Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi
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Cites methods from "Efficient four fragment cloning for..."
...Construction of single and double FgPTR2 disruption mutants The uracil-specific excision reagent (USER) cloning system (New England Biolabs) was used to generate the PTR2 binary deletion vectors for Fusarium graminearum transformation as described previously (Frandsen et al. 2008)....
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...The amplified fragments were introduced into the pRF-HU2 (Frandsen et al. 2008) and U-GO vector (Josefsen et al. 2012) flanking the hygromycin or geneticin resistance cassette, respectively into Escherichia coli JM109....
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...Blotting was carried out on EcoRI, SacI, SnaBI, and HindIII (New England Biolabs) digested genomic DNA using a gene specific probe for the hygromycin resistance cassette, which was generated from pRF-HU2 (Frandsen et al. 2008) plasmids using the primers HygF and HygR (Table S1)....
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References
2,471 citations
"Efficient four fragment cloning for..." refers methods in this paper
...Several laboratories have solved this problem by dividing the replacement constructs into two, a technique known as bipartite gene-targeting or split-marker recombination [6- 8]. In this technique, the two HRS's are fused with two thirds of either the 3' or 5'end of the selection marker gene, by fusion-PCR [ 9 ]....
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1,488 citations
"Efficient four fragment cloning for..." refers background in this paper
...The use of Proofreading DNA polymerase is essential when making targeted genome modifications in fungi, due to the close spacing of fungal genes [26], which often means that the HRS extends into neighbouring genes or their regulatory sequences....
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1,185 citations
"Efficient four fragment cloning for..." refers methods in this paper
...Examples are the Xi-cloning, InFusion cloning, Ligase independent cloning (LIC-PCR), Recombinational cloning and USER Friendly cloning techniques [16-20]....
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1,074 citations
"Efficient four fragment cloning for..." refers background or methods in this paper
...crassa, Colot and coworks [1], also allows for efficient four fragment cloning....
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...Contrary to Saccharomyces cerevisiae, where 30 bp is sufficient, many filamentous fungi require longer HRS [1], eg Fusarium graminearum needs 400 bp [2] 1500 bp is reported for Aspergillus niger [3] and around 1000 bp for Neurospora crassa [4]....
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...Vector construction for targeted replacement of genes is reduced to design of two primer pairs, which will permit automation of the experimental design as required for high-throughput knockout projects [1]....
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...Recombinational cloning of the two required HRS with a selection marker gene and a vector backbone is carried out in yeast, followed by PCR amplification of the two HRS and selection marker gene [1]....
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893 citations
"Efficient four fragment cloning for..." refers background in this paper
...The Agrobacterium tumefaciens mediated transformation (ATMT) technology [10] has the advantage of being independent of protoplast formation and can be used directly on a wide variety of fungal species and tissue types [11]....
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