Split selectable markers.

Selectable markers are widely used in transgenesis and genome editing for selecting engineered cells with a desired genotype but the variety of markers is limited. Here we present split selectable markers that each allow for selection of multiple unlinked transgenes in the context of lentivirus-mediated transgenesis as well as CRISPR-Cas-mediated knock-ins. Split marker gene segments fused to protein splicing elements called inteins can be separately co-segregated with different transgenic vectors, and rejoin via protein trans-splicing to reconstitute a full-length marker protein in host cells receiving all intended vectors. Using a lentiviral system, we create and validate 2-split Hygromycin, Puromycin, Neomycin and Blasticidin resistance genes as well as mScarlet fluorescent proteins. By combining split points, we create 3- and 6-split Hygromycin resistance genes, demonstrating that higher-degree split markers can be generated by a chaining design. We adapt the split marker system for selecting biallelically engineered cells after CRISPR gene editing. Future engineering of split markers may allow selection of a higher number of genetic modifications in target cells

Mutant loxP vectors for selectable marker recycle and conditional knock-outs

BACKGROUND: Gene disruption by targeted integration of transfected constructs becomes increasingly popular for studies of gene function. The chicken B cell line DT40 has been widely used as a model for gene knock-outs due to its high targeted integration activity. Disruption of multiple genes and complementation of the phenotypes is, however, restricted by the number of available selectable marker genes. It is therefore highly desirable to recycle the selectable markers using a site-specific recombination system like Cre/loxP. RESULTS: We constructed three plasmid vectors (neoR, puroR and bsr), which carry selectable marker genes flanked by two different mutant loxP sites. After stable transfection, the marker genes can be excised from the genome by transient induction of Cre recombinase expression. This excision converts the two mutant loxP sites to an inactive double-mutant loxP. Furthermore we constructed a versatile expression vector to clone cDNA expression cassettes between mutant loxP sites. This vector can also be used to design knock-out constructs in which the floxed marker gene is combined with a cDNA expression cassette. This construct enables gene knock-out and complementation in a single step. Gene expression can subsequently be terminated by the Cre mediated deletion of the cDNA expression cassette. This strategy is powerful for analyzing essential genes, whose disruption brings lethality to the mutant cell. CONCLUSIONS: Mutant loxP vectors have been developed for the recycle of selectable markers and conditional gene knock-out approaches. As the marker and the cDNA expression cassettes are driven by the universally active and evolutionary conserved β-actin promoter, they can be used for the selection of stable transfectants in a wide range of cell lines

RNA BASED PLASMID SELECTION SYSTEM FOR ANTIBIOTIC-FREE PLASMID DNA VECTOR PRODUCTION

Antibiotic resistance markers, typically kanamycin resistance (kanR), allow selective retention of plasmid DNA during bacterial fermentation and are the most commonly utilized selectable markers. However, to ensure safety, regulatory agencies recommend elimination of antibiotic resistance markers from therapeutic and vaccine plasmid DNA vectors. The presence of an antibiotic resistance gene in the plasmid backbone is considered undesirable by regulatory agencies, due to: 1) the potential transfer of antibiotic resistance to endogenous microbial flora; and 2) the potential activation and transcription of the genes from mammalian promoters after cellular incorporation into the genome. Here, we describe the development and application of a novel antibiotic-free (AF) selection system. Vectors with this selection system incorporate and express a 150 bp RNA-OUT antisense RNA. RNA-OUT represses expression of a counter-selectable marker (SacB) from the host chromosome. SacB encodes a levansucrase, which is toxic in the presence of sucrose. Sucrose selectable DNA vaccine vectors combine antibiotic-free selection with highly productive fermentation manufacturing (\u3e1 g/L plasmid DNA yields), while improving in vivo expression of encoded proteins. The RNA-OUT selectable marker can be used to retrofit existing kanR DNA vaccine plasmids into antibiotic-free vectors. Interestingly, a minimum vector size for high yield plasmid production was identified; strategies to ensure high yield production of small plasmids are reported. These vectors are safer, more potent alternatives for DNA therapy or vaccination

Global discovery of adaptive mutations.

Although modern DNA sequencing enables rapid identification of genetic variation, characterizing the phenotypic consequences of individual mutations remains a labor-intensive task. Here we describe array-based discovery of adaptive mutations (ADAM), a technology that searches an entire bacterial genome for mutations that contribute to selectable phenotypic variation between an evolved strain and its parent. We found that ADAM identified adaptive mutations in laboratory-evolved Escherichia coli strains with high sensitivity and specificity

Construction of a novel fungal gus expression plasmid, and its evaluation in Aspergillus nidulans : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University

A GUS expression plasmid, pFunGus, was constructed containing a multi-cloning site for the insertion of gene regulatory elements, to be used in fungal reporter gene studies. A derivative of pFunGus (pFG-gpd) was constructed by the insertion of the gpdA promoter (glyceradehyde-3-phosphatc dehydrogenase) into the multi-cloning site of pFunGus for the assessment of the plasmid's transformation and expression properties in Aspergillus niduans. The correct construction of pFunGus and pFG-gpd was verified by analytical restriction digests and by its property of GUS expression in A. nidulans. The plasmid was integrated into the A. nidulans genome via cotransformation with the phleomycin resistance plasmid, pAN8-l. Transformation frequencies of between 3 and 250 transformants per µg of pAN8-l DNA were obtained. Initial screening for cotransformation yielded no pFG-gpd transformants. Attempts to improve cotransformation frequencies by optimisation of cotransformation conditions were unsuccessful. However, large scale screenings of transformants lead to cotransformants being isolated at a very low cotransformation frequency. Approximately 0.45% of pAN8-l transformants possessed the GUS phenotype. The eight pFG-gpd transformants obtained were analysed by Southern hybridisation. Six out of the eight transformants had a single copy integration. Of the remaining two transformants, one had three copies integrated at separate locations, one of which was disrupted, and the other had four copies integrated as tandem repeats, one of which was disrupted. All the transforming DNA appeared to be integrated ectopically. The physiology of the transformants was assessed by dry weight increase, colony extension and total protein content. These showed that the transformants biology was not significantly compromised by the transforming DNA. Finally, high levels of GUS expression were observed in all pFG-gpd transformants and the GUS expression per copy of the GUS expression cassette integrated into the genome was constant. These results showed that the transformed gene copy number determined the levels of gene activity rather than the position of integration in the genome. Overall these results demonstrate the potential application of the versatile GUS expression plasmid, pFunGus for reporter gene studies in filamentous fungi

A series of vectors for fungal transformation

We report a new fungal selectable marker that confers resistance to chlorimuron ethyl, a sulfonylurea herbicide. This gene as well as genes that confer resistance to hygromycin and bialaphos have been engineered to be compact and to eliminate sites for most common restriction enzymes. These three selectable markers have been used to construct a series of vectors for fungal transformation

New Clox Systems for rapid and efficient gene disruption in Candida albicans

Acknowledgements: We are grateful to Janet Quinn, Lila Kastora, Joanna Potrykus, Michelle Leach, and others for sharing their experiences with the Clox cassettes. We thank Julia Kohler for her kind gift of the NAT1-flipper plasmid pJK863, Claudia Jacob for her advice with In-fusion cloning, and our colleagues in the Aberdeen Fungal Group for numerous stimulating discussions. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. The sequences of all Clox cassettes are available in GenBank: URA3-Clox (loxP-URA3-MET3p-cre-loxP): GenBank accession number KC999858. NAT1-Clox (loxP-NAT1-MET3p-cre-loxP): GenBank accession number KC999859. LAL (loxP-ARG4-loxP): GenBank accession number DQ015897. LHL (loxP-HIS1-loxP): GenBank accession number DQ015898. LUL (loxP-URA3-loxP): GenBank accession number DQ015899. Funding: This work was supported by the Wellcome Trust (www.wellcome.ac.uk): S.S., F.C.O., N.A.R.G., A.J.P.B. (080088); N.A.R.G., A.J.P.B. (097377). The authors also received support from the European Research Council [http://erc.europa.eu/]: DSC. ERB, AJPB (STRIFE Advanced Grant; C-2009-AdG-249793). The European Commission also provided funding [http://ec.europa.eu/research/fp7]: I.B., A.J.P.B. (FINSysB MC-ITN; PITN-GA-2008-214004). Also the UK Biotechnology and Biological Research Council provided support [www.bbsrc.ac.uk]: N.A.R.G., A.J.P.B. (Research Grant; BB/F00513X/1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

Reclamation of ampicillin sensitivity for the genetic manipulation of Legionella pneumophila

Research on Legionella pneumophila, the causative agent of Legionnaires' disease, has been hampered due to the lack of selectable markers for genetic manipulation. We report the construction of a mutant strain of L. pneumophila lacking loxA, a chromosomally encoded β-lactamase, that has enhanced sensitivity to ampicillin. Also described are a method for converting Legionella strains to ampicillin sensitivity and conditions for utilizing bla as a selectable marker

Integration of DNA into bacterial chromosomes from plasmids without a counter-selection marker.

Most bacteria can only be transformed with circular plasmids, so robust DNA integration methods for these rely upon selection of single-crossover clones followed by counter-selection of double-crossover clones. To overcome the limited availability of heterologous counter-selection markers, here we explore novel DNA integration strategies that do not employ them, and instead exploit (i) activation or inactivation of genes leading to a selectable phenotype, and (ii) asymmetrical regions of homology to control the order of recombination events. We focus here on the industrial biofuel-producing bacterium Clostridium acetobutylicum, which previously lacked robust integration tools, but the approach we have developed is broadly applicable. Large sequences can be delivered in a series of steps, as we demonstrate by inserting the chromosome of phage lambda (minus a region apparently unstable in Escherichia coli in our cloning context) into the chromosome of C. acetobutylicum in three steps. This work should open the way to reliable integration of DNA including large synthetic constructs in diverse microorganisms. © 2011 The Author(s)

A conditional marker gene allowing both positive and negative selection in plants

Selectable markers enable transgenic plants or cells to be identified after transformation. They can be divided into positive and negative markers conferring a selective advantage or disadvantage, respectively. We present a marker gene, dao1, encoding D-amino acid oxidase (DAAO, EC 1.4.3.3) that can be used for either positive or negative selection, depending on the substrate. DAAO catalyzes the oxidative deamination of a range of D-amino acids. Selection is based on differences in the toxicity of different D-amino acids and their metabolites to plants. Thus, D-alanine and D-serine are toxic to plants, but are metabolized by DAAO into nontoxic products, whereas D-isoleucine and D-valine have low toxicity, but are metabolized by DAAO into the toxic keto acids 3-methyl-2-oxopentanoate and 3-methyl-2-oxobutanoate, respectively. Hence, both positive and negative selection is possible with the same marker gene. The marker has been successfully established in Arabidopsis thaliana, and proven to be versatile, rapidly yielding unambiguous results, and allowing selection immediately after germination