• Nanoplasmids™

    NTC's newest and best-in-class vectors

  • RNA-OUT Retrofit

    The antibiotic-free, sucrose selectable marker,  RNA-OUT, is now available for use in many types of plasmid-based vectors, including: Lenti vectors, retroviral, AAV, helper plasmids, sleeping beauty, DNA vaccines, therapeutic plasmids, mRNA vectors, CAR-T cell vectors, etc.  RNA-OUT is compliant with regulatory agency preferred antibiotic-free plasmid backbones, and has already been approved and used in several first-in-human clinical trials.

     

    NTC now offers its RNA-OUT Retrofit services, which includes verification and plasmid DNA production.  Send us 10 micrograms of your plasmid (on filter paper or in liquid form) and NTC will swap your current marker (such as AmpR or KanR) for the RNA-OUT one, verify the construct, and return the desired quantity of the amplified product, along with the host strain.  Its small size increases potency and reduces the cost of producing plasmid copy number equivalents.

     

    RNA-OUT replaces antibiotics with sucrose selection.  It is regulatory agency compliant, smaller in size (150bp) than conventional markers, is easily swappable into most types of vectors and is therefore ideal for vector products that are destined for clinical use.

    Antibiotic Free RNA-OUT Selectable Marker

    Replaces antibiotic selection markers such as AmpR and KanR with Sucrose Selection

  • Mammalian Expression Vectors

    Enhanced recombinant protein expression in mammalian cells and tissues: superior genetic immunization

    Mammalian Expression Vectors

    A typical advanced NTC vector, NTC8685-EGFP, with the following features: markerless (sucrose selection), enhanced transcription (SV40/CMV), enhanced translation (CMV/HTLV-1-R), enhanced translation (VA1), enhanced plasmid production (PAS-BH).

    NTC Vector Advantages

     

    • DNA Vaccination

     

    • Therapeutic Vector

     

    • Native Expression

     

    • Secreted (TPA tag)

     

    • Minimal Backbone

     

    • Reporter (EGFP)

     

    • Regulatory Compliance

     

    • Simultaneous Cloning

  • DNA Vaccine Vectors

    NTC's DNA Vaccines: Gold Standard Vectors, Optimized Gene(s) of Interest/Antigens, Precision Cloning,

    Quality pDNA Manufacturing and Formulation, Tech Transfer

    DNA Vaccine Vectors

    DNA Vaccine Vectors,  Mammalian plasmid vector

    NTC's leading vector system offers the following improvments in DNA backbone architecture:

    •  Reduced genome (only essential sequences: increases potency, decreases recombination potential)

    •  Trafficking (DNA to the nucleus, protein to selected presentation pathways)

    •  Optimized expression via transcriptional and translational enhancement and a synthetic intron

    •  Antibiotic free (meets regulatory demands)

    •  Selected co-stimulators (activates antigen presentation, prevents tolerization)

    •  Highest yield in the fermentor (increases purity, cost effectiveness)

    A typical advanced NTC vector, NTC8685-EGFP, with the following features: markerless (sucrose selection), enhanced transcription (SV40/CMV), enhanced translation (CMV/HTLV-1-R), enhanced translation (VA1), enhanced plasmid production (PAS-BH).

    NTC's, rapidly deployable set of DNA vaccination tools can be readily adapted to any antigens in a two-week, "precision cloning" turnaround service.

    DNA Vaccine References

    pDNAVACCultra family:  High throughput intracellular targeting DNA vaccine plasmids.

    Williams, J.A., Luke, J., Johnson, L., Hodgson, C.P. (2006) Vaccine 24:4671-4679.

    Improved antibiotic-free DNA vaccine vectors utilizing a novel RNA based plasmid selection system.

    Luke, J. Carnes, A.E., Hodgson, C.P., Williams, J.A. (2009)Vaccine 27(46):6454-9.

    Plasmid DNA vaccine vector design: Impact on efficacy, safety and upstream production.

    Williams, J.A., Carnes, A.E., Hodgson, C.P. (2009) Biotechnol. Adv. 27:353-370.

    Coexpressed RIG-I Agonist Enhances Humoral Immune Response to Influenza DNA.

    Luke, J.M., Simon, G.G., Soderholm, J., Errett, J.S., August, J.T., Gale, J., Hodgson, C.P., Williams, J.A. (2011) Vaccine.  J. Virol. 85:1370-83.

  • Recombineering Vectors

    NTC's Recombineering Vectors: Chromosome Engineering in E. coli

    Recombineering plasmid vector

    Vector Specifications:

     

    pKD46-recA encodes constitutively expressed Escherichia coli recA+ along with wild type rpsL. Plasmid borne rpsL converts streptomycin resistant rpsL strains such as DH10B into streptomycin sensitive strains, allowing counterselection-based plasmid elimination (Imam et al., 2000).

    Recombineering plasmid vector RecA Pa

    Vector Specifications:

     

    pKD46-recApa encodes constitutively expressed Pseudomonas aeruginosa recA+. The RecApa protein induces hyper recombination in E. coli, in the absence of SOS induction, and presence or absence of E. coli RecA protein.

     

    Features:


    • Homologous Recombination-based technology to modify the host chromosome

    • Use PCR or donor cell DNA to directly and precisely alter plasmids, BACS, or host genomic DNA

    • Arabinose-inducible exo, bet, and gam and orf 60a genes

    • Conditional temperature sensitive (ts) vector that is maintained at 30ºC, and lost at 42ºC

    • Counter-selection against plasmid in streptomycin resistant (rpsL-based) host strains (pKD46-recA)

    • Polylinkers for addition of new genes into vector (pKD46-recApa)

    Description:

     

    Recombineering (recombinogenic engineering) is a homologous recombination-based technology used to modify DNA. Target DNA molecules (plasmids, BAC vectors, or the host chromosome) are precisely altered by homologous recombination in host cells which express recombineering enzymes. Recombineering in E. coli often utilize the phage λ Red recombination functions (Murphy, 1998; Datsenko and Wanner, 2000).  The λ genes involved in Red recombination are exo, bet, and gam. The exo (Reda) gene product has 5′ to 3′ exonuclease activity, and the bet (Redb) gene product is a single-strand DNA binding protein that promotes annealing. The gam gene product inhibits the RecBCD nuclease preventing linear DNA (i.e. PCR product) degradation.

     

    Both vectors have been validated for use in PCR- or genomic DNA-mediated recombineering applications

  • Antibiotic Free Vectors

    Antibiotic-Free RNA-OUT Marker for Mammalian Expression and DNA Vaccine Vectors

    Antibiotic Free Vectors, Antisense RNA-OUT marker for plasmid selection

    NTC Vector Advantages:

    • DNA Vaccination

    • Therapeutic Vector

    • Native Expression

    • Secreted (TPA tag)

    • Minimal Backbone

    • Reporter (EGFP)

    • Regulatory Compliance

    • Simultaneous Cloning

    Features:
    • Replaces KanR resistance gene with antisense RNA-OUT, 150bp RNA marker
    • Kit includes strain for selection with RNA-OUT
    • Enhanced expression with optimized, chimeric promoter-intron (SV40-CMV-HTLV-1 R-U5-synthetic intron)
    • Complies with regulatory guidance
    • Increased vaccine potency and safety by replacement of KanR gene
    • Improved DNA backbone for maximum plasmid production in
    E. coli.

    NTC offers a retrofitting service, directly replacing the Kan gene in your current vectors with the RNA-OUT cassette. Natx.com/cloning_qc_retrofit.html

    In keeping with regulatory agency guidance recommending removal of antibiotic resistance genes from vectors used in humans, NTC developed its leading antibiotic free selection system (Fig.1) based on sucrose. The KanR gene is replaced with a 150bp RNA-OUT antisense RNA, which represses expression of the (SacB) gene, located in the host chromosome. This innovation has the additional advantages of: shrinking the plasmid backbone (making it more potent); and decreasing the cost of production through high yield replication in E. coli.

  • Rig-I Activating Vectors

    RIG-I Activating Vectors: NTC8685-eRNA41H-EGFP

    Coexpressed RIG-I Agonist Enhances Humoral Immune Response to Influenza DNA Vaccine, Luke, J.M., Simon, G.G., Soderholm, J., Errett, J.S., August, J.T., Gale, J., Hodgson, C.P., Williams, J.A. J. Virol. 85:1370-83, (2011).

    Features

    • Enhanced antigen expression in mammalian cells

    • Compliance with FDA guidance

    • Improved antibody response

    • Improved antibody avidity

    • Kanamycin or RNA-OUT (antibiotic free) selectable markers

    • Improved DNA backbone for maximum plasmid production (>2.6g/L)

    • Enhanced co-stimulatory RNAe™ activates RIG-1 pathway, Type I interferon production

    • Optimized, chimeric promoter-intron (SV40-CMV-HTLV-1 R-U5-synthetic intron)

    • Superior E. coli plasmid production yields (>2.6g/L)

    • Optional TPA secretion tag

    Rig-I Activation on IFNβ Promoter Luciferase

    Methods to improve DNA vaccine-induced adaptive immunity are needed.  This may be accomplished using plasmid vector backbone-encoded innate immunity agonists. Retinoic-acid-inducible gene 1 (RIG-I) and melanoma differentiation-associated gene 5 (mda5) are critical cytoplasmic double stranded RNA (dsRNA) pattern receptors required for innate immune activation in response to viral infection. Activation of RIG-I and mda5 leads to type I interferon (IFN) and inflammatory cytokine production through IPS-1 activation. Since type I interferon enhances antigen-specific immune responses, we hypothesized that DNA vaccines coexpressing a RIG-I RNA agonist (eRNA) would generate superior immune responses to the encoded antigen. Indeed, expression of  RIG-I activating immunostimulatory RNA from the vector backbone (Fig 1) may be used to activate innate immunity and improve adaptive immune responses (Luke et al., 2011).

     

    Improved Innate and Adaptive Immune Responses

     

  • pVEX™ E. coli Expression Vectors

    NTC pVEX™ Advantages:

    • Highest yield recombinant Protein Production (up to 20g/L)

    • Vaccine Antigen Production

    • Native or Tagged Proteins

    • Thermostable Tag (TST)

          -Thermostable Antigen

          -Thermostable Adjuvant

    • C-tag Stability/Solubility

    • Broad Increased Temperature IPTG Inducible Promoter

    • Defined Media Fermentation

    • Fluorescent Protein Production (Correct Folding)

    • KanR or AmpR Selection

    • Simultaneous Cloning

    • High Throughput Type IIS Restriction Enzyme Cloning

    Pf MBP-HA2 is an effective immunogen
    E. coli expression vector, gel of recombinant NTC TST-Flagellin

    Gel = NTC developed TLR5 receptor agonist TST-Flagellin. lane 1 = uninduced total protein, lane 2 = induced total protein, lane 3 = uninduced soluble protein, lane 4 = induced soluble protein, lane 5 = induced total protein (large scale fermentation harvest), lane 6 = induced soluble protein (large scale fermentation harvest), lane 7 = induced soluble protein after 75ºC 40 min heat treatment (large scale fermentation harvest), M= Marker

    Thermostable antigen and thermostable adjuvant: flagellin, a TLR5 agonist, was fused to thermostable maltodextrin binding protein (MBP) from Pyrococcus furiosus (TST-flagellin).

     

    E. coli expressions vectors - pVEX vector for recombinant protein production

    NTC makes custom recombinant fluorescent proteins for flow cytometry and labelling applications.

     

    Custom proteins for flow cytometry and labelling applications from NTC

    pVEX™ E. coli Expression Vectors

  • Anti Silencing Vectors (ASEs)

    Anti-silencing vectors

    For prolonged expression of integrated genes in mammalian cells.

    ASEs Advantages

    • Maximal and prolonged expression after genome integration

    • Decreased gene silencing during cell line passage

    • Minimal prokaryotic backbone with antibiotic-free (AF) selectable marker

     

     

     

    • Swap your gene into the vector (replaces EGFP)*

     

    • Produce one milligram of plasmid DNA

     

    • Linearize 100 microgram (BspHI or XmnI)

     

    • Linearized vector, and remaining uncut vector is shipped to you for integration into your cell lines

     

    *Integrated cell lines containing the neo gene will be selectable with geneticin (other selectable marker swaps available).

     

     

    NTC’s ASE vector prep services:

     

    Gene silencing is a common phenomenon that is encountered when genes or vectors are integrated into the recombinant host cell chromatin.  NTC now introduces ASE elements to permit maximum gene expression long after integration.

    Anti Silencing Vectors (ASEs)

  • Gene Silencing Vectors

    Gene Silencing Vectors For Transient or Stable (Integrated) Expression of shRNAs

    shUT DOWN™,  Ultra Transient Transfection

    Gene silencing-transient transfection vector for gene silencing.

    shUT DOWN™,  Ultra Transient Transfection Advantages:

    • Antibiotic-free or Kanamycin selection:

    NTC8485E-U6-shRNA (antibiotic free, 2.7kb)

    NTC7485E-U6-shRNA  (Kan, 3.5kb)

    • Increased potency (reduced size)

    • HpaI-EcoRI cloning (NTC precision cloning service)

    • Green/White selection for inserts in E. coli

    • Promoter swap service

    • High fermentation yield plasmids (>1g/L)

    • Dimerization service for high yield/increased potency

  • Autolytic Cell Lines

    E. coli cell leaking plasmid DNA afterinduction of autolysis.

    Legend: brief treatment of plasmid-containing NTC-DH5-AL cells (with EDTA and Triton X100) partially disrupts outer and inner bacterial cell membranes, while chromosomally encoded lysozyme punctures the peptidoglycan cell wall, creating a molecular sieve, whereby plasmid DNA leaks from the cells, thus preventing viscous lysate, and leaving bacterial chromosomal DNA behind in the cell.

    Description:

    Nature Technology Corporation (NTC) has developed autolytic (AL) Escherichia coli host strains that express a chromosomal, integrated lysozyme gene (λR) to permeabilize the bacterial cell wall. Expression of the endolysin is induced during growth by either a heat inducible (ALHEAT) or IPTG inducible (ALIPTG) promoter. The endolysin remains in the cytoplasm, where it is separated from its peptidoglycan substrate in the cell wall; hence the cells remain alive and intact and can be harvested by the usual methods.  Cell lysis can then be performed using a freeze thaw cycle or with Triton X-100/EDTA lysis solutions.

    Features:

     

    • Simplified purification of recombinant proteins and plasmids

    • Stable, chromosomally integrated genes

    • Heat or IPTG inducible lysozyme

    • Derivatives of well-known strains: DH5a (plasmid production); or BL21 (recombinant proteins)

    • Can be used with standard antibiotic resistance markers

    NTC’s autolytic cell lines are derivatives of the popular DH5α and BL21 cells lines.

     

    • Simple, regulated E. coli autolysis using chromosomally integrated stable cell lines

    • Heat or IPTG-inducible phage λR endolysin

    • Heat inducible λR (tightly repressed at 30ºC, induced at 42ºC)

    • IPTG inducible λR (tightly repressed in glucose media, induced with IPTG)

    • Tight regulation prior to induction

    • High-level Endolysin production post-induction using optimized zwf- λR cistron

    • Derivatives of established strains for plasmid (DH5α) or protein (BL21) production

    • Streamlines and simplifies plasmid or protein extraction protocols

    • Compatible with a variety of plasmid selection markers including ampR, kanR, chlorR

  • dcm- Strains

    For easy purification of recombinant proteins and plasmid DNA

    NTC8685 is a highly effective, optimized DNA plasmid vector with sucrose selectable marker.

    Features:

     

    • Standard E. coli production host strains (DH5a, XL1-blue) modified to eliminate dcm methylation of plasmid DNA

     

    • Kanamycin-resistance or antibiotic-free (RNA-OUT) selection vectors (for 7-series or 8-series vectors, respectively)

     

    • Gram-plus per liter yields possible with KanR or antibiotic free (sucrose selection) strains

     

    • Increased expression in eukaryotic cells, combined with decreased immunogenicity, for gene therapy applications

     

    • Can be used with non-NTC Kanamycin-resistance vectors (use strain NTC48107 only)

    NTC7482-41H-VA2-HA plasmid (HA transgene version of NTC7482-41H-VA2-EGFP). Average_SD anti-HA2 total IgG, IgG1, and IgG2a titers at the indicated serum dilutions after prime-boost immunization of naked dcm+ (+) or dcm-(-) plasmid. The total IgG and IgG2a dcm+ versus dcm- titers were significantly different at a Pvalue 0.05 [Wilcoxon (Mann–Whitney) rank-sum test].

    •  Enhanced Gene expression

    •  Decreased immuno-genicity (for therapeutic, vs. vaccine use)

    •  Derivatives of well-known strains: DH5a and XL1-blue

    •  Can be used with standard antibiotic resistance markers

    The dcm methylase recognizes the internal cytosine residues in the recognition sequence 5'-CC*AGG-3' or 5'-CC*TGG-3'. This creates 5-methyl-cytosine (5mC), a common mammalian pattern (CG methylation) although the dcm methylated cytosine is in a different sequence context in bacteria. While plasmid production yields and quality are similar between dcm+ and dcm- host strains, CMV promoter expression is reduced by dcm methylation. Surprisingly, despite improved expression, dcm- plasmid DNA is less immunogenic. These results suggest  that, while dcm+ strains are optimal for DNA vaccination, that dcm- strains may be superior for gene therapy applications wherein reduced immunogenicity is desirable and for in vitro transient transfection applications such as AAV production where improved expression is beneficial. (Carnes et al. 2010, Biotechnol. Bioeng.).

     

    While DH5a is the production host of choice for most plasmid vectors, XL1Blue is a superior host for production of small (<3 kb) vectors (Carnes et al. 2010, J. Gene Med.). NTC offers dcm+ and dcm- versions of these cell lines for vaccine (dcm+) or therapy (dcm-) gene medicine plasmid production.

     

  • dcm- Strains2

VECTORS & CELL LINES

Nanoplasmids™

RNA-OUT Retrofit
Mammalian Expression Vectors

Antibiotic Free Vectors

DNA Vaccines

Rig-I Activating Vectors

Recombineering Vectors

pVEX™- E. coli Expression Vector

Anti-Silencing Elements

Gene Silencing

Transient

Retroviral, stable

Cell Lines

Autolytic E. coli Cell Lines

dcm- Strains

 

PLASMID MANUFACTURING

HyperGRO™, E. coli Cell paste

Contract Plasmid Manufacturing

Small Scale Large Scale

Additional Testing

Order Form

Linear Vectors

 

CLONING SERVICES & QC

Rapid VACC™ Precision Cloning

Plasmid Detox Services

Contract QC

Vector Retrofit Services

Swap Marker or Promoter

E. coli Genomic DNA Testing

Residual RNA qPCR

Residual RNA gel

Supercoil Assay

Lymulus Endotoxin Testing

 

PROTEIN PRODUCTS

Thermostable Protein Expression

HN-Flagellin Protein

HN-TST-HA2 Protein

HN-TST-Flagellin Protein

HYPERTAQ™ DNA Polymerase

Custom Recombinant Proteins

Fluorescent Proteins

TECHNOLOGY & CONSULTING

Process Development

Plasmid Fermentation & Purification

Protein Fermentation & Purification

High yield plasmid Fermentation

ABOUT NTC

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