Exogenous DNA

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Exogenous DNA strands (Red and Green) shown inside a cell's nucleus.

Exogenous DNA is DNA originating outside the organism of concern or study. [1] Exogenous DNA can be found naturally in the form of partially degraded fragments left over from dead cells. These DNA fragments may then become integrated into the chromosomes of nearby bacterial cells to undergo mutagenesis. [2] This process of altering bacteria is known as transformation. [3] Bacteria may also undergo artificial transformation through chemical and biological processes. The introduction of exogenous DNA into eukaryotic cells is known as transfection. [4] Exogenous DNA can also be artificially inserted into the genome, which revolutionized the process of genetic modification in animals. By microinjecting an artificial transgene into the nucleus of an animal embryo, the exogenous DNA is allowed to merge the cell's existing DNA to create a genetically modified, transgenic animal. [5] The creation of transgenic animals also leads into the study of altering sperm cells with exogenous DNA. [6]

Contents

History

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]

Applications

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]

See also

Related Research Articles

<span class="mw-page-title-main">Genetically modified organism</span> Organisms whose genetic material has been altered using genetic engineering methods

A genetically modified organism (GMO) is any organism whose genetic material has been altered using genetic engineering techniques. The exact definition of a genetically modified organism and what constitutes genetic engineering varies, with the most common being an organism altered in a way that "does not occur naturally by mating and/or natural recombination". A wide variety of organisms have been genetically modified (GM), from animals to plants and microorganisms. Genes have been transferred within the same species, across species, and even across kingdoms. New genes can be introduced, or endogenous genes can be enhanced, altered, or knocked out.

Molecular biology is a branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions.

<span class="mw-page-title-main">Molecular genetics</span> Scientific study of genes at the molecular level

Molecular genetics is a sub-field of biology that addresses how differences in the structures or expression of DNA molecules manifests as variation among organisms. Molecular genetics often applies an "investigative approach" to determine the structure and/or function of genes in an organism's genome using genetic screens. The field of study is based on the merging of several sub-fields in biology: classical Mendelian inheritance, cellular biology, molecular biology, biochemistry, and biotechnology. Researchers search for mutations in a gene or induce mutations in a gene to link a gene sequence to a specific phenotype. Molecular genetics is a powerful methodology for linking mutations to genetic conditions that may aid the search for treatments/cures for various genetics diseases.

<span class="mw-page-title-main">Transformation (genetics)</span> Genetic alteration of a cell by uptake of genetic material from the environment

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 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.

<span class="mw-page-title-main">Transduction (genetics)</span> Transfer process in 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.

<span class="mw-page-title-main">Transfer DNA</span> Type of DNA in bacterial genomes

The transfer DNA is the transferred DNA of the tumor-inducing (Ti) plasmid of some species of bacteria such as Agrobacterium tumefaciens and Agrobacterium rhizogenes . The T-DNA is transferred from bacterium into the host plant's nuclear DNA genome. The capability of this specialized tumor-inducing (Ti) plasmid is attributed to two essential regions required for DNA transfer to the host cell. The T-DNA is bordered by 25-base-pair repeats on each end. Transfer is initiated at the right border and terminated at the left border and requires the vir genes of the Ti plasmid.

<span class="mw-page-title-main">Gene gun</span> Device used in genetic engineering

In genetic engineering, a gene gun or biolistic particle delivery system is a device used to deliver exogenous DNA (transgenes), RNA, or protein to cells. By coating particles of a heavy metal with a gene of interest and firing these micro-projectiles into cells using mechanical force, an integration of desired genetic information can be introduced into desired cells. The technique involved with such micro-projectile delivery of DNA is often referred to as biolistics, short for "biological ballistics".

A DNA construct is an artificially-designed segment of DNA borne on a vector that can be used to incorporate genetic material into a target tissue or cell. A DNA construct contains a DNA insert, called a transgene, delivered via a transformation vector which allows the insert sequence to be replicated and/or expressed in the target cell. This gene can be cloned from a naturally occurring gene, or synthetically constructed. The vector can be delivered using physical, chemical or viral methods. Typically, the vectors used in DNA constructs contain an origin of replication, a multiple cloning site, and a selectable marker. Certain vectors can carry additional regulatory elements based on the expression system involved.

<span class="mw-page-title-main">RecA</span> DNA repair protein

RecA is a 38 kilodalton protein essential for the repair and maintenance of DNA. A RecA structural and functional homolog has been found in every species in which one has been seriously sought and serves as an archetype for this class of homologous DNA repair proteins. The homologous protein is called RAD51 in eukaryotes and RadA in archaea.

A transgene is a gene that has been transferred naturally, or by any of a number of genetic engineering techniques, from one organism to another. The introduction of a transgene, in a process known as transgenesis, has the potential to change the phenotype of an organism. Transgene describes a segment of DNA containing a gene sequence that has been isolated from one organism and is introduced into a different organism. This non-native segment of DNA may either retain the ability to produce RNA or protein in the transgenic organism or alter the normal function of the transgenic organism's genetic code. In general, the DNA is incorporated into the organism's germ line. For example, in higher vertebrates this can be accomplished by injecting the foreign DNA into the nucleus of a fertilized ovum. This technique is routinely used to introduce human disease genes or other genes of interest into strains of laboratory mice to study the function or pathology involved with that particular gene.

<span class="mw-page-title-main">Gene delivery</span> Introduction of foreign genetic material into host cells

Gene delivery is the process of introducing foreign genetic material, such as DNA or RNA, into host cells. Gene delivery must reach the genome of the host cell to induce gene expression. Successful gene delivery requires the foreign gene delivery to remain stable within the host cell and can either integrate into the genome or replicate independently of it. This requires foreign DNA to be synthesized as part of a vector, which is designed to enter the desired host cell and deliver the transgene to that cell's genome. Vectors utilized as the method for gene delivery can be divided into two categories, recombinant viruses and synthetic vectors.

<span class="mw-page-title-main">Transplastomic plant</span>

A transplastomic plant is a genetically modified plant in which genes are inactivated, modified or new foreign genes are inserted into the DNA of plastids like the chloroplast instead of nuclear DNA.

Sperm-mediated gene transfer (SMGT) is a transgenic technique that transfers genes based on the ability of sperm cells to spontaneously bind to and internalize exogenous DNA and transport it into an oocyte during fertilization to produce genetically modified animals.1 Exogenous DNA refers to DNA that originates outside of the organism. Transgenic animals have been obtained using SMGT, but the efficiency of this technique is low. Low efficiency is mainly due to low uptake of exogenous DNA by the spermatozoa, reducing the chances of fertilizing the oocytes with transfected spermatozoa.2 In order to successfully produce transgenic animals by SMGT, the spermatozoa must attach the exogenous DNA into the head and these transfected spermatozoa must maintain their functionality to fertilize the oocyte.2 Genetically modified animals produced by SMGT are useful for research in biomedical, agricultural, and veterinary fields of study. SMGT could also be useful in generating animals as models for human diseases or lead to future discoveries relating to human gene therapy.

In molecular cloning, a vector is any particle used as a vehicle to artificially carry a foreign nucleic sequence – usually DNA – into another cell, where it can be replicated and/or expressed. A vector containing foreign DNA is termed recombinant DNA. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Of these, the most commonly used vectors are plasmids. Common to all engineered vectors are an origin of replication, a multicloning site, and a selectable marker.

<span class="mw-page-title-main">Genetically modified animal</span> Animal that has been genetically modified

Genetically modified animals are animals that have been genetically modified for a variety of purposes including producing drugs, enhancing yields, increasing resistance to disease, etc. The vast majority of genetically modified animals are at the research stage while the number close to entering the market remains small.

<span class="mw-page-title-main">Genetically modified fish</span>

Genetically modified fish are organisms from the taxonomic clade which includes the classes Agnatha, Chondrichthyes and Osteichthyes whose genetic material (DNA) has been altered using genetic engineering techniques. In most cases, the aim is to introduce a new trait to the fish which does not occur naturally in the species, i.e. transgenesis.

<span class="mw-page-title-main">Molecular cloning</span> Set of methods in molecular biology


Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms. The use of the word cloning refers to the fact that the method involves the replication of one molecule to produce a population of cells with identical DNA molecules. Molecular cloning generally uses DNA sequences from two different organisms: the species that is the source of the DNA to be cloned, and the species that will serve as the living host for replication of the recombinant DNA. Molecular cloning methods are central to many contemporary areas of modern biology and medicine.

<span class="mw-page-title-main">History of genetic engineering</span>

Genetic engineering is the science of manipulating genetic material of an organism. The first artificial genetic modification accomplished using biotechnology was transgenesis, the process of transferring genes from one organism to another, first accomplished by Herbert Boyer and Stanley Cohen in 1973. It was the result of a series of advancements in techniques that allowed the direct modification of the genome. Important advances included the discovery of restriction enzymes and DNA ligases, the ability to design plasmids and technologies like polymerase chain reaction and sequencing. Transformation of the DNA into a host organism was accomplished with the invention of biolistics, Agrobacterium-mediated recombination and microinjection. The first genetically modified animal was a mouse created in 1974 by Rudolf Jaenisch. In 1976 the technology was commercialised, with the advent of genetically modified bacteria that produced somatostatin, followed by insulin in 1978. In 1983 an antibiotic resistant gene was inserted into tobacco, leading to the first genetically engineered plant. Advances followed that allowed scientists to manipulate and add genes to a variety of different organisms and induce a range of different effects. Plants were first commercialized with virus resistant tobacco released in China in 1992. The first genetically modified food was the Flavr Savr tomato marketed in 1994. By 2010, 29 countries had planted commercialized biotech crops. In 2000 a paper published in Science introduced golden rice, the first food developed with increased nutrient value.

<span class="mw-page-title-main">Genetic engineering techniques</span> Methods used to change the DNA of organisms

Genetic engineering techniques allow the modification of animal and plant genomes. Techniques have been devised to insert, delete, and modify DNA at multiple levels, ranging from a specific base pair in a specific gene to entire genes. There are a number of steps that are followed before a genetically modified organism (GMO) is created. Genetic engineers must first choose what gene they wish to insert, modify, or delete. The gene must then be isolated and incorporated, along with other genetic elements, into a suitable vector. This vector is then used to insert the gene into the host genome, creating a transgenic or edited organism.

References

  1. ↑ "Exogenous DNA definition". groups.molbiosci.northwestern.edu. Retrieved 2021-11-20.
  2. ↑ Hakansson, Anders P.; Marks, Laura R.; Roche-Hakansson, Hazeline (2015-01-01), Brown, Jeremy; Hammerschmidt, Sven; Orihuela, Carlos (eds.), "Chapter 7 - Pneumococcal Genetic Transformation During Colonization and Biofilm Formation", Streptococcus Pneumoniae, Amsterdam: Academic Press, pp. 129–142, ISBN   978-0-12-410530-0 , retrieved 2021-10-28
  3. 1 2 3 4 Farley, George E.; (5-1-1969) "Transformation of mammalian cells by exogenous DNA" University of Nebraska Medical Center, pp. 3-8, retrieved 2021-10-28
  4. 1 2 "Dorlands Medical Dictionary:transfection". 2009-02-13. Archived from the original on 2009-02-13. Retrieved 2021-11-20.
  5. 1 2 Pritchett-Corning, Kathleen R.; Landel, Carlisle P. (2015-01-01), Fox, James G.; Anderson, Lynn C.; Otto, Glen M.; Pritchett-Corning, Kathleen R. (eds.), "Chapter 32 - Genetically Modified Animals", Laboratory Animal Medicine (Third Edition), American College of Laboratory Animal Medicine, Boston: Academic Press, pp. 1417–1440, ISBN   978-0-12-409527-4 , retrieved 2021-10-28
  6. 1 2 Lavitrano, Marialuisa; Giovannoni, Roberto; Cerrito, Maria Grazia (2013). "Methods for Sperm-Mediated Gene Transfer". Spermatogenesis. Methods in Molecular Biology. Vol. 927. pp. 519–529. doi:10.1007/978-1-62703-038-0_44. ISBN   978-1-62703-037-3. ISSN   1940-6029. PMID   22992941.
  7. ↑ Griffith, Fred. (1928). "The Significance of Pneumococcal Types". The Journal of Hygiene. 27 (2): 113–159. doi:10.1017/s0022172400031879. ISSN   0022-1724. PMC   2167760 . PMID   20474956.
  8. 1 2 3 "Bacterial Transformation and Competent Cells–A Brief Introduction - US". www.thermofisher.com. Retrieved 2021-10-28.
  9. ↑ Chen, Inês; Dubnau, David (2004). "DNA uptake during bacterial transformation". Nature Reviews Microbiology. 2 (3): 241–249. doi:10.1038/nrmicro844. ISSN   1740-1534. PMID   15083159. S2CID   205499369.
  10. ↑ Wertz, John E.; Low, K. Brooks (2017-01-01), "Genetics, Microbial (general)☆", Reference Module in Biomedical Sciences, Elsevier, ISBN   978-0-12-801238-3 , retrieved 2021-10-28
  11. ↑ Uren, A. G.; Kool, J.; Berns, A.; van Lohuizen, M. (2005). "Retroviral insertional mutagenesis: past, present and future". Oncogene. 24 (52): 7656–7672. doi:10.1038/sj.onc.1209043. ISSN   1476-5594. PMID   16299527. S2CID   14441244.
  12. ↑ "MeSH Browser". meshb.nlm.nih.gov. Retrieved 2021-11-20.
  13. ↑ Jordan, Martin; Schallhorn, Annette; Wurm, Florian M (1996). "Transfecting Mammalian Cells: Optimization of Critical Parameters Affecting Calcium-Phosphate Precipitate Formation". Nucleic Acids Research. 24 (4): 596–601. doi:10.1093/nar/24.4.596. PMC   145683 . PMID   8604299 . Retrieved 2021-11-20.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  14. ↑ Sharei, Armon; Zoldan, Janet; Adamo, Andrea; Sim, Woo Young; Cho, Nahyun; Jackson, Emily; Mao, Shirley; Schneider, Sabine; Han, Min-Joon; Lytton-Jean, Abigail; Basto, Pamela A. (2013-02-05). "A vector-free microfluidic platform for intracellular delivery". Proceedings of the National Academy of Sciences of the United States of America. 110 (6): 2082–2087. Bibcode:2013PNAS..110.2082S. doi: 10.1073/pnas.1218705110 . ISSN   0027-8424. PMC   3568376 . PMID   23341631.
  15. ↑ "Transgenesis - an overview | ScienceDirect Topics". www.sciencedirect.com. Retrieved 2021-10-28.
  16. ↑ Costantini, F. (2001-01-01), "Transgenic Animals", in Maloy, Stanley; Hughes, Kelly (eds.), Brenner's Encyclopedia of Genetics (Second Edition), San Diego: Academic Press, pp. 117–123, ISBN   978-0-08-096156-9 , retrieved 2021-11-20
  17. ↑ De Windt, Leon J.; Doevendans, Pieter A.; Chien, Kenneth R. (2004-01-01), Chien, KENNETH R. (ed.), "chapter 4 - Generation and Cloning of Genetically Modified Animals", Molecular Basis of Cardiovascular Disease (Second Edition), Philadelphia: W.B. Saunders, pp. 49–71, ISBN   978-0-7216-9428-3 , retrieved 2021-10-28
  18. ↑ Costantini, F. (2001-01-01), "Transgenic Animals", in Maloy, Stanley; Hughes, Kelly (eds.), Brenner's Encyclopedia of Genetics (Second Edition), San Diego: Academic Press, pp. 117–123, ISBN   978-0-08-096156-9 , retrieved 2021-10-28
  19. ↑ Lavitrano, Marialuisa & French, De & Zani, M & Frati, Luigi & Spadafora, Corrado. (1992). "The Interaction between exogenous DNA and sperm cells." Molecular reproduction and development. 31. 161-9. 10.1002/mrd.1080310302. retrieved 2021-10-28
  20. ↑ García-Vázquez, Francisco Alberto; Ruiz, Salvador; Grullón, Luis Alberto; de Ondiz, Aitor; Gutiérrez-Adán, Alfonso; Gadea, Joaquín (December 2011). "Factors affecting porcine sperm mediated gene transfer". Research in Veterinary Science. 91 (3): 446–453. doi:10.1016/j.rvsc.2010.09.015. ISSN   1532-2661. PMID   20980036.
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