Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi
Citations
461 citations
Cites methods from "Efficient four fragment cloning for..."
...dahliae were generated by cloning of the Ave1 flanking sequences in pRF-HU2 (51)....
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288 citations
Cites methods from "Efficient four fragment cloning for..."
...PCR products were subsequently cloned into pRF-HU2 (Frandsen et al. 2008)....
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202 citations
Cites background or methods from "Efficient four fragment cloning for..."
...The two flanking fragments were introduced into pRFHU2 following the USER protocol described by [87] and the resulting plasmid was introduced into E....
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...This plasmid derives from plasmid pRFHU2 [87] and contains the flanking regions of the two genes surrounding the hygromycin resistant cassette in the T-DNA region of the plasmid....
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179 citations
Cites methods from "Efficient four fragment cloning for..."
...Both DNA fragments, corresponding to promoter and terminator regions, and the digested pRFHU2 vector (Frandsen et al. 2008) were mixed together and were treated with the USER enzyme (New England Biolabs) to obtain plasmids pRFHU2-patK, pRFHU2-patL, pRFHU2-patN, and pRFHU2-pksCT....
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162 citations
Cites background from "Efficient four fragment cloning for..."
...the number of nts lying between the restriction and nicking site may be increased and/or varied, which might be beneficial when having more than one USER cassette in a single vector and attempting simultaneous PCR product insertion into the different sites (13)....
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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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