Genetic engineering techniques Article

From Wikipedia, the free encyclopedia
Jump to navigation Jump to search

Genetic engineering can be accomplished using multiple techniques. 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 organism, creating the GMO.

The ability to genetically engineer organisms is built on years of research and discovery on how genes function and how we can manipulate them.Humans have been manipulating genetics since early domestication attempts around 12,000 BC. Following the discovery of genes by Gregor Mendel and the proof that they were involved in inheritance tools were developed that allowed there direct manipulation. Important advances included the discovery of restriction enzymes and DNA ligases and the development of polymerase chain reaction and sequencing.

This allowed the gene of interest to be isolated and then incorporated into a vector. Often a promoter and terminator region was added as well as a selectable marker gene. The gene may be modified further at this point to make it express more efficiently. This vector is then inserted into the host organism's genome. For animals, the gene is typically inserted into embryonic stem cells, while in plants it can be inserted into any tissue that can be cultured into a fully developed plant. Common techniques include microinjection, virus-mediated, Agrobacterium-mediated or biolistics. Further tests are carried out on the resulting organism to ensure stable integration, inheritance and expression. First generation offspring are heterozygous, requiring them to be inbred to create the homozygous pattern necessary for stable inheritance. Homozygosity must be confirmed in second generation specimens.

Traditional techniques inserted the genes randomly into the hosts genome. Advances have allowed genes to be inserted at specific locations within a genome, which reduces the unintended side effects of random insertion. Early targeting systems relied on meganucleases and zinc finger nucleases. Since 2009 more accurate and easier systems to implement have been developed. Transcription activator-like effector nucleases (TALENs) and the Cas9-guideRNA system (adapted from CRISPR) are the two most commonly used. They may potentially be useful in gene therapy and other procedures that require accurate or high through put targeting.

History

Many different discoveries and advancements lead to the development of genetic engineering. Human-directed genetic manipulation began with the domestication of plants and animals through artificial selection in about 12,000 BC. [1]:1 Various techniques were developed to aid in breeding and selection. Hybridization was one way rapid changes in an organisms makeup could be introduced. Hybridization most likely first occurred when humans first grew similar, yet slightly different plants in close proximity. [2]:32 Some plants were able to be propagated by vegetative cloning. [2]:31

Genetic inheritance was first discovered by Gregor Mendel in 1865, following experiments crossing peas. [3] In 1928 Frederick Griffith proved the existence of a "transforming principle" involved in inheritance, which was identified as DNA in 1944 by Oswald Avery, Colin MacLeod, and Maclyn McCarty. Frederick Sanger developed a method for sequencing DNA in 1977, greatly increasing the genetic information available to researchers.

After discovering the existence and properties of DNA, tools had to be developed that allowed it to be manipulated. In 1970 Hamilton Smiths lab discovered restriction enzymes, enabling scientists to isolate genes from an organism's genome. [4] DNA ligases, which join broken DNA together, were discovered earlier in 1967. [5] By combining the two enzymes it became possible to "cut and paste" DNA sequences to create recombinant DNA. Plasmids, discovered in 1952, [6] became important tools for transferring information between cells and replicating DNA sequences. Polymerase chain reaction (PCR), developed by Kary Mullis in 1983, allowed small sections of DNA to be amplified (replicated) and aided identification and isolation of genetic material.

As well as manipulating DNA, techniques had to be developed for its insertion into an organism's genome. Griffith's experiment had already shown that some bacteria had the ability to naturally uptake and express foreign DNA. Artificial competence was induced in Escherichia coli in 1970 by treating them with calcium chloride solution (CaCl2). [7] Transformation using electroporation was developed in the late 1980s, increasing the efficiency and bacterial range. [8] In 1907 a bacterium that caused plant tumors, Agrobacterium tumefaciens, had been discovered. In the early 1970s it was found that this bacteria inserted its DNA into plants using a Ti plasmid. [9] By removing the genes in the plasmid that caused the tumor 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. [10]

Choosing target genes

The first step is to identify the target gene or genes to insert into the host organism. This is driven by the goal for the resultant organism. In some cases only one or two genes are affected. For more complex objectives entire biosynthetic pathways involving multiple genes may be involved. Once found genes and other genetic information from a wide range of organisms can be inserted into bacteria for storage and modification, creating genetically modified bacteria in the process. Bacteria are cheap, easy to grow, clonal, multiply quickly, relatively easy to transform and can be stored at -80 °C almost indefinitely. Once a gene is isolated it can be stored inside the bacteria providing an unlimited supply for research. [11]

Genetic screens can be carried out to determine potential genes followed by other tests identify the best candidates. A simple screen involves randomly mutating DNA with chemicals or radiation and then selecting those that display the desired trait. For organisms where mutation is not practical, scientist instead look for individuals among the population who present the characteristic through naturally-occurring mutations. Processes that look at a phenotype and then try and identify the gene responsible are called forward genetics. The gene then needs to be mapped by comparing the inheritance of the phenotype with known genetic markers. Genes that are close together are likely to be inherited together. [12]

Another option is reverse genetics. This approach involves targeting a specific gene with a mutation and then observing what phenotype develops. [12] The mutation can be designed to inactivate the gene or only allow it to become active only under certain conditions. Conditional mutations are useful for identifying genes that are normally lethal if non-functional. [13] As genes with similar functions share similar sequences ( homologous) it is possible to predict the likely function of a gene by comparing its sequence to that of well-studied genes from model organisms. [12] The development of microarrays, transcriptomes and genome sequencing has made it much easier to find desirable genes. [14]

The bacteria Bacillus thuringiensis was first discovered in 1901 as the causative agent in the death of silkworms. Due to these insecticidal properties the bacteria was used as a biological insecticide, developed commercially in 1938. The cry proteins were discovered to provide the insecticidal activity in 1956 and by the 1980s scientists had successfully cloned the gene that codes for this protein and expressed it in plants. [15] Luck may also be involved. [16] The gene that provides resistance to the glyphosate herbicide was found, after seven years searching, in bacteria that were surviving in the outflow pipe of a Monsanto RoundUp manufacturing facility. [17] In animals the majority of genes used are growth hormone genes. [18]

Gene manipulation

All genetic engineering processes involve the modification of DNA. Traditionally DNA was isolated from the cells of organisms. Later, genes came to be cloned from a DNA segment after the creation of a DNA library or artificially synthesised. Once isolated, additional genetic elements are added to the gene to allow it to be expressed in the host organism and to aid selection.

Extraction from cells

First the cell must be gently opened, exposing the DNA without causing too much damage to it. The methods used vary depending on the type of cell. Once open, the DNA must be separated from the other cellular components. A ruptured cell contains proteins and other cell debris. By mixing with phenol and/or chloroform, followed by centrifuging, the nucleic acids can be separated from this debris into an upper aqueous phase. This aqueous phase can be removed and further purified if necessary by repeating the phenol-chloroform steps. The nucleic acids can then be precipitated from the aqueous solution using ethanol or isopropanol. Any RNA can be removed by adding a ribonuclease that will degrade it. Many companies now sell kits that simplify the process. [19]

Gene isolation

The gene of interest must be separated from the extracted DNA. If the sequence is not known then a common method is to break the DNA up with a random digestion method. This is usually accomplished using restriction enzymes (enzymes that cut DNA). A partial restriction digest cuts only some of the restriction sites, resulting in overlapping DNA fragment segments. The DNA fragments are put into individual plasmid vectors and grown inside bacteria. Once in the bacteria the plasmid is copied as the bacteria divides. To determine if a useful gene is present on a particular fragment the DNA library is screened for the desired phenotype. If the phenotype is detected then it is possible that the bacteria contains the target gene.

If the gene does not have a detectable phenotype or a DNA library does not contain the correct gene, other methods must be used to isolate it. If the position of the gene can be determined using molecular markers then chromosome walking is one way to isolate the correct DNA fragment. If the gene expresses close homology to a known gene in another species, then it could be isolated by searching for genes in the library that closely match the known gene. [20]

For known DNA sequences, restriction enzymes that cut the DNA on either side of the gene can be used. Gel electrophoresis then sorts the fragments according to length. [21] Some gels can separate sequences that differ by a single base-pair. The DNA can be visualised by staining it with ethidium bromide and photographing under UV light. A marker with fragments of known lengths can be laid alongside the DNA to estimate the size of each band. The DNA band at the correct size should contain the gene, where it can be excised from the gel. [19]:40–41 Another technique to isolate genes of known sequences involves polymerase chain reaction (PCR). [22] PCR is a powerful tool that can amplify a given sequence, which can then be isolated through gel electrophoresis. Its effectiveness drops with larger genes and it has the potential to introduce errors into the sequence.

It is possible to artificially synthesise genes. [23] Some synthetic sequences are available commercially, forgoing many of these early steps. [24]

Modification

The gene to be inserted must be combined with other genetic elements in order for it to work properly. The gene can be modified at this stage for better expression or effectiveness. As well as the gene to be inserted most constructs contain a promoter and terminator region as well as a selectable marker gene. The promoter region initiates transcription of the gene and can be used to control the location and level of gene expression, while the terminator region ends transcription. A selectable marker, which in most cases confers antibiotic resistance to the organism it is expressed in, is used to determine which cells are transformed with the new gene. The constructs are made using recombinant DNA techniques, such as restriction digests, ligations and molecular cloning. [25]

Inserting DNA into the host genome

Once the gene is constructed it must be stably integrated into the target organisms genome or exist as extrachromosomal DNA. There are a number of techniques available for inserting the gene into the host genome and they vary depending on the type of organism targeted. In multicellular eukaryotes, if the transgene is incorporated into the host's germline cells, the resulting host cell can pass the transgene to its progeny. If the transgene is incorporated into somatic cells, the transgene can not be inherited. [26]

Transformation

Bacterial transformation involves moving a gene from one bacteria to another. It is integrated into the recipients plasmid. and can then be expressed by the new host.

Transformation is the direct alteration of a cells genetic components by passing the genetic material through the cell membrane. About 1% of bacteria are naturally able to take up foreign DNA, but this ability can be induced in other bacteria. [27] Stressing the bacteria with a heat shock or electroporation can make the cell membrane permeable to DNA that may then incorporate into the genome or exist as extrachromosomal DNA. Typically the cells are incubated in a solution containing divalent cations (often calcium chloride) under cold conditions, before being exposed to a heat pulse (heat shock). Calcium chloride partially disrupts the cell membrane, which allows the recombinant DNA to enter the host cell. It is suggested that exposing the cells to divalent cations in cold condition may change or weaken the cell surface structure, making it more permeable to DNA. The heat-pulse is thought to create a thermal imbalance across the cell membrane, which forces the DNA to enter the cells through either cell pores or the damaged cell wall. Electroporation is another method of promoting competence. In this method the cells are briefly shocked with an electric field of 10-20 kV/cm, which is thought to create holes in the cell membrane through which the plasmid DNA may enter. After the electric shock, the holes are rapidly closed by the cell's membrane-repair mechanisms. Up-taken DNA can either integrate with the bacterials genome or, more commonly, exist as extrachromosomal DNA.

A gene gun uses biolistics to insert DNA into plant tissue
A. tumefaciens attaching itself to a carrot cell

In plants the DNA is often inserted using Agrobacterium-mediated recombination, [28] taking advantage of the Agrobacteriums T-DNA sequence that allows natural insertion of genetic material into plant cells. [29] Plant tissue are cut into small pieces and soaked in a fluid containing suspended Agrobacterium. The bacteria will attach to many of the plant cells exposed by the cuts. The bacteria uses conjugation to transfer a DNA segment called T-DNA from its plasmid into the plant. The transferred DNA is piloted to the plant cell nucleus and integrated into the host plants genomic DNA.The plasmid T-DNA is integrated semi-randomly into the genome of the host cell. [30]

By modifying the plasmid to express the gene of interest, researchers can insert their chosen gene stably into the plants genome. The only essential parts of the T-DNA are its two small (25 base pair) border repeats, at least one of which is needed for plant transformation. [31] [32] The genes to be introduced into the plant are cloned into a plant transformation vector that contains the T-DNA region of the plasmid. An alternative method is agroinfiltration. [33] [34]

Another method used to transform plant cells is biolistics, where particles of gold or tungsten are coated with DNA and then shot into young plant cells or plant embryos. [35] Some genetic material enters the cells and transforms them. This method can be used on plants that are not susceptible to Agrobacterium infection and also allows transformation of plant plastids. Plants cells can also be transformed using electroporation, which uses an electric shock to make the cell membrane permeable to plasmid DNA. Due to the damage caused to the cells and DNA the transformation efficiency of biolistics and electroporation is lower than agrobacterial transformation. [36]

Transfection

Transformation has a different meaning in relation to animals, indicating progression to a cancerous state, so the process used to insert foreign DNA into animal cells is usually called transfection. [37] There are many ways to directly introduce DNA into animal cells in vitro. Often these cells are stem cells that are used for gene therapy. Chemical based methods uses natural or synthetic compounds to form particles that facilitate the transfer of genes into cells. [38] These synthetic vectors have the ability to bind DNA and accommodate large genetic transfers. [39] One of the simplest methods involves using calcium phosphate to bind the DNA and then exposing it to cultured cells. The solution, along with the DNA, is encaspulated by the cells and a small amount of DNA can be integrated into the genome. [40] Liposomes and polymers can be used as vectors to deliver DNA into cultured animal cells. Positively charged liposomes bind with DNA, while polymers can designed that interact with DNA. [38] They form lipoplexes and polyplexes respectively, which are then up-taken by the cells. Other techniques include using electroporation and biolistics. [41]

To create transgenic animals the DNA must be inserted into viable embryos or eggs. This is usually accomplished using microinjection, where DNA is injected through the cell's nuclear envelope directly into the nucleus. [27] Superovulated fertilised eggs are collected at the single cell stage and cultured in vitro. When the pronuclei from the sperm head and egg are visible through the protoplasm the genetic material is injected into one of them. The oocyte is then implanted in the oviduct of a pseudopregnant animal. [42] Another method is Embryonic Stem Cell-Mediated Gene Transfer. The gene is transfected into embryonic stem cells and then they are inserted into mouse blastocysts that are then implanted into foster mothers. The resulting offspring are chimeric, and further mating can produce mice fully transgenic with the gene of interest. [43]

Transduction

Transduction is the process by which foreign DNA is introduced into a cell by a virus or viral vector. [44] Genetically modified viruses can be used as viral vectors to transfer target genes to another organism in gene therapy. [45] First the virulent genes are removed from the virus and the target genes are inserted instead. The sequences that allow the virus to insert the genes into the host organism must be left intact. Popular virus vectors are developed from retroviruses or adenoviruses. Other viruses used as vectors include, lentiviruses, pox viruses and herpes viruses. The type of virus used will depend on the cells targeted and whether the DNA is to be altered permanently or temporarily.

Regeneration

As often only a single cell is transformed with genetic material, the organism must be regenerated from that single cell. In plants this is accomplished through the use of tissue c ulture. [46] [47] Each plant species has different requirements for successful regeneration. If successful, the technique produces an adult plant that contains the transgene in every cell.[ citation needed] In animals it is necessary to ensure that the inserted DNA is present in the embryonic stem cells. [28] Offspring can be screened for the gene. All offspring from the first generation are heterozygous for the inserted gene and must be inbred to produce a homozygous specimen.[ citation needed] Bacteria consist of a single cell and reproduce clonally so regeneration is not necessary. Selectable markers are used to easily differentiate transformed from untransformed cells.

Cells that have been successfully transformed with the DNA contain the marker gene, while those not transformed will not. By growing the cells in the presence of an antibiotic or chemical that selects or marks the cells expressing that gene, it is possible to separate modified from unmodified cells. Another screening method involves a DNA probe that sticks only to the inserted gene. These markers are usually present in the transgenic organism, although a number of strategies have been developed that can remove the selectable marker from the mature transgenic plant. [48]

Confirmation

Finding that a recombinant organism contains the inserted genes is not usually sufficient to ensure that they will be appropriately expressed in the intended tissues. Further testing using PCR, Southern hybridization, and DNA sequencing is conducted to confirm that an organism contains the new gene. [49] These tests can also confirm the chromosomal location and copy number of the inserted gene. Once confirmed methods that look for and measure the gene products (RNA and protein) are also used to assess gene expression, transcription, RNA processing patterns and expression and localization of protein product(s). These include northern hybridisation, quantitative RT-PCR, Western blot, immunofluorescence, ELISA and phenotypic analysis. [50] When appropriate, the organism's offspring are studied to confirm that the transgene and associated phenotype are stably inherited.

Gene targeting

Traditional methods of genetic engineering generally inserts the new genetic material randomly within the host genome. This can impair or alter other genes within the organism. Methods were sort which targeted the inserted genes to specific sites within an organism genome. Early methods to target genes to certain sites within a genome relied on homologous recombination. [51] By creating DNA constructs that contain a template that matches the targeted genome sequence it is possible that the HR processes within the cell will insert the construct at the desired location. Using this method on embryonic stem cells led to the development of transgenic mice with targeted genes knocked out. It has also been possible to knock in genes or alter gene expression patterns. [52]

If a vital gene is knocked out it can prove lethal to the organism. In order to study the function of these genes site specific recombinases (SSR) were used. The two most common types are the Cre-LoxP and Flp-FRT systems. Cre recombinase is an enzyme that removes DNA by homologous recombination between binding sequences known as Lox-P sites. The Flip-FRT system operates in a similar way, with the Flip recombinase recognising FRT sequences. By crossing an organism containing the recombinase sites flanking the gene of interest with an organism that express the SSR under control of tissue specific promoters, it is possible to knock out or switch on genes only in certain cells. This has also been used to remove marker genes from transgenic animals. Further modifications of these systems allowed researchers to induce recombination only under certain conditions, allowing genes to be knocked out or expressed at desired times or stages of development. [52]

Genome editing uses artificially engineered nucleases that create specific double-stranded breaks at desired locations in the genome.The breaks are subject to cellular DNA repair processes that can be exploited for targeted gene replacement at high frequencies. If donor DNA is present with the correct end sequences then homologous recombination will add the new genetic material containing the transgene at that site. [53] There are four families of engineered nucleases: meganucleases, [54] [55] zinc finger nucleases, [56] [57] transcription activator-like effector nucleases (TALENs), [58] [59] and the Cas9-guideRNA system (adapted from CRISPR). [60] [61] TALEN and CRISPR are the two most commonly used. [62] Recent advances have looked at combining multiple systems to try and exploit the best features of both. Fusing meganucleases with TALENs "megaTALs" combine the ease of engineering and high DNA binding specificity of a TAL effector with the high cleavage efficiency of meganucleases. [63] Researchers are also looking at ways of targeting gene placement without causing double stranded breaks. [62]

Meganucleases and Zinc finger nucleases

Meganucleases are endodeoxyribonucleases that function as restriction enzymes with long recognition sites, making them are more specific to their target site than other restriction enzymes. This increases their precision and reduces their toxicity as they will not target as many sites within a genome. The most studied meganucleases are the LAGLIDADG family, and their purpose is still unclear. [64] Zinc-finger nucleases (ZFNs) also cleave DNA at target-able sites. [53] By engineering the nucleases to target specific sites within the genome it is possible to insert break the DNA at a predefined location. [64] Another approach is to fuse two protein domains together to create chimeric meganucleases or ZFNs that have novel sites of cleavage or greater specificity. [65] [53]

Meganucleases are still quite susceptible to off-target binding, which makes them less attractive than other targeting mechanisms. [66] They are smaller than zinc fingers, raising the possibility that they could be used in viral vectors. [66] ZFNs have greater specificity, but even the best designed one can still bind to non-specific sequences. They are also unable to bind to DNA with high chromotin density. While a certain amount of off-target cleavage is acceptable for creating transgenic model organisms they are not practical for human gene therapy treatments. [64]

TALEN and CRISPR

Transcription activator-like effectors (TALE) were discovered in the Xanthomonas bacteria in 2009. They bind to with great specificity to genes within the plant host and initiate transcription of the genes, which alters the environment to suit thebacteria. Engineering TALEs to contain the Fok1 nuclease created a new tool for genome editing, the TAL effector nuclease (TALEN). They have the greatest specificity of all the current engineered nucleases, making them less toxic to the organism. Due to a presence of up to 33.5 repeats they are difficult to construct through standard PCR and rely on the more complicated method of Golden gate cloning. [62]

In 2011 another major breakthrough was developed. Clustered regularly interspaced palindromic repeats (CRISPR) were discovered in bacteria and archaea. It was used to increase immunity to invading viruses by cleaving viral DNA and inserting pieces of that DNA into their own genome. They then transcribe this DNA into RNA and combined it with Cas9 proteins to make double strand breaks in the invading viral DNA. By pairing Cas9 with a guide RNA double stranded breaks could be induced at specific points within an organisms genome. Although they lack the specificity of TALENs the major advantage of this technology is the simplicity of the design and the ease of vector construction. It also allows multiple sites to be targeted simultaneously, allowing the addition of multiple genes at once. CRISPR-Cpf1 is an updated system that requires a shorter guide RNA and leaves overhangs when cleaving the DNA that can make inserting genes easier. [62]

See also

References

  1. ^ Root C (2007). Domestication. Greenwood Publishing Groups. ISBN  9780313339875.
  2. ^ a b Kingsbury N (15 October 2009). Hybrid: The History and Science of Plant Breeding. University of Chicago Press. ISBN  978-0-226-43705-7.
  3. ^ Hartl DL, Orel V (June 1992). "What did Gregor Mendel think he discovered?". Genetics. 131 (2): 245–53. PMID  1644269.
  4. ^ Roberts RJ (April 2005). "How restriction enzymes became the workhorses of molecular biology". Proceedings of the National Academy of Sciences of the United States of America. 102 (17): 5905–8. Bibcode: 2005PNAS..102.5905R. doi: 10.1073/pnas.0500923102. PMC  1087929. PMID  15840723.
  5. ^ Weiss B, Richardson CC (April 1967). "Enzymatic breakage and joining of deoxyribonucleic acid, I. Repair of single-strand breaks in DNA by an enzyme system from Escherichia coli infected with T4 bacteriophage". Proceedings of the National Academy of Sciences of the United States of America. 57 (4): 1021–8. Bibcode: 1967PNAS...57.1021W. doi: 10.1073/pnas.57.4.1021. PMC  224649. PMID  5340583.
  6. ^ Lederberg J (October 1952). "Cell genetics and hereditary symbiosis". Physiological Reviews. 32 (4): 403–30. doi: 10.1152/physrev.1952.32.4.403. PMID  13003535.
  7. ^ Mandel M, Higa A (October 1970). "Calcium-dependent bacteriophage DNA infection". Journal of Molecular Biology. 53 (1): 159–62. doi: 10.1016/0022-2836(70)90051-3. PMID  4922220.
  8. ^ Wirth R, Friesenegger A, Fiedler S (March 1989). "Transformation of various species of gram-negative bacteria belonging to 11 different genera by electroporation". Molecular & General Genetics. 216 (1): 175–7. doi: 10.1007/BF00332248. PMID  2659971.
  9. ^ Nester, Eugene. "Agrobacterium: The Natural Genetic Engineer (100 Years Later)". Retrieved 14 January 2011.
  10. ^ Zambryski P, Joos H, Genetello C, Leemans J, Montagu MV, Schell J (1983). "Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity". The EMBO Journal. 2 (12): 2143–50. PMC  555426. PMID  16453482.
  11. ^ "Rediscovering Biology - Online Textbook: Unit 13 Genetically Modified Organisms". www.learner.org. Retrieved 2017-08-18.
  12. ^ a b c Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P (2002). "Studying Gene Expression and Function". Molecular Biology of the Cell. New York: Garland Science.
  13. ^ Griffiths AJ, Miller JH, Suzuki DT, Lewontin RC, Gelbart WM (2000). "Mutant types". An Introduction to Genetic Analysis (7th ed.).
  14. ^ Koh H, Kwon S, Thomson M (2015-08-26). Current Technologies in Plant Molecular Breeding: A Guide Book of Plant Molecular Breeding for Researchers. Springer. p. 242. ISBN  9789401799966.
  15. ^ Roh JY, Choi JY, Li MS, Jin BR, Je YH (April 2007). "Bacillus thuringiensis as a specific, safe, and effective tool for insect pest control". Journal of Microbiology and Biotechnology. 17 (4): 547–59. PMID  18051264.
  16. ^ "How to Make a GMO". Science in the News. 2015-08-09. Retrieved 2017-11-08.
  17. ^ Adler J (May 2011). "The growing menace from superweeds". Scientific American. Scientific American. 304 (5): 74–9. Bibcode: 2011SciAm.304e..74A. doi: 10.1038/scientificamerican0511-74. PMID  21595408.
  18. ^ Food and Agricultural Organisation of the United Nations. "The process of genetic modification".
  19. ^ a b Nicholl ST (29 May 2008). An Introduction to Genetic Engineering. Cambridge University Press. ISBN  978-1-139-47178-7.
  20. ^ Michels CA (10 June 2002). Genetic Techniques for Biological Research: A Case Study Approach. John Wiley & Sons. pp. 85–88. ISBN  978-0-471-89919-8.
  21. ^ Alberts B, et al. (2002). "8". Isolating, Cloning, and Sequencing DNA (4th ed.). New York: Garland Science.
  22. ^ Kaufman RI, Nixon BT (July 1996). "Use of PCR to isolate genes encoding sigma54-dependent activators from diverse bacteria". Journal of Bacteriology. 178 (13): 3967–70. doi: 10.1128/jb.178.13.3967-3970.1996. PMC  232662. PMID  8682806.
  23. ^ Liang J, Luo Y, Zhao H (2011). "Synthetic biology: putting synthesis into biology". Wiley Interdisciplinary Reviews: Systems Biology and Medicine. 3 (1): 7–20. doi: 10.1002/wsbm.104. PMC  3057768.
  24. ^ "GeneArt Gene Synthesis". www.thermofisher.com. Retrieved 2017-11-09.
  25. ^ Berg P, Mertz JE (January 2010). "Personal reflections on the origins and emergence of recombinant DNA technology". Genetics. 184 (1): 9–17. doi: 10.1534/genetics.109.112144. PMC  2815933. PMID  20061565.
  26. ^ Nayerossadat N, Maedeh T, Ali PA (6 July 2012). "Viral and nonviral delivery systems for gene delivery". Advanced Biomedical Research. 1: 27. doi: 10.4103/2277-9175.98152. PMC  3507026. PMID  23210086.
  27. ^ a b Chen I, Dubnau D (March 2004). "DNA uptake during bacterial transformation". Nature Reviews. Microbiology. 2 (3): 241–9. doi: 10.1038/nrmicro844. PMID  15083159.
  28. ^ a b National Research Council (US) Committee on Identifying and Assessing Unintended Effects of Genetically Engineered Foods on Human Health (2004-01-01). Methods and Mechanisms for Genetic Manipulation of Plants, Animals, and Microorganisms. National Academies Press (US).
  29. ^ Gelvin SB (March 2003). "Agrobacterium-mediated plant transformation: the biology behind the "gene-jockeying" tool". Microbiology and Molecular Biology Reviews. 67 (1): 16–37, table of contents. doi: 10.1128/MMBR.67.1.16-37.2003. PMC  150518. PMID  12626681.
  30. ^ Francis KE, Spiker S (February 2005). "Identification of Arabidopsis thaliana transformants without selection reveals a high occurrence of silenced T-DNA integrations". The Plant Journal. 41 (3): 464–77. doi: 10.1111/j.1365-313x.2004.02312.x. PMID  15659104.
  31. ^ Schell J, Van Montagu M (1977). "The Ti-Plasmid of Agrobacterium Tumefaciens, A Natural Vector for the Introduction of NIF Genes in Plants?". In Hollaender A, Burris RH, Day PR, Hardy RW, Helinski DR, Lamborg MR, Owens L, Valentine RC. Genetic Engineering for Nitrogen Fixation. Basic Life Sciences. 9. pp. 159–79. doi: 10.1007/978-1-4684-0880-5_12. ISBN  978-1-4684-0882-9. PMID  336023.
  32. ^ Joos H, Timmerman B, Montagu MV, Schell J (1983). "Genetic analysis of transfer and stabilization of Agrobacterium DNA in plant cells". The EMBO Journal. 2 (12): 2151–60. PMC  555427. PMID  16453483.
  33. ^ Thomson JA. "Genetic Engineering of Plants" (PDF). Biotechnology. Encyclopedia of Life Support Systems. 3. Retrieved 17 July 2016.
  34. ^ Leuzinger K, Dent M, Hurtado J, Stahnke J, Lai H, Zhou X, Chen Q (July 2013). "Efficient agroinfiltration of plants for high-level transient expression of recombinant proteins". Journal of Visualized Experiments. 77 (77). doi: 10.3791/50521. PMC  3846102. PMID  23913006.
  35. ^ Head G, Hull RH, Tzotzos GT (2009). Genetically Modified Plants: Assessing Safety and Managing Risk. London: Academic Pr. p. 244. ISBN  0-12-374106-8.
  36. ^ Darbani B, Farajnia S, Toorchi M, Zakerbostanabad S, Noeparvar S, Neal Stewart C (March 2008). "DNA-Delivery Methods to Produce Transgenic Plants". Biotechnology (Faisalabad). 7 (3): 385–402. doi: 10.3923/biotech.2008.385.402.
  37. ^ Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P (2002). Molecular Biology of the Cell. New York: Garland Science. p. G:35. ISBN  978-0-8153-4072-0.
  38. ^ a b Kamimura K, Suda T, Zhang G, Liu D (October 2011). "Advances in Gene Delivery Systems". Pharmaceutical Medicine. 25 (5): 293–306. doi: 10.2165/11594020-000000000-00000. PMC  3245684. PMID  22200988.
  39. ^ Pack DW, Hoffman AS, Pun S, Stayton PS (July 2005). "Design and development of polymers for gene delivery". Nature Reviews. Drug Discovery. 4 (7): 581–93. doi: 10.1038/nrd1775. PMID  16052241.
  40. ^ "Lecture 8 genetic engineering of animal cells". www.slideshare.net. Retrieved 2018-07-18.
  41. ^ Biocyclopedia.com. "Gene transfer (transfection) methods in animals | Genetic Engineering and Biotechnology Gene Transfer Methods and Transgenic Organisms | Genetics, Biotechnology, Molecular Biology, Botany | Biocyclopedia.com". biocyclopedia.com. Retrieved 2018-07-18.
  42. ^ Mullin A. "Pronuclear Injection". Tulane University.
  43. ^ "Embryonic Stem Cell-Mediated Gene Transfer - transgenicanimals". sites.google.com. Retrieved 2018-07-19.
  44. ^ Transduction, Genetic at the US National Library of Medicine Medical Subject Headings (MeSH)
  45. ^ "Stemcells Redirection". stemcells.nih.gov.
  46. ^ Tuomela M, Stanescu I, Krohn K (October 2005). "Validation overview of bio-analytical methods". Gene Therapy. 12 Suppl 1 (S1): S131–8. doi: 10.1038/sj.gt.3302627. PMID  16231045.
  47. ^ Narayanaswamy S (1994). Plant Cell and Tissue Culture. Tata McGraw-Hill Education. pp. vi. ISBN  9780074602775.
  48. ^ Hohn B, Levy AA, Puchta H (April 2001). "Elimination of selection markers from transgenic plants". Current Opinion in Biotechnology. 12 (2): 139–43. doi: 10.1016/S0958-1669(00)00188-9. PMID  11287227.
  49. ^ Setlow JK (2002-10-31). Genetic Engineering: Principles and Methods. Springer Science & Business Media. p. 109. ISBN  9780306472800.
  50. ^ Deepak S, Kottapalli K, Rakwal R, Oros G, Rangappa K, Iwahashi H, Masuo Y, Agrawal G (June 2007). "Real-Time PCR: Revolutionizing Detection and Expression Analysis of Genes". Current Genomics. 8 (4): 234–51. doi: 10.2174/138920207781386960. PMC  2430684. PMID  18645596.
  51. ^ Lodish H, Berk A, Zipursky SL, Matsudaira P, Baltimore D, Darnell J (2000). "Chapter 8.5: Gene Replacement and Transgenic Animals: DNA Is Transferred into Eukaryotic Cells in Various Ways". Molecular Cell Biology (4th ed.). W. H. Freeman and Company. ISBN  0-7167-3136-3.
  52. ^ a b Rocha-Martins, Maurício; Cavalheiro, Gabriel R.; Matos-Rodrigues, Gabriel E.; Martins, Rodrigo a. P.; Rocha-Martins, Maurício; Cavalheiro, Gabriel R.; Matos-Rodrigues, Gabriel E.; Martins, Rodrigo a. P. (2015-8). "From Gene Targeting to Genome Editing: Transgenic animals applications and beyond". Anais da Academia Brasileira de Ciências. 87 (2): 1323–1348. doi: 10.1590/0001-3765201520140710. ISSN  0001-3765. Check date values in: |date= ( help)
  53. ^ a b c Carroll D (August 2011). "Genome engineering with zinc-finger nucleases". Genetics. 188 (4): 773–82. doi: 10.1534/genetics.111.131433. PMC  3176093. PMID  21828278.
  54. ^ Grizot S, Smith J, Daboussi F, Prieto J, Redondo P, Merino N, Villate M, Thomas S, Lemaire L, Montoya G, Blanco FJ, Pâques F, Duchateau P (September 2009). "Efficient targeting of a SCID gene by an engineered single-chain homing endonuclease". Nucleic Acids Research. 37 (16): 5405–19. doi: 10.1093/nar/gkp548. PMC  2760784. PMID  19584299.
  55. ^ Gao H, Smith J, Yang M, Jones S, Djukanovic V, Nicholson MG, West A, Bidney D, Falco SC, Jantz D, Lyznik LA (January 2010). "Heritable targeted mutagenesis in maize using a designed endonuclease". The Plant Journal. 61 (1): 176–87. doi: 10.1111/j.1365-313X.2009.04041.x. PMID  19811621.
  56. ^ Townsend JA, Wright DA, Winfrey RJ, Fu F, Maeder ML, Joung JK, Voytas DF (May 2009). "High-frequency modification of plant genes using engineered zinc-finger nucleases". Nature. 459 (7245): 442–5. Bibcode: 2009Natur.459..442T. doi: 10.1038/nature07845. PMC  2743854. PMID  19404258.
  57. ^ Shukla VK, Doyon Y, Miller JC, DeKelver RC, Moehle EA, Worden SE, Mitchell JC, Arnold NL, Gopalan S, Meng X, Choi VM, Rock JM, Wu YY, Katibah GE, Zhifang G, McCaskill D, Simpson MA, Blakeslee B, Greenwalt SA, Butler HJ, Hinkley SJ, Zhang L, Rebar EJ, Gregory PD, Urnov FD (May 2009). "Precise genome modification in the crop species Zea mays using zinc-finger nucleases". Nature. 459 (7245): 437–41. Bibcode: 2009Natur.459..437S. doi: 10.1038/nature07992. PMID  19404259.
  58. ^ Christian M, Cermak T, Doyle EL, Schmidt C, Zhang F, Hummel A, Bogdanove AJ, Voytas DF (October 2010). "Targeting DNA double-strand breaks with TAL effector nucleases". Genetics. 186 (2): 757–61. doi: 10.1534/genetics.110.120717. PMC  2942870. PMID  20660643.
  59. ^ Li T, Huang S, Jiang WZ, Wright D, Spalding MH, Weeks DP, Yang B (January 2011). "TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain". Nucleic Acids Research. 39 (1): 359–72. doi: 10.1093/nar/gkq704. PMC  3017587. PMID  20699274.
  60. ^ Esvelt KM, Wang HH (2013). "Genome-scale engineering for systems and synthetic biology". Molecular Systems Biology. 9: 641. doi: 10.1038/msb.2012.66. PMC  3564264. PMID  23340847.
  61. ^ Tan WS, Carlson DF, Walton MW, Fahrenkrug SC, Hackett PB (2012). "Precision editing of large animal genomes". Advances in Genetics. Advances in Genetics. 80: 37–97. doi: 10.1016/B978-0-12-404742-6.00002-8. ISBN  9780124047426. PMC  3683964. PMID  23084873.
  62. ^ a b c d Malzahn A, Lowder L, Qi Y (2017-04-24). "Plant genome editing with TALEN and CRISPR". Cell & Bioscience. 7: 21. doi: 10.1186/s13578-017-0148-4. PMC  5404292. PMID  28451378.
  63. ^ Boissel S, Jarjour J, Astrakhan A, Adey A, Gouble A, Duchateau P, Shendure J, Stoddard BL, Certo MT, Baker D, Scharenberg AM (February 2014). "megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering". Nucleic Acids Research. 42 (4): 2591–601. doi: 10.1093/nar/gkt1224. PMC  3936731. PMID  24285304.
  64. ^ a b c Silva G, Poirot L, Galetto R, Smith J, Montoya G, Duchateau P, Pâques F (February 2011). "Meganucleases and other tools for targeted genome engineering: perspectives and challenges for gene therapy". Current Gene Therapy. 11 (1): 11–27. doi: 10.2174/156652311794520111. PMC  3267165. PMID  21182466.
  65. ^ Silva GH, Belfort M, Wende W, Pingoud A (August 2006). "From monomeric to homodimeric endonucleases and back: engineering novel specificity of LAGLIDADG enzymes". Journal of Molecular Biology. 361 (4): 744–54. doi: 10.1016/j.jmb.2006.06.063. PMID  16872628.
  66. ^ a b Dunn DA, Pinkert CA (2014). "Gene Editing". Transgenic Animal Technology (3rd ed.). pp. 229–248. doi: 10.1016/B978-0-12-410490-7.00008-6. ISBN  978-0-12-410490-7.