "transformation" is typically used to describe non-viral DNA transfer in bacteria and non-animal eukaryotic cells, including plant cells. In animal cells, transfection is the preferred term as transformation is also used to refer to progression to a cancerous state (carcinogenesis) in these cells. Transduction is often used to describe virus-mediated gene transfer into eukaryotic cells.

Polyethylene glycol

Polyethylene glycol

Polyethylene glycol (PEG; ) is a polyether compound derived from petroleum with many applications, from industrial manufacturing to medicine. PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE), depending on its molecular weight. The structure of PEG is commonly expressed as H−(O−CH2−CH2)n−OH.

Electroporation

Electroporation

Electroporation, or electropermeabilization, is a microbiology technique in which an electrical field is applied to cells in order to increase the permeability of the cell membrane, allowing chemicals, drugs, electrode arrays or DNA to be introduced into the cell. In microbiology, the process of electroporation is often used to transform bacteria, yeast, or plant protoplasts by introducing new coding DNA. If bacteria and plasmids are mixed together, the plasmids can be transferred into the bacteria after electroporation, though depending on what is being transferred, cell-penetrating peptides or CellSqueeze could also be used. Electroporation works by passing thousands of volts across suspended cells in an electroporation cuvette. Afterwards, the cells have to be handled carefully until they have had a chance to divide, producing new cells that contain reproduced plasmids. This process is approximately ten times more effective in increasing cell membrane's permeability than chemical transformation.

Retrovirus

Retrovirus

A retrovirus is a type of virus that inserts a DNA copy of its RNA genome into the DNA of a host cell that it invades, thus changing the genome of that cell. After invading a host cell's cytoplasm, the virus uses its own reverse transcriptase enzyme to produce DNA from its RNA genome, the reverse of the usual pattern, thus retro (backwards). The new DNA is then incorporated into the host cell genome by an integrase enzyme, at which point the retroviral DNA is referred to as a provirus. The host cell then treats the viral DNA as part of its own genome, transcribing and translating the viral genes along with the cell's own genes, producing the proteins required to assemble new copies of the virus. Many retroviruses cause serious diseases in humans, other mammals, and birds.

Oncogene

Oncogene

An oncogene is a gene that has the potential to cause cancer. In tumor cells, these genes are often mutated, or expressed at high levels.

Precipitation (chemistry)

Precipitation (chemistry)

In an aqueous solution, precipitation is the process of transforming a dissolved substance into an insoluble solid from a super-saturated solution. The solid formed is called the precipitate. In case of an inorganic chemical reaction leading to precipitation, the chemical reagent causing the solid to form is called the precipitant.

Microinjection

Microinjection

Microinjection is the use of a glass micropipette to inject a liquid substance at a microscopic or borderline macroscopic level. The target is often a living cell but may also include intercellular space. Microinjection is a simple mechanical process usually involving an inverted microscope with a magnification power of around 200x.

Transduction (genetics)

Transduction (genetics)

Transduction is the process by which foreign DNA is introduced into a cell by a virus or viral vector. An example is the viral transfer of DNA from one bacterium to another and hence an example of horizontal gene transfer. Transduction does not require physical contact between the cell donating the DNA and the cell receiving the DNA, and it is DNase resistant. Transduction is a common tool used by molecular biologists to stably introduce a foreign gene into a host cell's genome.

Regulation of gene expression

Regulation of gene expression

Regulation of gene expression, or gene regulation, includes a wide range of mechanisms that are used by cells to increase or decrease the production of specific gene products. Sophisticated programs of gene expression are widely observed in biology, for example to trigger developmental pathways, respond to environmental stimuli, or adapt to new food sources. Virtually any step of gene expression can be modulated, from transcriptional initiation, to RNA processing, and to the post-translational modification of a protein. Often, one gene regulator controls another, and so on, in a gene regulatory network.