Marker removal from actinomycetes genome using Flp recombinase

Fedoryshyn, Marta; Petzke, Lutz; Welle, Elisabeth; Bechthold, Andreas; Luzhetskyy, Andriy

doi:10.1016/j.gene.2008.04.011

Cited by 52 publications

(26 citation statements)

References 29 publications

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“…These obstacles could be overcome if the resistance markers are eliminated after mutant selection, which can be achieved via SSR. Therefore, removal of a selectable marker from the genome using SSR has been frequently used in plants, mouse cell lines, yeast and recently in actinomycetes (Branda and Dymecki 2004;Schweizer 2003;Fedoryshyn et al 2008a;Zelyas et al 2009). SSR mediated excision of the antibiotic resistance cassette leaves a ''scar'' in the chromosome, meaning a single target site (e. g. loxP site in the case of Cre).…”

Section: Construction Of Unmarked Mutantsmentioning

confidence: 99%

Actinomycetes genome engineering approaches

Siegl

Luzhetskyy

2012

Antonie van Leeuwenhoek

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This review provides an overview of new technologies for DNA manipulations in actinomycetes exploiting recombinogenic engineering (Flp-FRT, Cre-loxP, Dre-rox, Tn5, GusA and I-SceI systems). We will describe some new vectors recently developed for engineering of complex phenotypes in actinomycetes. Several site-specific recombinases, transposons, reporter genes and I-SceI endonuclease have been utilized for genome manipulation in actinomycetes. Novel molecular tools will help to overcome many technical difficulties and will encourage new efforts to address the function of actinomycete genes.

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Section: Construction Of Unmarked Mutantsmentioning

confidence: 99%

Actinomycetes genome engineering approaches

Siegl

Luzhetskyy

2012

Antonie van Leeuwenhoek

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“…Codon distribution and genome GC content could play a role in successful heterologous gene expression in different organisms (Gustafsson et al 2004) because rare codon gene expression can lead to incorporation of wrong amino acids, termination of translation or frame shift errors (Calderone et al 1996). Changing the codon usage or the GC content increased heterologous gene expression in bacterial, animal, and plants (Wohlleben et al 1988;Adang et al 1993;Song and Niederweis 2007;Fedoryshyn et al 2008). The GC content of the native yeast FLP recombinase (39%) is different from that of the poplar lines used in our experiments (45.3%).…”

Section: Excisionmentioning

confidence: 99%

Elimination of marker genes and targeted integration via FLP/FRT recombination system from yeast in hybrid aspen (Populus tremula L. × P. tremuloides Michx.)

Fladung

Schenk

Polak

et al. 2009

Tree Genetics & Genomes

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Marker gene elimination was investigated in hybrid aspen (Populus tremula L. × Populus tremuloides Michx.) using the FLP/FRT recombination system. The construct contained the FLP recombinase under control of a heat inducible promoter, the antibiotic resistance gene nptII driven by the CaMV 35S promoter, and a promoterless uidA gene. The construct was integrated into poplar via Agrobacterium-mediated transformation. The active FLP recombinase excised the nptII marker gene and combined the promoterless uidA gene with the CaMV 35S promoter to form an active uidA gene. For targeted transgene integration, two constructs were used. The first one carried FLP under control of the heat-inducible Gmhsp17.5-E promoter from soybean as well as an active nptII gene flanked by two FRT sites; the second contained the promoterless bar selection marker gene also flanked by two FRT sites. Following transformation and induction of FLP, the enzyme mediated a site-specific recombination at the FRT sites of both constructs. This recombination leads to an excision of the FLP and nptII gene from the first as well as an excision of the promoterless bar gene from the second construct. The promoterless bar gene reintegrated exactly at the former position of the FLP and nptII genes in the first construct to form an active bar gene. The FLP/FRT recombination system from yeast forms a promising basis for the production of antibiotic-free transgenic plants and a useful tool for directed integration of transgenes into plant genomes.

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“…has been attempted in the past unsuccessfully (Khodakaramian et al, 2006;Fedoryshyn et al, 2008). The G + C content of the native flp gene is only 38% while that of Streptomyces genomes is close to 70% (Kieser et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

“…It is possible that the large number of rare tRNAs required for the expression of flp results in ribosome bypassing (the production of a shortened version of the protein) or inhibition of growth (Lindsley et al, 2003;Zahn, 1996). However, a totally synthetic version of flp containing fewer rare codons for S. coelicolor was generated recently and shown to produce functional FLP in S. coelicolor, S. lividans and Saccharotrix espanaensis (Fedoryshyn et al, 2008).…”

Section: Introductionmentioning

confidence: 99%