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Shuttle vector

A shuttle vector is a specialized plasmid designed to replicate and function in two or more distinct host organisms, typically a prokaryote such as Escherichia coli and a eukaryote such as yeast, enabling the transfer of genetic material between incompatible systems.^[1]^[2] This dual-host capability facilitates efficient DNA manipulation, amplification in fast-growing bacterial cells, and subsequent expression or analysis in more complex eukaryotic environments.^[1]^[3] Shuttle vectors are constructed by incorporating multiple origins of replication (ori), one tailored to each host, along with species-specific selectable markers to ensure propagation and selection.^[2] For instance, bacterial origins like the ColE1 ori allow high-copy replication in E. coli, while eukaryotic elements such as autonomously replicating sequences (ARS) and centromeres (CEN) enable stable maintenance in yeast.^[1] Additional features often include multiple cloning sites for inserting genes of interest, antibiotic resistance genes for prokaryotic selection (e.g., ampicillin resistance via β-lactamase), and auxotrophic markers for eukaryotic selection (e.g., TRP1 for tryptophan prototrophy in yeast).^[3] These components make shuttle vectors versatile tools for genetic engineering, avoiding the need for host-specific redesign.^[2] In molecular biology and biotechnology, shuttle vectors play a crucial role in applications ranging from gene cloning and mutagenesis to the production of viral vectors for gene therapy.^[3] Common examples include yeast-E. coli shuttle vectors like YEp (yeast episomal plasmids) derived from the 2-micron circle, which support high-level expression, and YCp (yeast centromeric plasmids) for low-copy stability.^[3] They are also employed in plant biotechnology for shuttling between E. coli and Agrobacterium tumefaciens, and in mammalian systems to bridge bacterial propagation with transfection-based delivery.^[2] By streamlining workflows across species barriers, shuttle vectors have significantly advanced research in recombinant DNA technology and synthetic biology.^[1]

Definition and Characteristics

Definition

A shuttle vector is a hybrid plasmid engineered to replicate autonomously in two or more distinct host organisms, typically a prokaryote such as Escherichia coli and a eukaryote such as yeast (Saccharomyces cerevisiae) or mammalian cells.^[3] This dual-host capability enables the vector to serve as a versatile carrier for genetic material across species boundaries that would otherwise be incompatible due to differences in cellular machinery.^[4] The primary purpose of a shuttle vector is to facilitate the transfer and maintenance of recombinant DNA between these host systems without the need for extensive in vitro manipulation at each step, allowing efficient propagation, modification, and analysis of DNA constructs.^[5] In contrast to standard plasmids, which are restricted to replication within a single host species owing to their singular origin of replication, shuttle vectors incorporate multiple origins of replication (ori) tailored to function in each respective host.^[2] This design leverages the ease of genetic manipulation in prokaryotic systems like E. coli for initial cloning and amplification, followed by seamless introduction into eukaryotic hosts for further study.^[6] At its core, the replication mechanism of a shuttle vector depends on host-specific replication machinery that recognizes and utilizes heterologous ori sequences present in the plasmid, ensuring stable maintenance and copy number control in each environment.^[3] Shuttle vectors emerged in the late 1970s and early 1980s amid the rise of recombinant DNA technology, providing a critical tool for bridging prokaryotic and eukaryotic genetic systems.^[7]

Key Components

Shuttle vectors are engineered with a prokaryotic origin of replication to enable efficient propagation in bacterial hosts such as Escherichia coli. A common example is the ColE1 origin derived from plasmids like pBR322, which supports high-copy-number replication (typically 15–20 copies per cell) and allows for easy amplification and manipulation of the vector DNA in prokaryotic systems.^[8] This origin functions by initiating bidirectional DNA synthesis from a specific RNA primer, ensuring stable maintenance during bacterial cell division without requiring additional viral proteins.^[9] To facilitate replication in eukaryotic hosts, shuttle vectors incorporate an eukaryotic origin of replication tailored to the target organism. For yeast systems, autonomously replicating sequences (ARS), such as those from the yeast 2-micron plasmid or chromosomal fragments like the one in YRp7, allow extrachromosomal maintenance with copy numbers of 5–10 per cell.^[8] In mammalian cells, the SV40 origin of replication enables episomal propagation by recruiting host replication machinery, often requiring co-expression of SV40 large T antigen for initiation, resulting in high-level amplification during transient transfection.^[9] These origins ensure the vector persists independently of the host genome, supporting iterative transfer between prokaryotic and eukaryotic environments. Selectable markers are essential for identifying and maintaining transformed cells in each host, typically including dual systems for independent selection. In bacterial hosts, antibiotic resistance genes like ampR (conferring ampicillin resistance) allow growth on selective media, while in yeast, auxotrophic markers such as URA3 (for uracil prototrophy) or LEU2 (for leucine prototrophy) complement host mutations, enabling selection without antibiotics.^[8] For mammalian hosts, markers like neomycin resistance (neoR) under control of SV40 promoters permit selection with G418, ensuring only successfully transfected cells survive.^[9] These markers are positioned to avoid disrupting replication origins, providing robust dual-host compatibility. Promoter and terminator sequences in shuttle vectors are designed for cross-host functionality to drive expression of selectable markers and inserted genes. Bacterial promoters, such as those upstream of ampR, ensure transcription in prokaryotes, while eukaryotic elements like the SV40 early promoter or yeast-specific upstream activating sequences (UAS) support expression in non-bacterial systems.^[9] Terminators, including polyadenylation signals from SV40 for mammalian hosts or yeast transcriptional terminators, prevent read-through and stabilize transcripts, thereby maintaining efficient gene expression across hosts without interference from host-specific transcription factors. A multiple cloning site (MCS) provides a polylinker region with unique restriction enzyme sites for straightforward insertion of foreign DNA. In vectors like YIp5 or pSV2 derivatives, the MCS is strategically located downstream of promoters but upstream of terminators, minimizing disruption to essential replication and selection elements.^[8] This design allows precise integration of genes of interest while preserving the vector's shuttling capability, with sites such as EcoRI, BamHI, and SalI commonly included for versatility.^[9]

History and Development

Origins

The development of shuttle vectors emerged in the 1970s amid the rapid expansion of recombinant DNA technology, which began with foundational experiments demonstrating the cloning of DNA fragments into bacterial plasmids. Following the 1972 construction of the first recombinant DNA molecule by Paul Berg and colleagues, which joined SV40 viral DNA to lambda phage DNA, and the 1973 work by Stanley Cohen and Herbert Boyer on plasmid-based gene transfer in Escherichia coli, researchers recognized the potential to extend these techniques across prokaryotic and eukaryotic hosts to facilitate genetic manipulation in more complex systems.^[10]^[11] Initial concepts for shuttle vectors drew inspiration from studies of naturally occurring plasmids exhibiting broad host ranges, such as the tumor-inducing (Ti) plasmid in Agrobacterium tumefaciens, which enables DNA transfer between bacterial and plant cells. These natural systems highlighted the feasibility of DNA propagation across species boundaries, prompting molecular biologists to engineer artificial vectors that could replicate in both bacterial and eukaryotic environments to overcome limitations in host-specific cloning.^[2]^[12] Theoretical proposals for shuttle vectors specifically designed to operate between E. coli and Saccharomyces cerevisiae crystallized between 1978 and 1980, fueled by advances in yeast genetics that demanded efficient tools for gene isolation and manipulation. The seminal 1978 experiment by Hinnen, Hicks, and Fink demonstrated yeast transformation using a chimeric ColE1 plasmid carrying the yeast LEU2 gene, marking the first instance of foreign DNA integration into the yeast genome and laying the groundwork for hybrid vector systems. This was rapidly advanced in 1979 by Struhl, Stinchcomb, Scherer, and Davis, who constructed yeast/E. coli shuttle vectors (YEp series) capable of high-frequency autonomous replication in yeast, achieving transformation efficiencies of 5,000–20,000 colonies per microgram of DNA through the incorporation of yeast-derived replication origins.^[13]^[14]^[15] A primary early challenge in these designs was the instability of eukaryotic sequences when propagated in bacterial hosts, where factors like restriction-modification systems and recombination machinery often led to deletions, rearrangements, or loss of inserts, necessitating targeted engineering to stabilize chimeric plasmids for reliable shuttling.^[16]^[17]

Milestones

The development of shuttle vectors began in the late 1970s with foundational work in yeast systems. In 1978, Hinnen et al. published the first yeast/E. coli shuttle vector, YIp5, derived from pBR322 and incorporating the yeast LEU2 selectable marker for integrative transformation, enabling propagation and selection in both hosts. This marked a pivotal advance in recombinant DNA technology, allowing efficient cloning in E. coli followed by transfer to Saccharomyces cerevisiae. By 1979, Struhl et al. introduced the URA3 marker into similar shuttle constructs, further standardizing selectable elements for yeast transformation and gene isolation. The late 1970s saw rapid refinements, particularly in episomal maintenance. In 1979, Broach et al. developed high-copy episomal shuttle vectors like YEp13 by integrating the 2-micron plasmid origin of replication (ori) into bacterial backbones, achieving stable, high-copy propagation in yeast without chromosomal integration. YEp24, specifically constructed with a 2.2 kb EcoRI fragment from the 2-micron plasmid and the URA3 gene, became a widely adopted tool for overexpression studies due to its autonomous replication.^[18] Concurrently, expansion into mammalian systems occurred; in 1982, Southern and Berg created pSV2-neo, an SV40-based E. coli-mammalian shuttle vector expressing the neomycin resistance gene under SV40 promoter control, compatible with COS cells and CHO lines for transient and stable transfection.^[19] The 1990s brought enhancements for stability in eukaryotic hosts. Researchers incorporated centromeric (CEN) sequences into shuttle vectors, improving mitotic segregation; for instance, Clarke and Carbon's 1980s work on yeast CEN-ARS elements was extended in the 1990s to mammalian-compatible designs, such as those using human alphoid DNA for artificial chromosomes in shuttle systems, enabling low-copy, stable maintenance in human cell lines. This addressed limitations of episomal vectors in higher eukaryotes, reducing loss during cell division. Post-2000 advancements integrated shuttle vectors with emerging genome editing tools. In the 2010s, shuttle vectors were adapted for CRISPR/Cas9 delivery, incorporating Cas9 and guide RNA cassettes into compatible backbones for efficient, targeted editing in various systems while maintaining bacterial propagation for vector production. These hybrid constructs facilitated precise gene modifications across prokaryotic and eukaryotic hosts, bridging classical shuttling with modern synthetic biology.^[20]

Design and Construction

Plasmid Backbone Selection

The selection of an appropriate plasmid backbone forms the foundational step in shuttle vector design, providing essential elements for replication and selection primarily in the bacterial host, such as Escherichia coli, while allowing space for integration of host-specific modules. Key criteria include high structural and segregational stability to ensure reliable maintenance across generations, controlled copy number to balance yield and avoid metabolic burden on the host, and a compact size ideally between 3 and 10 kb, which minimizes instability risks when additional shuttle elements are incorporated.^[21]^[22] These attributes enable the backbone to serve as a robust scaffold without overwhelming the host's replication machinery or introducing recombination hotspots.^[23] Widely adopted backbones derive from well-characterized plasmids like pBR322 or the pUC series, which incorporate the ColE1 origin of replication for medium- to high-copy propagation in E. coli and the ampicillin resistance (ampR) marker for straightforward selection.^[2]^[24] These plasmids, originating from early cloning vectors, offer predictable behavior due to their extensively mapped genetics, including defined restriction sites that facilitate engineering while maintaining overall stability.^[5] For instance, pUC backbones support copy numbers of 500-700 per cell, aiding high-yield plasmid preparation before transfer to secondary hosts.^[2] Compatibility with the secondary host, such as yeast or mammalian cells, requires screening the backbone for absence of deleterious sequences, including cryptic promoters or elements that could trigger toxicity, immune responses, or replication interference in the target organism.^[25] This evaluation often involves sequence analysis to avoid restriction sites prone to cleavage by host enzymes or motifs that might express unintended transcripts.^[26] Once selected, the backbone undergoes initial modifications, such as linearization with restriction endonucleases at unique sites and subsequent dephosphorylation using calf intestinal phosphatase (CIP) to inhibit recircularization and promote efficient ligation of inserts.^[27] These steps typically yield transformation frequencies of 10^5 to 10^7 colonies per microgram of DNA in E. coli, establishing a high-efficiency platform for further assembly.^[27]

Shuttle Elements Integration

Shuttle elements, such as origins of replication and selectable markers for non-bacterial hosts, are integrated into the bacterial plasmid backbone to enable propagation across multiple species. Ligation-based assembly is a foundational technique for this integration, involving the use of compatible restriction enzymes to excise and insert eukaryotic origin fragments. For instance, the ARS1 sequence from Saccharomyces cerevisiae was originally cloned as an approximately 0.85 kb EcoRI-HindIII fragment into the bacterial plasmid pBR322, creating the early shuttle vector YRp7 through standard ligation with T4 DNA ligase following end-compatible digestion.^[28] Similarly, for mammalian systems, the oriP element from Epstein-Barr virus (approximately 1.8 kb) is inserted via ligation into a bacterial backbone, often using enzymes like BamHI or EcoRI to ensure precise orientation and compatibility with the ColE1 origin.^[29] PCR-mediated cloning offers a more modern and flexible approach for amplifying and inserting shuttle elements, allowing for the generation of tailored fragments with added restriction sites for directional cloning. Shuttle elements like ARS1 from yeast or oriP from Epstein-Barr virus (often co-amplified with the EBNA-1 trans-acting factor) are PCR-amplified using high-fidelity polymerases, incorporating flanking sequences for enzymes such as EcoRI and BamHI to facilitate insertion into the linearized bacterial backbone via ligation or recombination. This method was employed, for example, in constructing Haemophilus influenzae shuttle vectors by PCR-amplifying a native origin fragment and ligating it into the EcoRV site of pGP704, ensuring high-fidelity integration without unwanted mutations.^[30] In contemporary synthetic biology, seamless assembly techniques such as Gibson assembly, Golden Gate cloning, or recombination-based methods (e.g., using yeast homologous recombination or TAR cloning) are commonly used to integrate shuttle elements without relying on restriction enzymes, reducing scar sequences and enabling complex multi-part assemblies. Additionally, de novo DNA synthesis allows for the direct chemical assembly of entire minimal shuttle vectors, as demonstrated in 2025 designs for Saccharomyces cerevisiae centromeric and integrating vectors that minimize non-essential sequences for improved stability and efficiency.^[31] Following assembly, verification of the integrated shuttle elements is essential to confirm structural integrity and functionality in both host systems. Restriction mapping involves digesting the construct with enzymes like EcoRI, HindIII, or BamHI to produce a predicted fragment pattern matching the expected map, as routinely applied to yeast-E. coli shuttle vectors like the pRS series. Sanger sequencing of junctions and key regions verifies precise insertion without deletions or rearrangements. Functional assays assess replication and selection in each host; for example, transformation efficiency is evaluated by electroporation or chemical competence, yielding greater than 10^4 colony-forming units (CFU) per μg DNA in both E. coli and yeast strains, indicating successful multi-host propagation.^[32]^[33] To enhance long-term stability and reduce risks such as homologous recombination or excision, shuttle elements are strategically positioned during construction. The multiple cloning site (MCS) is typically placed downstream of bacterial and eukaryotic promoters to avoid interference with replication origins or selection markers, minimizing unintended recombination events between repeated sequences. For instance, in optimized yeast shuttle vectors, ARS1 and CEN elements are located upstream of the MCS, with selectable markers like URA3 or LEU2 flanking the insert region to promote structural maintenance over multiple generations without selective pressure.^[23]^[34]

Applications

Cloning and Propagation

Shuttle vectors facilitate DNA cloning by allowing the insertion of target DNA sequences into their multiple cloning site (MCS), typically through restriction enzyme digestion of both the insert and vector followed by ligation using T4 DNA ligase. The recombinant construct is then transformed into a primary host, such as Escherichia coli, where it undergoes initial amplification due to the vector's bacterial origin of replication. This step leverages E. coli's rapid growth and high transformation efficiency to produce sufficient plasmid DNA for further use.^[31] The propagation cycle begins with high-yield amplification in bacteria, where shuttle vectors achieve copy numbers often exceeding 400 per cell, enabling robust plasmid maintenance and extraction. Purified plasmid DNA is subsequently transferred to a secondary host, such as Saccharomyces cerevisiae, via electroporation, lithium acetate-mediated transformation, or spheroplast fusion, allowing replication in the eukaryotic environment through integrated yeast-specific origins like ARS or 2μ sequences. This shuttling process avoids recloning, streamlining experiments across prokaryotic and eukaryotic systems.^[35]^[36]^[31] Maintenance of shuttle vectors during serial passaging requires selective pressure to prevent loss; in bacterial hosts, antibiotics like ampicillin are added to the growth medium (e.g., LB broth), while in yeast, auxotrophic markers such as URA3 necessitate uracil-deficient synthetic complete (SC) media (e.g., SC -ura plates or liquid). These protocols ensure stable retention over multiple generations, with yeast selections often using dropout media tailored to the marker, such as -his or -leu for HIS3 or LEU2 complements.^[36]^[31] Bacterial hosts provide plasmid yields of 1-10 mg/L of culture under standard conditions, supporting downstream transfer to eukaryotic systems with ample DNA quantities and minimal purification steps. This efficiency is particularly valuable for large-scale cloning, where high bacterial productivity offsets lower eukaryotic yields.^[37]

Gene Expression Studies

Shuttle vectors enable promoter shuttling by incorporating hybrid or interchangeable promoter elements that drive gene expression in diverse host organisms, allowing researchers to compare transcriptional efficiency across prokaryotic and eukaryotic systems. For instance, vectors combining the GAL1 promoter, which is inducible by galactose in yeast, with the cytomegalovirus (CMV) promoter, active in mammalian cells, facilitate the propagation of constructs in Escherichia coli for initial cloning before transfer to yeast or mammalian hosts for expression analysis. This approach has been demonstrated in dual-expression plasmids like pYM101, where the same gene cassette yields detectable β-galactosidase activity in both Saccharomyces cerevisiae (under GAL1 control) and mammalian cell lines (under CMV control).^[38] Such comparisons inform the design of cross-species expression studies. In reporter gene assays, shuttle vectors integrate reporter genes such as lacZ (encoding β-galactosidase) or gfp (encoding green fluorescent protein) downstream of target promoters to quantify transcription and translation efficiency between prokaryotic and eukaryotic environments. These vectors, often based on E. coli-yeast or E. coli-bacterial scaffolds, allow initial assembly in bacteria followed by functional testing in the secondary host, where differences in mRNA stability and protein folding become apparent. For example, in multidrug-resistant Acinetobacter baumannii, shuttle vectors like pLPV2Z and pLPV3Z with lacZ or gfp reporters showed dose-dependent induction under stress conditions like DNA damage, with gfp enabling real-time visualization via fluorescence microscopy and lacZ providing enzymatic quantification.^[39] Similarly, E. coli-mycobacteria shuttle vectors with lacZ fusions have quantified promoter activity in operon studies.^[40] This methodology underscores the vectors' utility in dissecting host-dependent bottlenecks in gene expression. Functional complementation using shuttle vectors tests whether a gene from one organism can restore wild-type phenotypes in mutants of another, leveraging the vectors' ability to propagate and express across kingdoms. A classic application involves introducing human genes into yeast mutants via E. coli-yeast shuttle vectors; for example, human topoisomerase IIβ, expressed from a GAL1-controlled cassette, fully complemented temperature-sensitive top2 mutants in S. cerevisiae, rescuing growth at restrictive temperatures. Human topoisomerase IIα can also complement these mutants, indicating functional conservation across isoforms.^[41]^[42] In another study, the human RCC1 gene complemented prp20 mutants in yeast, restoring nuclear protein import and chromosome condensation, with expression levels achieving near-wild-type rescue efficiency.^[43] These assays not only validate orthologous functions but also reveal evolutionary divergences in protein interactions, with shuttle vectors enabling rapid iteration between bacterial cloning and eukaryotic testing. Shuttle vectors support post-translational analysis by allowing initial propagation in bacteria for high-fidelity construct preparation, followed by expression in eukaryotic hosts capable of modifications absent in prokaryotes, such as glycosylation. In Pichia pastoris, E. coli-yeast shuttle vectors like pPIC9K and pPICZα integrate genes into the genome, enabling secretion of glycoproteins with N-linked mannose chains (Man_8-14GlcNAc₂), which are critical for protein stability and activity. This workflow has been pivotal in studying therapeutic proteins, where eukaryotic glycosylation prevents immunogenicity issues seen in prokaryotic systems, achieving yields up to several grams per liter with native-like modifications.^[44]^[45] By bridging bacterial ease with eukaryotic machinery, these vectors provide a streamlined platform for dissecting modification impacts on protein function. Shuttle vectors are also used in plant biotechnology for shuttling between E. coli and Agrobacterium tumefaciens to facilitate T-DNA transfer for plant transformation, and in mammalian systems to amplify constructs in bacteria before transfection for gene therapy applications, such as producing viral vectors.^[2]

Examples and Variants

Common Shuttle Vectors

One of the most widely used shuttle vectors for Escherichia coli and Saccharomyces cerevisiae is YEp351, an episomal plasmid developed in the 1980s that facilitates high-copy-number expression in yeast.^[46] This vector incorporates the 2-micron origin of replication (ori) from yeast for autonomous replication and maintenance at elevated copy numbers, typically 50-100 per cell, enabling robust gene expression studies.^[47] It also features the ampicillin resistance gene (ampR) for selection in E. coli and the LEU2 auxotrophic marker for selection in leucine-deficient yeast strains, allowing seamless shuttling between bacterial propagation and yeast functional assays.^[46] Another common yeast-E. coli shuttle vector is pRS416, a low-copy centromeric plasmid designed for stable maintenance and genomic integration studies in S. cerevisiae. pRS416 includes the autonomously replicating sequence (ARS1) combined with a centromere element (CEN4) to mimic chromosomal segregation and limit copy number to 1-2 per cell, reducing variability in expression levels compared to episomal vectors.^[48] The vector carries ampR for E. coli selection and the URA3 marker for uracil prototrophy in yeast, with a multiple cloning site derived from pBluescript for straightforward insertional mutagenesis and library construction.^[49] Its design supports precise analysis of gene function without the artifacts of plasmid instability.^[32] For mammalian systems, pcDNA3.1 serves as a versatile E. coli-mammalian shuttle vector optimized for transient and stable transfection in primate cell lines.^[50] This 5.4 kb plasmid uses the cytomegalovirus (CMV) immediate-early promoter to drive high-level expression of inserted genes and includes the SV40 origin of replication, enabling episomal propagation in cells expressing the SV40 large T antigen, such as COS-7 (African green monkey kidney) or engineered HEK293 lines.^[50] Selection in E. coli relies on ampR, while mammalian cells are selected via neomycin resistance (NeoR) conferred by the SV40 promoter, making it ideal for rapid protein production and functional validation in HEK293 cells through transient transfection protocols.^[51]

Specialized Variants

Specialized variants of shuttle vectors have been developed to accommodate non-standard host combinations, enabling replication and function in unique prokaryotic-prokaryotic, viral-hybrid, or cross-kingdom systems beyond typical bacterial-eukaryotic pairings. These adaptations often incorporate specific origins of replication (oris) tailored to the target hosts, facilitating specialized applications in microbiology, gene therapy, and synthetic biology.^[52] Bacterial-bacterial shuttle vectors are designed for propagation between Gram-negative and Gram-positive bacteria, such as Escherichia coli and Bacillus subtilis. These vectors typically combine a ColE1 or p15A ori for high-efficiency replication in E. coli with a Gram-positive-compatible ori, like that from pAMβ1, which originates from Enterococcus faecalis and supports stable maintenance in B. subtilis. For instance, early shuttle vectors derived from pUB110 (a B. subtilis plasmid) and pBR322 (E. coli) feature multiple cloning sites and antibiotic resistance markers for seamless transfer and selection in both hosts, allowing genetic manipulations in diverse bacterial species without eukaryotic intermediates. Such vectors, including pGK12 derivatives, have been instrumental in studying Gram-positive gene regulation and metabolic engineering.^[53]^[54] Viral hybrid shuttle vectors leverage viral elements for shuttling between mammalian cells and insect cells, particularly in gene therapy production pipelines. Adeno-associated virus (AAV)-based plasmids, such as pAAV-CMV, contain the AAV inverted terminal repeats (ITRs) and a cytomegalovirus (CMV) promoter for transgene expression in mammalian cells, while relying on bacterial oris for initial propagation in E. coli. These plasmids are transfected into insect cells (e.g., Sf9) via baculovirus systems to produce AAV particles, which then transduce mammalian targets like human hepatocytes or neurons for therapeutic delivery. This hybrid approach enhances scalability in manufacturing recombinant AAV vectors for treating genetic disorders, with yields improved through optimized insect cell expression.^[55]^[56] In the 2020s, modern lentiviral shuttle vectors integrated with CRISPR/Cas9 cassettes have emerged for bacterial propagation and delivery to human induced pluripotent stem (iPS) cells. These vectors, such as all-in-one lentiviral plasmids (e.g., lentiCRISPR v2 derivatives), feature a bacterial ori (e.g., pUC) for high-copy amplification in E. coli, alongside HIV-1-derived elements for packaging into lentiviral particles in HEK293 producer cells. The resulting virions efficiently transduce iPS cells, enabling precise genome editing for disease modeling and regenerative medicine, with Cas9 and guide RNA expressed from a single cassette under constitutive promoters. This adaptation supports multiplexed CRISPR screens in stem cell lineages while maintaining biosafety through self-inactivating designs.^[57]^[58]

Advantages and Limitations

Advantages

Shuttle vectors enhance efficiency in multi-host molecular biology workflows by allowing initial DNA manipulation and amplification in a prokaryotic host like Escherichia coli, followed by direct transfer to eukaryotic hosts such as yeast or mammalian cells, thereby eliminating the need for multiple intermediate cloning steps that can extend experimental timelines from weeks to days.^[5]^[15] This streamlined process leverages the high transformation efficiency and ease of genetic engineering in bacterial systems, enabling rapid propagation and modification before introduction into more complex hosts where direct cloning is less efficient.^[3] Their cost-effectiveness stems from the ability to produce large quantities of DNA at low cost through bacterial amplification, as shuttle vectors often incorporate high-copy-number origins like ColE1, yielding hundreds of copies per cell (e.g., 500-700 for pUC derivatives).^[59]^[60] This scalable production reduces the reliance on expensive eukaryotic culturing for plasmid preparation, making shuttle vectors particularly advantageous for resource-limited labs conducting iterative experiments.^[5] Shuttle vectors provide versatility for comparative studies of gene function across biological kingdoms, as they support replication and expression in diverse hosts, revealing host-specific regulatory effects on the same genetic construct.^[3] For instance, a gene inserted into a yeast-E. coli shuttle vector can be analyzed for prokaryotic versus eukaryotic phenotypes without re-cloning, facilitating insights into evolutionary adaptations or pathway differences.^[3] Stability is improved through dual selectable markers tailored to each host, which minimize insert loss during propagation by enforcing maintenance under selective pressure in both bacterial and eukaryotic environments.^[15] Centromeric elements in yeast-compatible shuttles, for example, ensure low-copy, high-segregational stability (approximately 1 copy per cell), while 2μ-based systems maintain 40–80 copies per cell, reducing the risk of plasmid segregation errors over multiple generations.^[15]

Limitations

Shuttle vectors face significant size constraints, with total construct lengths typically ranging from 4 to 10 kb, though larger constructs (over 15 kb) are possible but often result in reduced transformation efficiency and increased instability in the secondary host, particularly in yeast or mammalian systems where the additional origins of replication and selection markers already increase the baseline size. For instance, standard yeast-bacteria shuttle vectors range from 4 to 10 kb, incorporating essential elements like ARS/CEN sequences and bacterial ColE1 origins, beyond which insert capacity diminishes due to packaging and propagation inefficiencies.^[31] Host-specific instability poses another key limitation, as eukaryotic elements integrated into shuttle vectors are prone to rearrangements and deletions when propagated in bacterial hosts like Escherichia coli. This instability arises from bacterial recombination machinery acting on repetitive or AT-rich eukaryotic sequences, leading to structural mutations at significant rates in multicopy plasmids without stabilizing modifications such as low-copy origins or recA-deficient strains. Such issues are exacerbated in vectors shuttling between bacteria and yeast, where failure to maintain sequence integrity during bacterial amplification can compromise downstream applications in the eukaryotic host.^[9]^[62]^[63] Expression discrepancies further hinder shuttle vector utility, as promoters optimized for one host frequently underperform or silence in the other due to divergent transcription factors and chromatin environments. For example, bacterial promoters like lac may drive robust expression in E. coli but fail in eukaryotic cells lacking compatible RNA polymerase machinery, while eukaryotic promoters such as CMV can be silenced in mammalian hosts through histone modifications and DNA methylation induced by heterochromatin spreading. These chromatin-mediated effects often result in variegated or transient expression, limiting reliable gene function across hosts.^[2]^[64]^[65] The dual-host replication capability of shuttle vectors also introduces biosafety concerns, particularly under genetically modified organism (GMO) regulations that scrutinize risks of unintended dissemination. In frameworks like the EU's Directive 2001/18/EC, these vectors are assessed for potential recombination with endogenous elements or shedding into the environment, which could enable horizontal gene transfer between prokaryotic and eukaryotic organisms and amplify containment challenges in clinical or agricultural settings. Mammalian-compatible shuttle vectors, for instance, heighten regulatory scrutiny due to possible integration risks in therapeutic applications.^[66]^[67]

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The ARS1 fragment is in a 0.85 kb EcoRI-HindIII subfragment. [5] A general purpose replicating Saccharomyces cerevisiae/Escherichia coli shuttle vector. [4] ...Missing: enzymes | Show results with:enzymes
[28]
Stable replication of plasmids derived from Epstein–Barr virus in ...
Feb 28, 1985 · We show that circular DNAs containing oriP, the EBNA-1 gene and a selectable marker replicate autonomously in cells derived from at least four developmental ...
[29]
Construction of a novel shuttle vector for use in Haemophilus ... - NIH
The shuttle vector, pEJ6, was constructed by ligating a PCR-amplified fragment of pLS88 into EcoRV-digested pGP704 (Fig. 1). pEJ6 was transferred by ...Missing: ARS1 oriP
[30]
New and Redesigned pRS Plasmid Shuttle Vectors for Genetic ...
May 1, 2012 · Saccharomyces cerevisiae, plasmid shuttle vector, auxotrophic marker, drug resistance marker, polylinker/multiple cloning site. Issue Section ...
[31]
Construction and Characterization of Shuttle Vectors for Succinic ...
In this paper, we report the development and characterization of E. coli-rumen bacteria shuttle vectors. After several shuttle vectors were constructed, their ...
[32]
New Shuttle Vector-Based Expression System To Generate ...
Four Staphylococcus aureus-Escherichia coli shuttle vectors were constructed for gene expression and production of tagged fusion proteins.
[33]
Minimal shuttle vectors for Saccharomyces cerevisiae - PMC - NIH
On average, 68 mutations were introduced per minimal shuttle vector, effectively removing unnecessary restriction sites while preserving the original ...
[34]
High copy number and highly stable Escherichia coli–Bacillus ... - NIH
Feb 7, 2020 · The best shuttle plasmids pUC980-1 and pUC980-2, which showed copies more than 450 per cell and segregational stabilities up to 98%, were ...
[35]
Plasmids 101: Yeast Vectors - Addgene Blog
Feb 25, 2014 · Yeast expression plasmids used in the lab typically contain all the necessary components to allow shuttling between E. coli and yeast cells. To ...
[36]
[PDF] Optimizing Plasmid Yields: Flask, Media, Shaking
The pDNA yields from E. coli cultivated in LB media stayed below 10 mg/L in all conditions, whereas the incubation in rich media resulted in 4-5 ...Missing: shuttle | Show results with:shuttle<|control11|><|separator|>
[37]
Promoters for shuttle expression vectors in yeasts/bacteria
Aug 16, 2024 · Yeast shuttle vectors express RFP in Escherichia coli. a Comparison of the yeast km.TEF1 promoter and the bacteriophage T7 RNAP promoter.
[38]
New Shuttle Vectors for Real-Time Gene Expression Analysis ... - NIH
To minimize plasmid size without affecting stability and antibiotic selection, only a small region of the pVRL1 ancestor was incorporated in pLPV vectors ...Results · Table 1 · Iron-Regulated Gene...Missing: positioning | Show results with:positioning
[39]
Assaying Promoter Activity Using LacZ and GFP as Reporters
Green fluorescent protein as a vital marker and reporter of gene ... Escherichia coli-mycobacteria shuttle vectors for operon and gene fusions to lacZ: the pJEM ...
[40]
Complementation of temperature-sensitive topoisomerase II ...
A shuttle vector with either human topoisomerase II beta, human topoisomerase II alpha or yeast topoisomerase II under the control of a GAL1 promoter was used.
[41]
Analysis of yeast prp20 mutations and functional complementation ...
Analysis of yeast prp20 mutations and functional complementation by the human homologue RCC1, a protein involved in the control of chromosome condensation.
[42]
Pichia pastoris: A highly successful expression system for optimal ...
Significant advantages of this system include proper protein folding, posttranslational modifications, and glycosylation ... pastoris are shuttle vectors ...
[43]
Overview of Protein Expression Systems | Thermo Fisher Scientific
Furthermore, these proteins have more native folding and post-translational modifications, such as glycosylation, as compared to other expression systems. In ...
[44]
Yeast/E. Coli Shuttle Vectors With Multiple Unique Restriction Sites
Two yeast/E. coli shuttle vectors have been constructed. The two vectors, YEp351 and YEp352, have the following properties: (1) they can replicate autonomously ...Missing: original paper
[45]
Vector Database - YEp351 - Addgene
(ATCC staff) This is a E.coli/yeast episomal shuttle vector with multiple restriction sites for cloning. (personal communication) Medium is 1227 LB plus ...Missing: markers | Show results with:markers
[46]
pRS416 Sequence and Map - Plasmids - SnapGene
Yeast centromere vector with a URA3 marker and an MCS derived from pBLUESCRIPT II. Sticky ends from different PfoI sites may not be compatible.Missing: shuttle | Show results with:shuttle
[47]
pRS416 - 87521 - ATCC
A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae.Missing: ARS CEN
[48]
[PDF] Mammalian Expression Vector User Manual (Version K)
Nov 10, 2010 · Description. pcDNA™3.1(+) and pcDNA™3.1(–) are 5.4 kb vectors derived from pcDNA™3 and designed for high-level stable and transient ...Missing: shuttle | Show results with:shuttle
[49]
pcDNA3.1(+) Sequence and Map - Plasmids - SnapGene
Mammalian expression vector with the CMV promoter. The MCS is in the forward (+) orientation. Sequence Author: Thermo Fisher (Invitrogen)Missing: E. coli shuttle
[50]
A series of shuttle vectors for Bacillus subtilis and Escherichia coli
Dec 1, 1992 · Shuttle vectors for Bacillus subtilis and E. coli, derived from pUB110 and pBR322, have multiple cloning sites, and are used for cloning, ...
[51]
New shuttle vectors for Bacillus subtilis and Escherichia coli which ...
The new shuttle vectors replicate in both E. coli and B. subtilis, have seven restriction sites, and allow for easy detection of inserted DNA.
[52]
Construction of compatible wide-host-range shuttle vectors for lactic ...
Construction of compatible wide-host-range shuttle vectors for lactic acid bacteria and Escherichia coli ... Bacillus subtilis (pAMbeta1, pWV01). These plasmids ...Missing: ori | Show results with:ori
[53]
Adeno-associated virus vector as a platform for gene therapy delivery
Abstract. Adeno-associated virus (AAV) vectors are the leading platform for gene delivery for the treatment of a variety of human diseases.Missing: pAAV- | Show results with:pAAV-
[54]
Production of AAV Vectors Using Suspension Mammalian and ...
Oct 28, 2019 · Adeno-associated viral (AAV) vectors are currently the most widely used gene transfer vectors in many gene therapies that are in development ...Missing: shuttle CMV
[55]
Lentiviral Vectors for Delivery of Gene-Editing Systems Based on ...
Lentiviral vector (LV) systems are one of the main delivery vehicles for the CRISPR/Cas systems due to (i) its ability to carry bulky and complex transgenes.
[56]
Lentivirus CRISPR Vector - VectorBuilder
A lentivirus CRISPR vector is a viral vehicle for introducing Cas9 and gRNA into cells, delivering all components for gene editing using a single vector.
[57]
Shuttle Vector - an overview | ScienceDirect Topics
Such systems have both advantages and disadvantages. The rapid replication of these vectors generates up to 105 vector molecules per cell in 48 hours. This ...
[58]
[PDF] Minimal integrating shuttle vectors for Saccharomyces ... - bioRxiv
Nov 5, 2024 · Current Protocols in Molecular Biology Kevin Struhl ... A shuttle vector series for precise genetic engineering of. Saccharomyces cerevisiae.
[59]
What is the upper limit on plasmid size that can be transformed into ...
Dec 18, 2024 · The size limit of plasmids that can be smoothly transformed into chemically competent TOP10 cells is generally around 15-20 kb, depending on the ...
[60]
Reduction in the structural instability of cloned eukaryotic tandem ...
Oct 27, 2014 · Here, we show an example of a clear difference in the instability of tandem-repeat DNA between different culturing temperatures.Missing: shuttle specific<|separator|>
[61]
Remarkable stability of an instability-prone lentiviral vector plasmid ...
It is noteworthy that the majority of reported instability occurrences are observed in attempts to clone eukaryotic DNA in constructs propagated in prokaryotic ...
[62]
The sound of silence: transgene silencing in mammalian cell ...
As silencing often correlates with specific chromatin modifications profiles of transgenes, epigenetic regulation putatively supports and reinforces transgene ...
[63]
A chromatin insulator protects retrovirus vectors from chromosomal ...
A chromatin insulator can improve the expression performance of a widely used class of gene therapy vectors by protecting these vectors from chromosomal ...
[64]
General Considerations on the Biosafety of Virus-derived Vectors ...
This introductory paper gathers general considerations on the biosafety of virus-derived vectors that are used in human gene therapy and/or vaccination.Missing: shuttle | Show results with:shuttle
[65]
Biosafety Issues of Unintended Horizontal Transfer of Recombinant ...
The unintended HGT of rDNA to both prokaryotes and eukaryotes could raise biosafety issues to both human and animal health and the environment.4.1 Risks Due To Hgt · 5. Hgt Of Rdna To Eukaryotes · 5.2 Dna In FoodMissing: shuttle dual