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Selectable marker

A selectable marker is a genetic element, typically a gene, incorporated into recombinant DNA vectors to confer a detectable phenotype that enables the artificial selection of host cells—such as bacteria, yeast, or plant cells—that have successfully integrated the exogenous DNA during transformation or transfection processes.^[1] These markers provide a survival or growth advantage under specific selective conditions, distinguishing transformed cells from the vast majority of non-transformed ones in a heterogeneous population.^[2] Common examples include antibiotic resistance genes, like the ampR gene encoding beta-lactamase for ampicillin resistance in bacterial cloning, which allow only recombinant cells to form colonies on media laced with the antibiotic.^[3] In genetic engineering and molecular biology, selectable markers are indispensable for applications ranging from plasmid propagation in Escherichia coli to the production of transgenic organisms, facilitating high-throughput screening after DNA introduction methods like electroporation or Agrobacterium-mediated transfer.^[4] They are categorized primarily as positive markers, which promote proliferation of successfully modified cells (e.g., herbicide resistance genes such as bar for phosphinothricin in plants), or negative markers, which permit targeted elimination of cells retaining the marker (e.g., the codA gene converting 5-fluorocytosine to toxic 5-fluorouracil).^[5] Advances have introduced auxotrophic markers relying on complementation of metabolic deficiencies and split-marker systems for multiplexed selection without traditional antibiotics, addressing limitations like off-target effects or regulatory hurdles in commercial biotechnology.^[6]^[4] While selectable markers have driven breakthroughs in recombinant protein production and genome editing—such as CRISPR-Cas9 workflows where markers confirm stable integration—their use, particularly antibiotic-based ones, has sparked debate over potential horizontal gene transfer risks to environmental microbes, prompting regulatory scrutiny and the pursuit of marker-free alternatives via site-specific recombination. Empirical studies indicate negligible real-world dissemination under controlled conditions, yet persistent concerns have influenced policy in genetically modified crop approvals.^[7]^[8]

Fundamentals

Definition and mechanism

Selectable markers are exogenous genetic elements, typically genes, introduced into host cells via recombinant DNA constructs to confer a detectable phenotype that distinguishes successfully transformed cells from non-transformed ones in a heterogeneous population.^[1] This phenotype most commonly manifests as survival or growth under conditions lethal or inhibitory to untransformed cells, thereby enabling selective enrichment of transformants.^[3] The operational mechanism centers on the stable integration and expression of the marker gene within the host genome or as an extrachromosomal element, such as a plasmid, following delivery methods like electroporation or Agrobacterium-mediated transfer.^[9] Upon exposure to a selective agent—such as a toxin or nutrient limitation—the expressed marker protein causally intervenes in the host's physiology: it may enzymatically degrade or efflux the toxin, or restore a blocked biosynthetic pathway, allowing metabolic competence and cell viability. Non-transformed cells, lacking this expression, experience unchecked toxicity or deprivation, leading to their death and thus purifying the population through differential survival rates empirically observed in transformation efficiency assays.^[3] This process underscores a direct causal linkage between marker gene transcription, translation, and phenotypic rescue, validated across prokaryotic and eukaryotic hosts where marker absence correlates with zero survival under selection, independent of the gene of interest.^[2] The efficiency stems from the marker's dominance over host susceptibility, minimizing escapees and ensuring high-fidelity identification without reliance on visual or auxiliary screening.^[1]

Types

Positive selectable markers

Positive selectable markers are genes introduced into host cells that confer a survival advantage under selective conditions detrimental to non-transformed cells, thereby enabling the identification and enrichment of successfully transformed populations through gain-of-function mechanisms. These markers typically encode enzymes or proteins that neutralize toxic substances, such as antibiotics or herbicides, or facilitate the metabolism of specific substrates unavailable to untransformed cells, ensuring that only recombinant cells proliferate.^[1]^[10] The dominance of these markers in selection arises from their ability to override endogenous cellular processes, imposing a positive selective pressure that favors transformed cells; for instance, in the presence of an antibiotic, only cells expressing a resistance gene can divide, leading to clonal expansion of desired genotypes. This contrasts with negative selection by emphasizing survival promotion rather than lethality induction, and it underpins routine genetic engineering workflows where initial transformation frequencies may be low (e.g., 10^6 to 10^9 transformants per microgram of DNA in competent Escherichia coli strains).^[11]^[8] In optimized bacterial transformation systems, positive selectable markers achieve screening efficiencies routinely surpassing 90%, as selective media suppress non-transformant growth to negligible levels, minimizing false positives and enabling rapid isolation of rare events without extensive screening. Empirical studies confirm this high stringency, with colony formation rates correlating directly to marker expression and selective agent concentration, though efficiency can vary with host strain competence and marker stability.^[12]^[13]

Negative selectable markers

Negative selectable markers, also termed counterselectable markers, are genetic elements engineered to induce cell death under defined selective conditions, facilitating the targeted elimination of cells that retain or express the marker. Unlike positive markers that promote survival, these enable counter-selection by enriching for populations that have lost the marker through recombination or excision events.^[14] The core mechanism relies on marker-mediated sensitivity to exogenous agents or intrinsic toxicity, where expression activates lethality pathways such as toxin production that disrupts vital cellular functions or enzymatic conversion of non-toxic substrates into cytotoxic metabolites. This process supports applications like post-integration marker removal in gene targeting, where initial transformants are subjected to conditions that kill cells retaining the negative marker, thereby isolating those with precise modifications. Empirical data from bacterial systems demonstrate high specificity, with survival frequencies under selective pressure validating the exclusion of undesired integrants.^[14] Effective deployment demands tight regulatory control over marker expression to prevent unintended lethality in desired cells, often achieved via promoter elements responsive to specific inducers or environmental cues. Studies in microbial genome editing highlight the causal link between marker retention and agent-induced death, with quantitative assays showing near-complete elimination of marked cells (e.g., >99% reduction in viable counts) when parameters like agent concentration and exposure duration are optimized.^[14]^[15]

Historical development

Origins in recombinant DNA technology

Selectable markers emerged as essential components in the foundational experiments of recombinant DNA technology during the early 1970s, driven by the need to identify and propagate rare bacterial transformants harboring engineered plasmids. In a landmark study published in November 1973, Stanley N. Cohen and colleagues at Stanford University constructed the first biologically functional recombinant bacterial plasmids in vitro by ligating EcoRI-generated DNA fragments from separate plasmids, including resistance determinants to tetracycline and kanamycin. These antibiotic resistance genes, such as the tetR locus from the pSC101 plasmid, served as the primary selectable markers, allowing researchers to distinguish transformed Escherichia coli cells—capable of growth on media containing the corresponding antibiotic—from the vast majority of non-transformed cells.^[16] This approach built on prior demonstrations of plasmid-mediated transformation but marked the first use of such markers in engineered chimeric DNA molecules, confirming stable inheritance and expression of foreign genetic material. The necessity for selectable markers stemmed directly from the inherently low efficiency of bacterial transformation protocols available at the time, which yielded transformation frequencies on the order of 10⁻⁵ to 10⁻⁶ per viable cell using calcium chloride-mediated uptake.^[16] Without a linked marker conferring a survival advantage, recombinant events—causally tied to successful plasmid uptake and replication—could not be reliably amplified amid background non-transformants, rendering cloning impractical. Empirical validation came from observing colony formation solely among marker-positive cells, which harbored the predicted recombinant plasmids as verified by resistance profiles and gel electrophoresis. This integration of selectable markers with restriction-ligation techniques, facilitated by Cohen's plasmid expertise and Herbert Boyer's provision of EcoRI enzyme, established the core methodology for DNA cloning, enabling subsequent molecular biology advances while highlighting the causal role of selection in isolating functional recombinants.^[16]

Advancements through the 1980s and 1990s

In the 1980s, selectable markers transitioned from primarily prokaryotic applications to eukaryotic systems, enabling broader genetic engineering efforts. The nptII gene, encoding neomycin phosphotransferase II and conferring resistance to aminoglycoside antibiotics such as kanamycin, became a cornerstone for plant transformation. Horsch et al. (1985) demonstrated its efficacy in tobacco (Nicotiana tabacum) via Agrobacterium tumefaciens-mediated delivery, where leaf disc explants exposed to kanamycin yielded stable transformants with integrated and expressed transgenes, marking a key step in achieving reliable selection in dicotyledonous plants. This adaptation addressed limitations of earlier bacterial markers by optimizing promoters like the cauliflower mosaic virus 35S for eukaryotic expression, facilitating gene transfer in species previously recalcitrant to transformation.^[17] By the 1990s, marker diversity expanded with the integration of herbicide resistance genes, reducing reliance on antibiotics amid emerging biosafety scrutiny over horizontal gene transfer risks to environmental microbes. The bar gene from Streptomyces hygroscopicus, which detoxifies phosphinothricin-based herbicides like bialaphos or glufosinate, was characterized in 1987 and applied as a selectable marker in crops such as rice, where protoplast-derived transformants survived herbicide exposure, yielding herbicide-tolerant regenerants at efficiencies suitable for commercial breeding.^[18]^[19] In parallel, auxotrophic complementation systems advanced for mammalian cells, exemplified by dihydrofolate reductase (DHFR) mutants in Chinese hamster ovary (CHO) lines selected via methotrexate amplification, and glutamine synthetase (GS) systems inhibiting growth with methionine sulfoximine (MSX); these metabolic markers supported high-yield protein production without antibiotics, as GS-knockout NS0 cells complemented with human GS achieved survival rates exceeding 90% under selective pressure. These developments reflected data-driven refinements, with non-antibiotic options like bar correlating with reduced ecological risks while maintaining transformation frequencies comparable to antibiotic systems (e.g., 10-50% recovery in optimized protocols). Biosafety evaluations in the 1990s highlighted antibiotic markers' potential for unintended dissemination, prompting regulatory preferences for alternatives in field trials, though empirical assessments confirmed low transfer probabilities under contained conditions.^[20]^[21]

Applications

In microbial and cell culture systems

In bacterial systems, selectable markers facilitate the stable maintenance of recombinant plasmids during continuous culture by enabling growth under selective pressure from antibiotics, distinguishing transformed cells from non-transformants and preventing plasmid loss over generations.^[22]^[11] This application underpins routine molecular cloning, where markers ensure propagation of expression vectors in hosts like Escherichia coli, supporting scalable production of recombinant proteins such as insulin analogs, with transformation efficiencies reaching up to 10^9 colony-forming units per microgram of DNA in optimized electroporation protocols.^[3]^[23] In yeast systems, such as Saccharomyces cerevisiae, auxotrophic markers complement host deficiencies to permit prototrophic growth on defined minimal media, essential for iterative strain engineering and high-density fermentations in industrial biotechnology.^[24] The URA3 marker, for instance, restores uracil biosynthesis in ura3 mutants, enabling efficient selection of transformants with reported plasmid retention rates exceeding 95% under non-selective conditions when initially established selectively.^[25] This supports applications like library screening for enzyme variants, where marker-based selection accelerates identification of high-producers from 10^6 to 10^8 transformants.^[26] For mammalian cell culture, selectable markers drive the isolation of stable transfectants in lines like CHO or HEK293, critical for biopharmaceutical production where transient expression yields are insufficient.^[27] Markers conferring resistance to agents like G418 allow enrichment of integrants, with selection efficiencies improving clone viability to 1-5% of transfected populations, facilitating high-throughput screening of expression libraries for therapeutic antibodies and yielding titers up to 5-10 g/L in fed-batch processes.^[6]^[28] Overall, these systems leverage markers for empirical advantages in library diversification and protein yield optimization, with marker choice influencing expression variability by up to 10-fold across clones.^[29]

In plant and animal genetic engineering

In plant genetic engineering, selectable markers such as herbicide resistance genes facilitate the identification and regeneration of transformed cells from explants like cotyledons or callus tissue, enabling the development of agronomically improved crops. The cp4-epsps gene, derived from Agrobacterium strain CP4 and conferring tolerance to glyphosate, serves as a prominent example; it was integral to the creation of Roundup Ready soybeans, which were first commercialized in the United States in 1996 by Monsanto.^[30] During transformation via Agrobacterium-mediated methods or particle bombardment, glyphosate application selectively kills non-transgenic cells, allowing efficient recovery of stable integrants and accelerating the breeding cycle from transformation to field-ready lines, often within 1-2 years compared to conventional methods.^[31] This approach has supported widespread adoption, with herbicide-tolerant soybeans occupying over 90% of U.S. acreage by the early 2000s, contributing to yield gains through superior weed control—estimated at 5-15% in glyphosate-tolerant varieties under high weed pressure—while facing regulatory approvals that delayed initial releases by several years in some jurisdictions.^[32] In animal genetic engineering, selectable markers enable the isolation of successfully modified embryonic stem cells, fibroblasts, or oocytes for producing transgenic livestock or biomedical models, particularly in pronuclear injection or CRISPR-assisted editing workflows. The neomycin phosphotransferase II (neo) gene, providing resistance to G418 antibiotic, is commonly co-introduced to select mammalian cell lines harboring transgenes, as demonstrated in the generation of knockout pigs via homologous recombination.^[33] For xenotransplantation, markers like hygromycin resistance cassettes integrated via transposons (e.g., Sleeping Beauty) have been used to produce multi-gene edited pigs, such as those with inactivated alpha-1,3-galactosyltransferase, by enriching cell populations post-transfection before somatic cell nuclear transfer.^[34] These systems expedite therapeutic transgenics, reducing the timeline for establishing founder lines from months to weeks and enabling models for organ compatibility testing, though stringent biosafety regulations have historically limited commercial applications to research herds.^[35]

Examples

Antibiotic resistance markers

The ampR gene, encoding β-lactamase, confers resistance to ampicillin by hydrolyzing the β-lactam ring, enabling selective growth of transformed Escherichia coli on media containing 50–100 μg/mL ampicillin.^[36] This marker was integrated into pBR322, a 4361 bp plasmid constructed in 1977 by combining elements from pMB1 and other sources, which also includes a tetracycline resistance gene for dual selection protocols isolating insertional mutants via marker inactivation.^[37] Transformants are identified by plating on agar with ampicillin, where non-transformed cells fail to form colonies due to cell wall disruption.^[38] The nptII gene, derived from transposon Tn5, encodes neomycin phosphotransferase II, an enzyme that phosphorylates and inactivates aminoglycoside antibiotics including kanamycin (typically at 50 μg/mL) and neomycin, facilitating selection in bacterial, yeast, and plant systems.^[39] In protocols, nptII-containing vectors yield transformants detectable within 24–48 hours on selective media, with expression driven by constitutive promoters like CaMV 35S in plants.^[40] Its efficacy stems from low endogenous activity in most hosts, ensuring high specificity for engineered cells.^[41] These genes proliferated in recombinant DNA applications from the late 1970s, underpinning cloning in vectors like pBR322 for gene isolation and expression studies.^[42] However, empirical assessments of horizontal gene transfer risks—evidenced by rare conjugation events in lab settings—prompted regulatory scrutiny, with bodies like EFSA concluding transfer frequencies below 10^{-9} per recipient but recommending phase-out in GM crops to mitigate potential bacterial resistance dissemination.^[43]^[44] By the 2000s, alternatives were favored in commercial approvals, such as EU directives limiting antibiotic markers in food/feed GMOs due to soil microbe exposure pathways.^[45]

Metabolic and auxotrophic markers

Auxotrophic markers function by complementing mutations in host genes essential for the biosynthesis of amino acids, nucleotides, or other metabolites, thereby restoring prototrophic growth on minimal media lacking the required nutrient.^[24] In the yeast Saccharomyces cerevisiae, the HIS3 gene encodes imidazoleglycerol-phosphate dehydratase, enabling histidine prototrophy in his3 mutants, while LEU2 encodes β-isopropylmalate dehydrogenase for leucine biosynthesis in leu2 strains.^[24] Selection occurs by plating transformants on histidine- or leucine-deficient media, where only cells harboring the functional marker gene survive and proliferate.^[46] These markers have been integrated into single-copy plasmids that complement deficiencies in HIS3, LEU2, or combinations thereof, facilitating stable maintenance without multicopy artifacts.^[46] Metabolic markers extend this principle by conferring the ability to metabolize non-standard substrates unavailable to wild-type cells, often involving enzymatic conversion to usable forms. The bacterial ptxD gene, encoding phosphite oxidoreductase (also termed phosphite dehydrogenase), oxidizes phosphite (Phi) to orthophosphate (Pi), allowing growth on Phi as the sole phosphorus source—a compound that inhibits wild-type organisms due to its toxicity and poor utilization.^[47] Originating from Pseudomonas species, ptxD has been codon-optimized for expression in chloroplasts of algae like Chlamydomonas reinhardtii and plants such as rapeseed, enabling direct selection on Phi-supplemented media without additional supplements.^[47]^[48] This system supports transformation efficiencies comparable to traditional markers, as verified in algal biotechnology where ptxD-expressing lines grew robustly on 1-5 mM Phi while untransformed controls failed.^[47] Empirical studies highlight advantages of these markers, including minimized biosafety risks from avoiding antibiotic resistance dissemination, which regulatory frameworks prioritize to prevent horizontal gene transfer to pathogens.^[21] Auxotrophic complementation imposes no extraneous metabolic burden beyond pathway restoration, unlike resistance genes requiring constant selective pressure, and facilitates marker excision for clean genomes via recombination.^[24] Similarly, ptxD-based selection circumvents antibiotic concerns while enabling crop protection traits, as Phi tolerance correlates with transgene stability in field trials up to 2024.^[48] These approaches have been validated across microbial, plant, and algal systems, supporting pursuits of marker-free engineering without compromising transformation yields.^[21]^[47]

Advantages and limitations

Efficiency and utility in transformation

Selectable markers enhance the efficiency of genetic transformation by imposing a selective pressure that causally filters for cells successfully incorporating exogenous DNA, reducing the reliance on low-probability uptake events typically ranging from 10^{-6} to 10^{-9} in bacterial systems without enrichment.^[13]^[49] This selection mechanism ensures that, among surviving cells on selective media, the proportion of true transformants approaches 100%, as non-transformants are eliminated, thereby minimizing false negatives and enabling reliable recovery from large populations where unassisted screening would be impractical due to overwhelming background growth.^[1]^[2] In practical terms, this utility scales biotechnological pipelines by facilitating high-throughput identification of stable integrants, as evidenced in the 1978 Genentech production of recombinant human insulin in Escherichia coli, where antibiotic resistance markers on the plasmid vector allowed selective propagation of insulin-gene-bearing cells from rare transformants.^[50]^[51] Similar causal efficacy underpins recombinant vaccine development, such as those involving viral antigens expressed in transformed microbial hosts, where markers enable efficient isolation and amplification of producer strains, supporting industrial-scale yields unattainable without targeted selection.^[3] The integration of selectable markers thus causally decouples transformation success from stochastic DNA uptake limitations, promoting scalability in applications like metabolic engineering and protein therapeutics by concentrating resources on verified positives, with empirical transformation efficiencies exceeding 10^8 colony-forming units per microgram of DNA in optimized competent cell protocols.^[52]^[53]

Potential risks and empirical assessments

Concerns have been expressed that antibiotic resistance selectable marker genes in genetically modified (GM) plants could transfer horizontally to bacterial pathogens via mechanisms such as natural transformation, potentially contributing to clinical antibiotic resistance.^[54]^[41] This apprehension has been amplified in public debates over GMOs, with critics positing that even rare transfers could disseminate resistance determinants in microbial communities exposed to decaying plant material in soil or the gut.^[55]^[56] Empirical assessments, however, demonstrate that horizontal gene transfer (HGT) from transgenic plant DNA to bacteria occurs at exceedingly low frequencies, typically ranging from 10^{-10} to 10^{-9} per recipient cell in controlled laboratory conditions involving plant-derived DNA, and often dropping to undetectable levels (e.g., below 10^{-13} per cell without sequence homology) due to barriers like rapid environmental DNA degradation, lack of bacterial uptake competence for plant DNA, and insufficient promoter activity in bacterial hosts.^[57]^[58] Field and microcosm studies simulating soil or phytosphere environments have similarly failed to detect significant HGT events from GM crops harboring antibiotic resistance markers, with transfer rates remaining orders of magnitude below natural bacterial-to-bacterial HGT baselines (10^{-8} to 10^{-1} per cell).^[59]^[60] Regulatory reviews by bodies such as the European Food Safety Authority (EFSA) and Australia's Office of the Gene Technology Regulator (OGTR) conclude that the probability of ecologically relevant transfer is negligible, as no increase in antibiotic resistance attributable to GM selectable markers has been observed despite over 25 years of commercial GM crop cultivation since 1996, encompassing trillions of meals consumed without linked superbug emergence.^[43]^[61] Anti-GMO perspectives persist in highlighting theoretical long-term uncertainties, such as cumulative effects in microbial ecosystems, yet these lack causal substantiation against the weight of longitudinal surveillance data favoring observed non-impact over unverified risks.^[61]^[62]

Recent advances

Development of marker-free approaches

The development of marker-free approaches in genetic transformation arose primarily from regulatory pressures in the early 2000s to minimize the use of antibiotic resistance markers in genetically modified organisms (GMOs), particularly due to concerns over potential transfer to pathogens. The European Union's Directive 2001/18/EC explicitly required risk assessments to consider antibiotic resistance genes in GMOs, prompting innovations to excise or avoid such markers post-selection for compliance and reduced environmental risks.^[63] This directive, along with calls from the European Parliament for phasing out antibiotic-resistant GMOs by 2005, accelerated research into excision systems, enabling transgenics with only the desired trait genes.^[64] Site-specific recombination systems, such as Cre-loxP derived from bacteriophage P1, emerged as a key post-2000 method for marker excision, where the selectable marker is flanked by loxP sites and transiently expressed Cre recombinase catalyzes precise removal.^[65] In plants, this approach achieved marker-free rice plants with enhanced grain quality traits by 2015, demonstrating excision efficiencies up to 90% in progeny after heat-inducible Cre activation.^[65] Similarly, co-transformation strategies, involving separate delivery of the marker and transgene via distinct T-DNAs or plasmids followed by segregation in subsequent generations, produced marker-free lines without recombination machinery, as reviewed in 2006 with applications in crops like tobacco and maize.^[66] These methods reduced residual foreign DNA to below detectable levels in verified lines, addressing stability concerns in self-pollinating species.^[67] Such innovations facilitated regulatory approval by eliminating selectable markers, thereby lowering perceived risks of horizontal gene transfer and enhancing public acceptance of GM crops in regions with stringent GMO policies.^[68] Empirical assessments in excised rice lines confirmed stable transgene inheritance without marker reinsertion, supporting scalability for commercial varieties while complying with post-2001 EU guidelines.^[68] Overall, these approaches shifted transformation protocols toward cleaner genomes, with excision rates improving from initial 10-20% in early trials to over 70% by the 2010s through optimized inducible promoters.^[69]

Novel non-antibiotic and counter-selectable systems

In the 2020s, innovations in selectable markers have emphasized plant-derived or chemically inducible systems to enhance biosafety and regulatory acceptance by avoiding antibiotic or herbicide resistance genes. The GASA6 gene, identified from Arabidopsis thaliana in 2021, functions as a gibberellin-responsive marker that promotes cell survival on sucrose-free media, leveraging glucose signaling pathways for non-antibiotic selection in plant transformation protocols. This system exploits GASA6's role in integrating gibberellin and abscisic acid signaling with carbon starvation responses, yielding transformation efficiencies comparable to traditional markers while remaining endogenous to plants. Phosphite-based selection via the bacterial ptxD gene represents another advance, as detailed in a 2021 review, where expression of phosphite dehydrogenase enables transgenic cells to oxidize phosphite (Phi) to phosphate, a usable nutrient unavailable to wild-type cells.^[70] This dominant positive selection system minimizes environmental release risks, with empirical tests showing low escape rates (under 0.1% in rice and cotton) and cost savings over antibiotic alternatives, as phosphite is inexpensive and non-toxic at selective concentrations.^[70] Applications extend to diverse crops, including rapeseed, where ptxD integration in 2024 conferred phosphite utilization without fitness penalties in field trials.^[48] Counter-selectable systems, which impose lethality unless the marker is excised or lost, facilitate marker-free outcomes and precise edits. The human PIGA gene, encoding a glycosyltransferase essential for GPI anchor biosynthesis, was adapted in 2021 as a counterselectable marker in CRISPR workflows; cells retaining PIGA become sensitive to proaerolysin toxin, enabling single-cell resolution selection for deletions with efficiencies exceeding 90% in mammalian lines.^[71] Though initially mammalian-focused, such toxin-based counters inform analogous yeast engineering by providing conditional lethality tied to enzymatic function loss.^[71] Recent 2024 assessments underscore these systems' utility in recalcitrant fruit trees like citrus and peach, where non-antibiotic markers such as ptxD and inducible auxotrophies have improved regeneration rates by 20-50% over antibiotic baselines, bypassing biosafety scrutiny while maintaining transgene stability in polyploid genomes.^[72] These advances prioritize empirical validation, with field data confirming negligible horizontal transfer risks and enhanced public acceptance for perennial crops.^[72]

References

[1]
Selectable Marker - an overview | ScienceDirect Topics
A selectable marker is defined as a genetic element that provides a survival advantage to transformed cells containing exogenous DNA, allowing for the ...
[2]
Overview on Current Selectable Marker Systems and ... - PubMed
Nov 6, 2024 · Selectable marker genes are useful for recognizing which cells have integrated specific sequences in their genome after genetic transformation ...
[3]
Selectable Marker - an overview | ScienceDirect Topics
Selectable markers are used to select for successful transformants, from untransformed cells, they provide a survival advantage to the cells containing ...
[4]
Split selectable markers - PubMed - NIH
Oct 31, 2019 · Selectable markers are widely used in transgenesis and genome editing for selecting engineered cells with a desired genotype but the variety ...<|separator|>
[5]
Selectable marker genes in transgenic plants - PubMed
Feb 5, 2004 · Positive selectable marker genes are defined as those that promote the growth of transformed tissue whereas negative selectable marker genes ...Missing: molecular | Show results with:molecular
[6]
Split selectable markers | Nature Communications
Oct 31, 2019 · Selectable markers are widely used in transgenesis and genome editing for selecting engineered cells with a desired genotype but the variety ...
[7]
Choice of selectable marker affects recombinant protein expression ...
May 27, 2021 · We show here that the choice of selectable marker gene has a significant impact on both the level of recombinant protein expression and the cell-to-cell ...
[8]
Overview on Current Selectable Marker Systems and Novel ... - NIH
Selectable marker genes are useful for recognizing which cells have integrated specific sequences in their genome after genetic transformation processes.
[9]
Advances in selectable marker genes for plant transformation
Nov 1, 2008 · This review article describes the developments made in the recent past on plant transformation systems using different selection methods
[10]
Selectable Marker - an overview | ScienceDirect Topics
A selectable marker is defined as an antibiotic-expressing gene, either endogenous or exogenous, used to identify positively transformed cells by conferring ...
[11]
Plasmids 101: Positive and Negative Selection for Plasmid Cloning
Aug 22, 2019 · In positive selection, only cells that contain a specific gene survive. The rest do not grow. While there are many ways to design your positive ...
[12]
https://goldbio.com/articles/article/Bacterial-Transformation-Deep-Dive
[13]
Bacterial Transformation Workflow | Thermo Fisher Scientific - ES
The transformation efficiency of competent cells is usually measured by the uptake of sub saturating amounts of a supercoiled intact plasmid, approximately 10– ...
[14]
Counterselectable Markers: Untapped Tools for Bacterial Genetics ...
Counterselectable markers are useful tools for basic genetics, for the study of pathogenesis, and for genome analysis.
[15]
Cytosine deaminase as a negative selectable marker for the ...
The codA gene of Escherichia coli encodes cytosine deaminase, and has previously been used as a negative selectable marker in a range of organisms that ...
[16]
Personal Reflections on the Origins and Emergence of Recombinant ...
With Cohen joining the collaborative effort, pSC101 was chosen as the cloning vector because it contained a readily selectable marker. After ligating the ...
[17]
Agrobacterium-mediated plant transformation: biology and ...
The neomycin phosphotransferase (NPTII) gene for kanamycin resistance and hygromycin phosphotransferase ... selectable marker gene systems in plants.
[18]
Characterization of the herbicide‐resistance gene bar ... - EMBO Press
A gene which confers resistance to the herbicide bialaphos (bar) has been characterized. The bar gene was originally cloned from Streptomyces hygroscopicus.Missing: introduction | Show results with:introduction
[19]
Use of bar as a selectable marker gene and for the production of ...
Use of bar as a selectable marker gene and for the production of herbicide-resistant rice plants from protoplasts. Research Articles; Published: March 1993.
[20]
Safety aspects of microorganisms deliberately released into the ...
In the 1990s, several studies looked at the persistence of GMMs introduced into the environment. Markers like antibiotic resistance, β-galactosidase, catechol 2 ...
[21]
Antibiotic-Free Selection in Biotherapeutics: Now and Forever - PMC
This short review will present examples of alternatives to antibiotic selection and their context of application to highlight their ineluctable invasion of the ...
[22]
New Generation of Plasmid Backbones Devoid of Antibiotic ... - NIH
Aug 30, 2011 · Genes conferring resistance to antibiotics are often used as plasmid selection markers and require growing the bacteria in an antibiotic- ...
[23]
Molecular Biology Reference - Addgene
Selectable Marker, Used to select for cells that have successfully taken up the plasmid and can be used to study the insert. This is different than selecting ...
[24]
Auxotrophic Yeast Strains in Fundamental and Applied Research
Strains that contain disfunctional chromosomal copies of both FUR1 and URA3 allow for the use of a plasmid-borne URA3 gene as a selectable marker, even in ...
[25]
URA3 | SGD - Saccharomyces Genome Database
Nov 17, 2005 · Since URA3 allows for both positive and negative selection, it has been developed as a genetic marker for DNA transformations and other genetic ...<|separator|>
[26]
the effect of common auxotrophic markers on complex traits in yeast
Feb 21, 2024 · Auxotrophic markers in yeast (such as leu2∆, ura3∆, his3∆1, met15∆, and trp1∆) are commonly used to assist in genetic engineering in ...
[27]
Plasmids 101: Mammalian Vectors - Addgene Blog
Mar 25, 2014 · Positive selection in mammalian cells ; Blasticidin, bsd. HeLa, NIH3T3, CHO, COS-1, 293HEK. Inhibits termination step of translation ; G418/ ...
[28]
Positive Selectable Markers for Use with Mammalian Cells in Culture
In this review, a brief chronology of the development of useful selectable marker systems will be presented. The earliest systems, which depended on recessive ...
[29]
Choice of selectable marker affects recombinant protein expression ...
Specifically, we observed that cell lines generated with the NeoR or BsdR selectable markers and selected in the antibiotics G418 or blasticidin, respectively, ...
[30]
Glyphosate‐resistant crops: history, status and future - Dill - 2005
Jan 20, 2005 · Glyphosate-resistant soybean was the first crop launched and marketed under the Roundup Ready brand in the USA in 1996. Since introduction, ...
[31]
The Development of Herbicide Resistance Crop Plants Using ... - NIH
Jun 12, 2021 · Notably, the ALS gene, ACCase gene, and EPSPS gene have provided a wealth of potential genes to produce herbicide-resistant crops. ALS (EC 4.1.
[32]
[PDF] Economic Benefits of Genetically-modified Herbicide-tolerant ...
The study identified the key producer impacts as improved yield, slightly increased fertilizer usage, increased seed costs, decreased tillage use, improved soil ...
[33]
A Site-Specific Recombinase-Based Method to Produce Antibiotic ...
Antibiotic selectable marker genes have been widely used to generate transgenic animals. Once transgenic animals have been obtained, the selectable marker is ...
[34]
Efficient production of multi-modified pigs for xenotransplantation by ...
Jun 29, 2016 · Xenotransplantation from pigs could alleviate the shortage of human tissues and organs for transplantation ... selectable marker (Suppl. Fig. 1A).
[35]
Genetic engineering of pigs for xenotransplantation to overcome ...
Dec 6, 2022 · Genetically modified pigs have been considered as potential animal donors for human xenotransplantation and have been widely used in preclinical research.
[36]
Plasmids 101: Choosing an Antibiotic Resistance Gene
Oct 10, 2023 · The AmpR (amp resistance) gene produces beta-lactamase to degrade amp and therefore prevent cell death. One of the cool things about amp is ...
[37]
PBR322 - an overview | ScienceDirect Topics
pBR322 was made by combining segments of DNA from other plasmids. It has the origin of plasmid replication of a clinically isolated plasmid, pMB1, which is ...
[38]
Plasmid DNA Vaccine vector design: impact on efficacy, safety ... - NIH
Sequences necessary for bacterial retention: Selectable markers. Antibiotic resistance markers are the most commonly utilized selectable markers. Alternative ...
[39]
Statement on the safe use of the nptII antibiotic resistance marker ...
The nptII gene codes for an aminoglycoside phosphotransferase conferring resistance to antibiotics such as kanamycin, neomycin, paromomycin, butirosin, ...
[40]
Kanamycin resistance as a selectable marker for plastid ... - PubMed
The kan gene from the bacterial transposon Tn5, encoding neomycin phosphotransferase (NPTII), was placed under control of plastid expression signals and cloned
[41]
Antibiotic resistance markers in genetically modified plants: a risk to ...
The most frequently used selectable marker in plant cell modification is the nptII gene, which encodes a neomycin phosphotransferase, an enzyme that ...
[42]
The art of vector engineering: towards the construction of next ...
Furthermore, there is a series of vectors called 'shuttle vectors' that contain two different origins and two different selection markers so they can be ...
[43]
EFSA provides scientific advice on the use of antibiotic resistance ...
Apr 19, 2004 · The GMO Panel concluded that the frequency of gene transfer is very low for all ARMGs considered. In its opinion, the GMO Panel took into ...
[44]
Opinion of the Scientific Panel on Genetically Modified Organisms
In this group the nptII gene, which confers kanamycin resistance, has a 13-year history of safe use in food crops and resistance to this group of antibiotics is ...<|separator|>
[45]
Health Considerations Regarding Horizontal Transfer of Microbial ...
The potential effects of horizontal gene transfer on human health are an important item in the safety assessment of genetically modified organisms.
[46]
Saccharomyces cerevisiae single-copy plasmids for auxotrophy ...
Sep 20, 2016 · The 23 single-copy plasmids complement for deficiencies in HIS3, LEU2, URA3, MET17 or LYS2 genes and in their combinations, to match the ...Missing: selectable | Show results with:selectable
[47]
The phosphite oxidoreductase gene, ptxD as a bio-contained ...
Dec 2, 2019 · In theory, ptxD could be used as a selectable marker for chloroplast engineering of other microalgal species, and for introduction of phosphite ...
[48]
The ptxD Gene Confers Rapeseed the Ability to Utilize Phosphite ...
Apr 1, 2024 · The ptxD gene, originating from bacteria, encodes the phosphite dehydrogenase enzyme that is responsible for converting phosphite (Phi) into orthophosphate (Pi ...
[49]
https://www.abcam.com/en-us/knowledge-center/cell-biology/bacterial-transformation-tool-molecular-biology
[50]
[PDF] Production of Insulin Using Recombinant DNA Technology
The vector also includes a selectable marker (e.g., antibiotic resistance gene) for identifying transformed cells. • Example: In E. coli, the insulin gene is ...
[51]
History of insulin - PMC - NIH
Jul 16, 2012 · In 1978, the first recombinant DNA human insulin was prepared by David Goeddel and his colleagues (of Genentech) by utilizing and combining ...Missing: selectable markers
[52]
Six Considerations for Competent Cell Selection - US
Transformation efficiency reflects the amount of supercoiled plasmid taken up by the competent cells; therefore, it directly impacts the cloning efficiency, ...
[53]
https://www.neb.com/en-us/tools-and-resources/usage-guidelines/chemical-transformation-tips
[54]
Horizontal transfer of antibiotic resistance genes into microflora and ...
Nov 18, 2022 · Despite all of the benefits of GMOs, there is great concern among scientists about the possible occurrence of horizontal gene transfer of ...
[55]
Antibiotic resistance and genetically modified plants - PMC - NIH
We should realize that the presence of resistance genes in GM crops represents a much larger burden to the microbial world than all prophylactic and therapeutic ...
[56]
Antibiotic resistance marker genes as environmental pollutants in ...
Antibiotic resistance genes may be considered as environmental pollutants if anthropogenic emission and manipulations increase their prevalence above usually ...
[57]
Horizontal gene transfer from genetically modified plants - NIH
The regulators must assess the risks to human and animal health and to the environment from releasing these GM plants. One such consideration, of many, is the ...
[58]
Horizontal gene transfers in the environment: natural transformation ...
Horizontal gene transfers among bacteria, such as natural transformation or conjugation, may have played an important role in bacterial evolution.
[59]
Horizontal gene transfer from transgenic plants to terrestrial bacteria
Few cases of HGT from plants to bacteria have been reported, suggesting low frequencies, but this is based on a small number of studies.
[60]
Horizontal gene transfer from transgenic plants to terrestrial bacteria
In this review we indicate why putative HGT from plants to terrestrial (soil and plant associated) bacteria has raised concern in biosafety evaluations.Abstract · Introduction · Mechanisms of horizontal... · Methods for the detection of...Missing: empirical | Show results with:empirical
[61]
[PDF] Risk Assessment Reference: Marker Genes in GM Plants
Miki, B., and McHugh, S. (2004). Selectable marker genes in transgenic plants: applications, alternatives and biosafety. Journal of Biotechnology 107, 193-232.<|separator|>
[62]
assessment of the risks associated with the use of antibiotic ...
We conclude that the risk of transfer of AR genes from GM plants to bacteria is remote, and that the hazard arising from any such gene transfer is, at worst, ...
[63]
[PDF] B DIRECTIVE 2001/18/EC OF THE EUROPEAN PARLIAMENT AND ...
Mar 21, 2008 · The issue of antibiotic-resistance genes should be taken into particular consideration when conducting the risk assessment of. GMOs containing ...
[64]
European Parliament Votes For Tighter Restrictions On GMOs
Apr 19, 2000 · They said they wanted a definite date of 2005 for phasing out the use of GMOs that are resistant to antibiotics, rather than simply phasing them ...
[65]
Development of Selectable Marker-Free Transgenic Rice Plants with ...
Jul 14, 2015 · Among these, the Cre/lox system from bacteriophage P1 has been most extensively used for the generation of marker-free plants. Moreover, ...
[66]
Methods to produce marker-free transgenic plants
Oct 30, 2006 · Co-transformation is a method for production of marker- free transformants based on Agrobacterium- or biolistics- mediated transformation in ...
[67]
Development of a Simple and Efficient System for Excising ... - NIH
In the co-transformation and segregation method, the selectable marker gene and desired transgene are co-transformed with two separate T-DNAs or plasmids.Missing: review | Show results with:review
[68]
A Prospective Review on Selectable Marker-Free Genome ...
Jun 9, 2022 · Plant genetic engineering would not have become possible without selectable markers. The selectable markers allow the transformed cells to ...
[69]
Construction of Marker-Free Genetically Modified Maize Using a ...
Here, we present an auto-elimination vector system that uses a heat-inducible Cre to eliminate the selectable marker from transgenic maize, without the need for ...
[70]
ptxD/Phi as alternative selectable marker system for genetic ...
Jul 27, 2021 · Antibiotic and herbicide resistance genes are the most common marker genes for plant transformation to improve crop yield and food quality.
[71]
Application of counter-selectable marker PIGA in engineering ...
Feb 16, 2021 · Such designer genomic manipulations rely on selectable markers to distinguish successfully targeted cells from a much larger number of wild-type ...Abstract · INTRODUCTION · RESULTS · DISCUSSION<|separator|>
[72]
Overview on Current Selectable Marker Systems and Novel ... - MDPI
Selectable marker genes are useful for recognizing which cells have integrated specific sequences in their genome after genetic transformation processes.