Inducible Selectable Marker Genes to Improve Aspergillus fumigatus Genetic Manipulation

The hygromycin B phosphotransferase gene from Escherichia coli and the pyrithiamine resistance gene from Aspergillus oryzae are two dominant selectable marker genes widely used to genetically manipulate several fungal species. Despite the recent development of CRISPR/Cas9 and marker-free systems, in vitro molecular tools to study Aspergillus fumigatus, which is a saprophytic fungus causing life-threatening diseases in immunocompromised hosts, still rely extensively on the use of dominant selectable markers. The limited number of drug selectable markers is already a critical aspect, but the possibility that their introduction into a microorganism could induce enhanced virulence or undesired effects on metabolic behavior constitutes another problem. In this context, here, we demonstrate that the use of ptrA in A. fumigatus leads to the secretion of a compound that allows the recovery of thiamine auxotrophy. In this study, we developed a simple modification of the two commonly used dominant markers in which the development of resistance can be controlled by the xylose-inducible promoter PxylP from Penicillium chrysogenum. This strategy provides an easy solution to avoid undesired side effects, since the marker expression can be readily silenced when not required.

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In Vitro Evolution of Itraconazole Resistance in Aspergillus fumigatus Involves Multiple Mechanisms of Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.11.4405-4413.2004 ◽

2004 ◽

Vol 48 (11) ◽

pp. 4405-4413 ◽

Cited By ~ 101

Author(s):

Márcia Eliana da Silva Ferreira ◽

José Luiz Capellaro ◽

Everaldo dos Reis Marques ◽

Iran Malavazi ◽

David Perlin ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Resistant Mutant ◽

Marker Genes ◽

Reverse Transcription Pcr ◽

Selectable Marker Genes ◽

Clinical Resistance ◽

Mutant Strains ◽

Drug Efflux Pumps ◽

High Level

ABSTRACT We investigated the evolution of resistance to the antifungal drug itraconazole in replicate populations of Aspergillus fumigatus that were founded from a strain with a genotype of sensitivity to a single drug and then propagated under uniform conditions. For each population, conidia were serially transferred 10 times to agar medium either with or without itraconazole. After 10 transfers in medium supplemented with itraconazole, 10 itraconazole-resistant mutant strains were isolated from two populations. These mutant strains had different growth rates and different levels of itraconazole resistance. Analysis of the ergosterol contents of these mutants showed that they accumulate ergosterol when they are grown in the presence of itraconazole. The replacement of the CYP51A gene of the wild-type strain changed the susceptibility pattern of this strain to one of itraconazole resistance only when CYP51A genes with N22D and M220I mutations were used as selectable marker genes. Real-time quantitative reverse transcription-PCR was used to assess the levels of expression of the Afumdr1, Afumdr2, Afumdr3, Afumdr4, AtrF transporter, CYP51A, and CYP51B genes in these mutant strains. Most mutants showed either constitutive high-level expression or induction upon exposure of Afumdr3, Afumdr4, and AtrF to itraconazole. Our results suggest that overexpression of drug efflux pumps and/or selection of drug target site mutations are at least partially responsible for itraconazole resistance and could be considered mechanisms for the emergence of clinical resistance to this drug.

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Poly-L-ornithine-mediated transformation of mammalian cells.

Molecular and Cellular Biology ◽

10.1128/mcb.7.6.2286 ◽

1987 ◽

Vol 7 (6) ◽

pp. 2286-2293 ◽

Cited By ~ 33

Author(s):

V C Bond ◽

B Wold

Keyword(s):

Cell Lines ◽

Mammalian Cells ◽

High Efficiency ◽

Protein Product ◽

Marker Genes ◽

Recipient Mouse ◽

L Cells ◽

Dna And Rna ◽

Selectable Marker Genes

Poly-L-ornithine has been used to introduce DNA and RNA into mammalian cells in culture. Ornithine-mediated DNA transfer has several interesting and potentially useful properties. The procedure is technically straightforward and is easily applied to either small or large numbers of recipient cells. The efficiency of transformation is high. Under optimal conditions, 1 to 2% of recipient mouse L cells take up and continue to express selectable marker genes. DNA content of transformants can be varied reproducibly, yielding cells with just one or two copies of the new gene under one set of conditions, while under a different set of conditions 25 to 50 copies are acquired. Cotransformation and expression of physically unlinked genes occur at high efficiency under conditions favoring multiple-copy transfer. Polyornithine promotes gene transfer into cell lines other than L cells. These include Friend erythroleukemia cells and NIH 3T3 cells. Both are transformed about 1 order of magnitude more efficiently by this procedure than by standard calcium phosphate products. However, the method does not abolish the large transformation efficiency differences between these cell lines that have been observed previously by other techniques. (vi) mRNA synthesized in vitro was also introduced into cells by this method. The RNA was translated resulting in a transient accumulation of the protein product.

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Genetic Engineering with Medicinal Plants: Selection of Fusion Products after Transformation with Dominant Selectable Marker Genes

Planta Medica ◽

10.1055/s-2006-961988 ◽

1989 ◽

Vol 55 (02) ◽

pp. 231-231

Author(s):

G. Lurz ◽

P. Bachmann ◽

R. Greinwald ◽

H. Neuhöfer ◽

F.-C. Czygan

Keyword(s):

Medicinal Plants ◽

Genetic Engineering ◽

Selectable Marker ◽

Marker Genes ◽

Selectable Marker Genes ◽

Dominant Selectable Marker ◽

Fusion Products ◽

Selection Of

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Heat shock induced excision of selectable marker genes in transgenic banana by the Cre-lox site-specific recombination system

Journal of Biotechnology ◽

10.1016/j.jbiotec.2011.07.031 ◽

2012 ◽

Vol 159 (4) ◽

pp. 265-273 ◽

Cited By ~ 23

Author(s):

Borys Chong-Pérez ◽

Rafael G. Kosky ◽

Maritza Reyes ◽

Luis Rojas ◽

Bárbara Ocaña ◽

...

Keyword(s):

Heat Shock ◽

Selectable Marker ◽

Marker Genes ◽

Site Specific ◽

Site Specific Recombination ◽

Selectable Marker Genes ◽

Recombination System ◽

Transgenic Banana ◽

Site Specific Recombination System

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Positive selectable marker genes for routine plant transformation

In Vitro Cellular & Developmental Biology - Plant ◽

10.1079/ivp2001272 ◽

2002 ◽

Vol 38 (2) ◽

pp. 125-128 ◽

Cited By ~ 16

Author(s):

Suprasanna Penna ◽

László Sági ◽

Rony Swennen

Keyword(s):

Plant Transformation ◽

Selectable Marker ◽

Marker Genes ◽

Selectable Marker Genes

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Glyphosate Resistance as a Novel Select-Agent-Compliant, Non-Antibiotic-Selectable Marker in Chromosomal Mutagenesis of the Essential Genes asd and dapB of Burkholderia pseudomallei

Applied and Environmental Microbiology ◽

10.1128/aem.00820-09 ◽

2009 ◽

Vol 75 (19) ◽

pp. 6062-6075 ◽

Cited By ~ 32

Author(s):

Michael H. Norris ◽

Yun Kang ◽

Diana Lu ◽

Bruce A. Wilcox ◽

Tung T. Hoang

Keyword(s):

Burkholderia Pseudomallei ◽

Selectable Marker ◽

Genetic Manipulation ◽

Chromosome 1 ◽

Chromosome 2 ◽

Gene Encoding ◽

Selectable Markers ◽

Genetic Manipulations ◽

Semialdehyde Dehydrogenase ◽

Allelic Replacement

ABSTRACT Genetic manipulation of the category B select agents Burkholderia pseudomallei and Burkholderia mallei has been stifled due to the lack of compliant selectable markers. Hence, there is a need for additional select-agent-compliant selectable markers. We engineered a selectable marker based on the gat gene (encoding glyphosate acetyltransferase), which confers resistance to the common herbicide glyphosate (GS). To show the ability of GS to inhibit bacterial growth, we determined the effective concentrations of GS against Escherichia coli and several Burkholderia species. Plasmids based on gat, flanked by unique flip recombination target (FRT) sequences, were constructed for allelic-replacement. Both allelic-replacement approaches, one using the counterselectable marker pheS and the gat-FRT cassette and one using the DNA incubation method with the gat-FRT cassette, were successfully utilized to create deletions in the asd and dapB genes of wild-type B. pseudomallei strains. The asd and dapB genes encode an aspartate-semialdehyde dehydrogenase (BPSS1704, chromosome 2) and dihydrodipicolinate reductase (BPSL2941, chromosome 1), respectively. Mutants unable to grow on media without diaminopimelate (DAP) and other amino acids of this pathway were PCR verified. These mutants displayed cellular morphologies consistent with the inability to cross-link peptidoglycan in the absence of DAP. The B. pseudomallei 1026b Δasd::gat-FRT mutant was complemented with the B. pseudomallei asd gene on a site-specific transposon, mini-Tn7-bar, by selecting for the bar gene (encoding bialaphos/PPT resistance) with PPT. We conclude that the gat gene is one of very few appropriate, effective, and beneficial compliant markers available for Burkholderia select-agent species. Together with the bar gene, the gat cassette will facilitate various genetic manipulations of Burkholderia select-agent species.

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