Team:Cornell/project/background/fungal genetic engineering

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Recent years have shown increasing interest in the genetic engineering of filamentous fungi. This interest has been supplemented by many developments in transformation systems. Currently, there are three main methods commonly used to transform fungal species with recombinant DNA [1]. The primary method is polyethylene glycol (PEG) transformation, whereby the fungal species is protoplasted to rid the cells of their cell walls. By exposing the fragile protoplasts to PEG and recombinant DNA the cells uptake the DNA. Integration of the DNA into the fungal genome can then occur via homologous recombination, random insertion, or restriction enzyme mediated integration (REMI) [2]. Typically, homologous recombination is the most efficient, though this varies by species. Transformation markers are typically used in order to select for transformants. In fungal strains, these typically include resistances for hygromycin and geneticin. <br> <br>
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Recent years have shown increasing interest in the genetic engineering of filamentous fungi. With this interest has come many developments in transformation methods. Currently, there are two main methods commonly used to transform fungal species with recombinant DNA: <i>Agrobacterium</i> mediated transformation and protoplasting [1].
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<i>Agrobacterium tumefaciens</i> mediated transformation (AMT) is a method by which the fungus is co-cultivated with <i>Agrobacterium</i> [3]. The <i>Agrobacterium</i> is cloned to carry specific plasmids, and passes on these plasmids through horizontal gene transfer, allowing a path through the cell wall. The fungus then integrates the plasmids of the <i>Agrobacterium</i>. <br> <br>
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<i>Ganoderma lucidum</i> and <i>Cochliobolus heterostrophus</i> were the fungal strains we used for genetic engineering. Protoplasting of both organisms was attempted but has thus far only been successful with <i>Cochliobolus heterostrophus</i>. The <i>Cochliobolus</i> protoplasts were then transformed with various constructs. The enzymes typically used for protoplasting <i>Ganoderma lucidum</i> are exclusively available in China, so we attempted to use alternate enzymes [4]. Future attempts at transforming <i>Ganoderma lucidum</i> will be done with <i>Agrobacterium</i> mediated integration.
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<i>Agrobacterium tumefaciens</i> mediated transformation (AMT) is a method by which <i>Agrobacterium</i> are utilized to transfect fungal cells [3]. Genetic constructs are first transformed into the T-DNA of <i>A. tumefaciens</i>. <i>A. tumefaciens</i>, when co-cultured with fungi, will then transfect the fungal cells, inserting the gene of interest into the fungal genetic material. This DNA can then be incorporated into the genome through random insertions or homologous recombination.
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                              <i>Cochliobolus heterostrophus</i>
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The more common method, however, involves protoplasting.  Protoplasts are formed using commercially available enzymes to remove the wall from fungal cells.  Once these fragile cells are formed, they can be transformed with polyethylene glycol (PEG). By exposing the fragile protoplasts to PEG and recombinant DNA the cells uptake the DNA. Integration of the DNA into the fungal genome can then occur via homologous recombination, random insertion, or restriction enzyme mediated integration (REMI) [2]. Typically, homologous recombination is the most efficient, though this varies by species.
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With both <i>Agrobacterium</i> mediated transformation and protoplasting, selection markers are used to select successful transformants. Two commonly used selection markers are the antibiotics hygromycin and geneticin, which are both effective against the strains used in our project: <i>Ganoderma lucidum</i> and <i>Cochliobolus heterostrophus</i>.
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<h3>Reference</h3>
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<h3>References</h3>
1. Weld, R. J., Plummer, K. M., Carpenter, M. A., & Ridgeway, H. J. (n.d.). <i>Appraoches to functional genomics in filamentous fungi</i>. N.p.: Nature.
1. Weld, R. J., Plummer, K. M., Carpenter, M. A., & Ridgeway, H. J. (n.d.). <i>Appraoches to functional genomics in filamentous fungi</i>. N.p.: Nature.

Latest revision as of 03:04, 29 October 2013

Cornell University Genetically Engineered Machines

Fungal Genetic Engineering


Recent years have shown increasing interest in the genetic engineering of filamentous fungi. With this interest has come many developments in transformation methods. Currently, there are two main methods commonly used to transform fungal species with recombinant DNA: Agrobacterium mediated transformation and protoplasting [1].

Agrobacterium tumefaciens mediated transformation (AMT) is a method by which Agrobacterium are utilized to transfect fungal cells [3]. Genetic constructs are first transformed into the T-DNA of A. tumefaciens. A. tumefaciens, when co-cultured with fungi, will then transfect the fungal cells, inserting the gene of interest into the fungal genetic material. This DNA can then be incorporated into the genome through random insertions or homologous recombination.

Cochliobolus heterostrophus
The more common method, however, involves protoplasting. Protoplasts are formed using commercially available enzymes to remove the wall from fungal cells. Once these fragile cells are formed, they can be transformed with polyethylene glycol (PEG). By exposing the fragile protoplasts to PEG and recombinant DNA the cells uptake the DNA. Integration of the DNA into the fungal genome can then occur via homologous recombination, random insertion, or restriction enzyme mediated integration (REMI) [2]. Typically, homologous recombination is the most efficient, though this varies by species.

With both Agrobacterium mediated transformation and protoplasting, selection markers are used to select successful transformants. Two commonly used selection markers are the antibiotics hygromycin and geneticin, which are both effective against the strains used in our project: Ganoderma lucidum and Cochliobolus heterostrophus.

References

1. Weld, R. J., Plummer, K. M., Carpenter, M. A., & Ridgeway, H. J. (n.d.). Appraoches to functional genomics in filamentous fungi. N.p.: Nature.

2. Turgeon, G. B., Condon, B., Liu, J., & Zhang, N. (2010). Protoplast Transformation of Filamentous Fungi (Vol. 638). N.p.: Molecular and Cell Biology for Fungi.

3. Feldmann, K. A., & Marks, D. M. (1986). Agrobacterium-mediated transformation of germinating seeds of Arabidopsi thaliana: A non-tissue culture approach. N.p.: Zoecon Research Institute.

4. Sun, L., Cai, H., Xu, W., Hu, Y., Gao, Y., & Lin, Z. (2001). Efficient Transformation of The Medicinal Mushroom Ganoderma Lucidum (19th ed.). N.p.: Plant Molecular Biology Reporter.