Techniques of genetic engineering

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

There are various techniques used to genetically engineer organisms. The first step involves choosing and isolating the gene that will be inserted into the genetically modified organism. Most genes transferred into plants provide protection against insects or tolerance to herbicides. In animals the majority of genes used are growth hormone genes. The gene can be isolated using restriction enzymes to cut DNA into fragments and gel electrophoresis to separate them out according to length. Polymerase chain reaction (PCR) can also be used to amplify up a gene segment, which can then be isolated through gel electrophoresis. The gene to be inserted into the genetically modified organism must be combined with other genetic elements in order for it to work properly. 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. The selectable marker, which in most cases confers antibiotic resistance to the organism it is expressed in, is needed 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. The manipulation of the DNA generally occurs within a plasmid.

The most common form of genetic engineering involves inserting new genetic material randomly within the host genome. Other techniques allow new genetic material to be inserted at a specific location in the host genome or generate mutations at desired genomic loci capable of knocking out endogenous genes. The technique of gene targeting uses homologous recombination to target desired changes to a specific endogenous gene. This tends to occur at a relatively low frequency in plants and animals and generally requires the use of selectable markers. The frequency of gene targeting can be greatly enhanced with the use of engineered nucleases such as zinc finger nucleases, engineered homing endonucleases, or nucleases created from TAL effectors. In addition to enhancing gene targeting, engineered nucleases can also be used to introduce mutations at endogenous genes that generate a gene knockout.

About 1% of bacteria are naturally able to take up foreign DNA but it can also be induced in other bacteria. DNA is generally inserted into animal cells using microinjection, where it can be injected through the cells nuclear envelope directly into the nucleus or through the use of viral vectors. In plants the DNA is generally inserted using Agrobacterium-mediated recombination or biolistics. As often only a single cell is transformed with genetic material the organism must be regenerated from that single cell. As bacteria consist of a single cell and reproduce clonally regeneration is not necessary. In plants this is accomplished through the use of tissue culture. Each plant species has different requirements for successful regeneration through tissue culture. If successful an adult plant is produced that contains the transgene in every cell. In animals it is necessary to ensure that the inserted DNA is present in the embryonic stem cells. Selectable markers are used to easily differentiate transformed from untransformed cells. 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. When the offspring is produced they can be screened for the presence of the gene. All offspring from the first generation will be heterozygous for the inserted gene and must be mated together to produce a homozygous animal.

Further testing uses PCR, Southern hybridization, and DNA sequencing is conducted to confirm that an organism contains the new gene. These tests can also confirm the chromosomal location and copy number of the inserted gene. The presence of the gene does not guarantee it will be expressed at appropriate levels in the target tissue so methods that look for and measure the gene products (RNA and protein) are also used. These include northern hybridization, quantitative RT-PCR, Western blot, immunofluorescence, ELISA and phenotypic analysis. For stable transformation the gene should be passed to the offspring in a Mendelian inheritance pattern, so the organism's offspring are also studied.

Gene isolation[edit]

The first step is to find and isolate the gene that will be inserted into the genetically modified organism. Finding the right gene to insert usually draws on years of scientific research into the identity and function of useful genes. Presently, most genes transferred into plants provide protection against insects or tolerance to herbicides.[1] For example, 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 an biological insecticide, commercially developed in 1938. The cry proteins were discovered to provide the insecticidal activity in 1956 and by the 1980s scientists had successfully cloned the gene coding for this protein and expressed it in plants.[2] The gene that provides resistance to the glyphosate herbicide was found, after seven years searching, in the outflow pipe of a Monsanto roundup manufacturing facility.[3] In animals the majority of genes used are growth hormone genes.[4]

The gene can be cloned from a DNA segment after the creation of a DNA library. The libraries generally cover the organisms genome multiple times and its size will depend on how large the genome is. The DNA is first digested with a random digestion method, commonly by cutting the DNA with restriction enzymes (enzymes that cut DNA). By only doing a partial restriction digest some of the restriction sites will not be cut, resulting in DNA fragment lengths that should overlap. The DNA fragments will then be put into individual plasmid vectors and grown inside bacteria. Once in the bacteria the plasmid will multiply as the bacteria divides producing multiple copies (clones) of the DNA fragment. To determine if a useful gene is present on a particular fragment the bacterial library are screened for the desired phenotype. If the phenotype is detected then it is possible that the bacteria contains a clone of the gene being searched for. If the gene does not have a detectable phenotype or a DNA library does not contain the correct clone other methods can be used to isolate the gene. If the position of the gene can be determined using molecular markers then chromosome walking is one way the DNA fragment containing the gene can be isolated. If the gene bears close homology to a know gene in another species then it could be isolated by searching for genes in the library that closely match the known gene.[5]

If the DNA sequence of the gene and the organism is known then it can be isolated relatively easily. Restriction enzymes can cut the DNA either side of the gene and gel electrophoresis can be used to separate the fragments according to length.[6] The DNA band at the correct size should be the one containing the gene, and it can then be excised from the gel. Polymerase chain reaction (PCR) can also be used to amplify up the gene, which can then be isolated the same way through gel electrophoresis.[7] It is also possible to have the gene artificially synthesized.[8]

Constructs[edit]

The gene to be inserted into the genetically modified organism must be combined with other genetic elements in order for it to work properly. The gene can also 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. The selectable marker, which in most cases confers antibiotic resistance to the organism it is expressed in, is needed 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.[9]

Gene targeting[edit]

The most common form of genetic engineering involves inserting new genetic material randomly within the host genome. Other techniques allow new genetic material to be inserted at a specific location in the host genome or generate mutations at desired genomic loci capable of knocking out endogenous genes. The technique of gene targeting uses homologous recombination to target desired changes to a specific endogenous gene. This tends to occur at a relatively low frequency in plants and animals and generally requires the use of selectable markers. The frequency of gene targeting can be greatly enhanced with the use of engineered nucleases such as zinc finger nucleases,[10] [11] engineered homing endonucleases,[12] [13] or nucleases created from TAL effectors.[14] [15] In addition to enhancing gene targeting, engineered nucleases can also be used to introduce mutations at endogenous genes that generate a gene knockout[16] .[17]

Transformation[edit]

A. tumefaciens attaching itself to a carrot cell

About 1% of bacteria are naturally able to take up foreign DNA but it can also be induced in other bacteria.[18] Stressing the bacteria for example, with a heat shock or an electric shock, can make the cell membrane permeable to DNA that may then incorporate into their genome or exist as extrachromosomal DNA. DNA is generally inserted into animal cells using microinjection, where it can be injected through the cells nuclear envelope directly into the nucleus or through the use of viral vectors. In plants the DNA is generally inserted using Agrobacterium-mediated recombination or biolistics.[19]

In Agrobacterium-mediated recombination the plasmid construct must also contain T-DNA. Agrobacterium naturally inserts DNA from a tumor inducing plasmid into any susceptible plant's genome it infects, causing crown gall disease. The T-DNA region of this plasmid is responsible for insertion of the DNA. The genes to be inserted are cloned into a binary vector, which contains T-DNA and can be grown in both E. Coli and Agrobacterium. Once the binary vector is constructed the plasmid is transformed into Agrobacterium containing no plasmids and plant cells are infected. The Agrobacterium will then naturally insert the genetic material into the plant cells.[20]

In biolistics particles of gold or tungsten are coated with DNA and then shot into young plant cells or plant embryos. Some genetic material will enter the cells and transform them. This method can be used on plants that are not susceptible to Agrobacterium infection and also allows transformation of plant plastids. Another transformation method for plant and animal cells is electroporation. Electroporation involves subjecting the plant or animal cell to an electric shock, which can make the cell membrane permeable to plasmid DNA. In some cases the electroporated cells will incorporate the DNA into their genome. Due to the damage caused to the cells and DNA the transformation efficiency of biolistics and electroporation is lower than agrobacterial mediated transformation and microinjection.[21]

Selection[edit]

Not all the organism's cells will be transformed with the new genetic material; in most cases a selectable marker is used to differentiate transformed from untransformed cells. If a cell has been successfully transformed with the DNA it will also contain the marker gene. 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 the transgenic events from the non-transgenic. Another method of screening involves using a DNA probe that will only stick to the inserted gene. A number of strategies have been developed that can remove the selectable marker from the mature transgenic plant.[22]

Regeneration[edit]

As often only a single cell is transformed with genetic material the organism must be regrown from that single cell. As bacteria consist of a single cell and reproduce clonally regeneration is not necessary. In plants this is accomplished through the use of tissue culture. Each plant species has different requirements for successful regeneration through tissue culture. If successful an adult plant is produced that contains the transgene in every cell. In animals it is necessary to ensure that the inserted DNA is present in the embryonic stem cells. When the offspring is produced they can be screened for the presence of the gene. All offspring from the first generation will be heterozygous for the inserted gene and must be mated together to produce a homozygous animal.

Confirmation[edit]

The finding that a recombinant organism contains the inserted genes is not usually sufficient to ensure that the genes will be expressed in an appropriate manner in the intended tissues of the recombinant organism. To examine the presence of the gene, further analysis frequently uses PCR, Southern hybridization, and DNA sequencing, which serve to determine the chromosomal location and copy number of the inserted gene. To examine expression of the trans-gene, an extensive analysis of transcription, RNA processing patterns, and the expression and localization of the protein product(s) is usually necessary, using methods including northern hybridization, quantitative RT-PCR, Western blot, immunofluorescence and phenotypic analysis. When appropriate, the organism's offspring are studied to confirm that the trans-gene and associated phenotype are stably inherited.

References[edit]

  1. ^ James, Clive (2008). "Global Status of Commercilized Biotech/GM Crops:2008". ISSA Brief No. 39. 
  2. ^ Roh JY, Choi JY, Li MS, Jin BR, Je YH (2007). "Bacillus thuringiensis as a specific, safe, and effective tool for insect pest control". J Microbiol Biotechnol 17 (4): 547–59. 
  3. ^ Jerry Adler (May 2011). The Growing Menace From Superweeds. Scientific American. 
  4. ^ Food and Agricultural Organisation of the United Nations. "The process of genetic modification". 
  5. ^ Corinne A. Michels (2002). "7". Genetic Techniques for Biological Research: A Case Study Approach. John Wiley & Sons. p. 85-88. ISBN 0-471-89919-4. 
  6. ^ Alberts B, Johnson A, Lewis J, et al. (2002). "8". Isolating, Cloning, and Sequencing DNA. (4th ed.). New York: Garland Science. 
  7. ^ R I Kaufman and B T Nixon (1996). "Use of PCR to isolate genes encoding sigma54-dependent activators from diverse bacteria.". J Bacteriol 178 (13): 3967–3970. 
  8. ^ Liang, J.; Luo, Y.; Zhao, H. (2011). "Synthetic biology: Putting synthesis into biology". Wiley Interdisciplinary Reviews: Systems Biology and Medicine 3: 7. doi:10.1002/wsbm.104.  edit
  9. ^ Berg, P.; Mertz, J. (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.  edit
  10. ^ Townsend JA, Wright DA, Winfrey RJ, et al. (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. 
  11. ^ Shukla VK, Doyon Y, Miller JC, et al. (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. 
  12. ^ Grizot S, Smith J, Daboussi F, et al. (September 2009). "Efficient targeting of a SCID gene by an engineered single-chain homing endonuclease". Nucleic Acids Res. 37 (16): 5405–19. doi:10.1093/nar/gkp548. PMC 2760784. PMID 19584299. 
  13. ^ Gao H, Smith J, Yang M, et al. (January 2010). "Heritable targeted mutagenesis in maize using a designed endonuclease". Plant J. 61 (1): 176–87. doi:10.1111/j.1365-313X.2009.04041.x. PMID 19811621. 
  14. ^ Christian M, Cermak T, Doyle EL, et al. (July 2010). "TAL Effector Nucleases Create Targeted DNA Double-strand Breaks". Genetics 186 (2): 757–61. doi:10.1534/genetics.110.120717. PMC 2942870. PMID 20660643. 
  15. ^ Li T, Huang S, Jiang WZ, et al. (August 2010). "TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain". Nucleic Acids Res 39 (1): 359–72. doi:10.1093/nar/gkq704. PMC 3017587. PMID 20699274. 
  16. ^ S.C. Ekker (2008). "Zinc finger-based knockout punches for zebrafish genes". Zebrafish 5 (2): 1121–3. doi:10.1089/zeb.2008.9988. PMC 2849655. PMID 18554175. 
  17. ^ Geurts AM, Cost GJ, Freyvert Y, et al. (July 2009). "Knockout rats via embryo microinjection of zinc-finger nucleases". Science 325 (5939): 433. Bibcode:2009Sci...325..433G. doi:10.1126/science.1172447. PMC 2831805. PMID 19628861. 
  18. ^ Chen I, Dubnau D (2004). "DNA uptake during bacterial transformation". Nature Reviews Microbiology 2 (3): 241–9. doi:10.1038/nrmicro844. PMID 15083159. 
  19. ^ Graham Head; Hull, Roger H; Tzotzos, George T. (2009). Genetically Modified Plants: Assessing Safety and Managing Risk. London: Academic Pr. p. 244. ISBN 0-12-374106-8. 
  20. ^ Gelvin, S. B. (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.  edit
  21. ^ Behrooz Darbani, Safar Farajnia, Mahmoud Toorchi, Saeed Zakerbostanabad, Shahin Noeparvar and C. Neal Stewart Jr. (2010). "DNA-Delivery Methods to Produce Transgenic Plants". Science Alert. 
  22. ^ Barbara Hohn, Avraham A Levy and Holger Puchta (2001). "Elimination of selection markers from transgenic plants". Current Opinion in Biotechnology 12 (2): 139–143. doi:10.1016/S0958-1669(00)00188-9. PMID 11287227.