NATX Plasmids, for: DNA Vaccines, Gene Therapeutics, Viral Vectors andBiophamacueticals
dcm- Strains
For easy purification of recombinant proteins and plasmid DNA

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Phone: (402) 323-6289 (natx)
Fax: (402) 323-6292
Email: support@natx.com

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X• E. coli Genomic DNA Testing
X• Residual RNA qPCR
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• 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
Fig 1
Fig 2
click image to enlarge

XXXDescription:
 
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.
XXXFeatures:

• 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)
XXXFigures

Figure 1. Plasmid dcm methylation reduced transgene expression. Expression of EGFP from various vectors in the HEK293 cell line is increased with dcm- plasmid.

A: Average +SD EGFP fluorescence (FU) from 18 and 48 h time points posttriplicate transfection of the indicated dcm+ or dcm- plasmids.


B: Average +SD EGFP fluorescence (FU) from 48 h time points posttriplicate transfection of the indicated dcm+ or dcm- -plasmids.


Figure 2. Plasmid dcm methylation increased transgene immunogenicity. Despite increased expression, dcm- influenza H5 HA plasmid had reduced antibody titers against the HA2 protein domain after naked DNA IM immunization.
A: Average +SD EGFP fluorescence from 48 h time point posttriplicate transfection of dcm+ or dcm- NTC8485-VA1-EGFP and NTC7482-41H-VA2-EGFP plasmids.


B: Immunogenicity (humoral immune response after prime boost vaccination) of dcm+ or dcm-
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].


Xdmc-E. coli Cell Lines Pricing


Strain Catalog Number Price
NTC4862 DH5a host strain for 8-series vectors selection, 0.5 mL NTC-DVU-CC1
$199.00
NTC48165 DH5a dcm- host strain for 8-series vectors selection, 0.5 mL
 NTC-DVU-CC2
$199.00
NTC48107 DH5a dcm-NTC48107 DH5a dcm- host strain for 7-series vectors, 0.5 mL NTC-DVU-CC3
$199.00
NTC54207 XL1-Blue dcm- host strain for 7-series vectors, 0.5 mL NTC-DVU-CC4
$199.00
NTC54208 XL1-Blue dcm- host strain for 8-series vectors selection, 0.5 mL NTC-DVU-CC5
$199.00
NTC5402 XL1-Blue host strain for 8-series vectors selection, 0.5 mL
 NTC-DVU-CC6
$199.00