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Production of Retroviral, Lentiviral and shRNA Vectors

One common challenge encountered in pDNA production is presence of stem-loop DNA structures such as viral Long Terminal Repeats (LTR) or short-hairpin RNA (shRNA) that are difficult to replicate and which render the vectors unstable, even in purpose-designed E. coli hosts such as Stbl3™. Clean Genome® E. coli dramatically improves production such of unstable pDNA vectors.

The Clean Genome® E. coli Out Performs Stbl3™ for Lentiviral Production. Chakiath & Esposito (2007) showed that “Lentiviral expression clones, which contain long direct repeats, often show dramatic instability in Escherichia coli.” Even hosts such as Stbl3™ that are specifically designed for cloning lentiviral direct repeats have proven inadequate. Chakiath & Esposito showed that Clean Genome® E. coli strain MDS™42, the foundation strain of the reduced genome platform, stabilizes lentiviral expression clones containing these repeats (Figure 1). In over 100 cloning reactions using MDS™42, these authors picked just two transformant colonies for analysis, and in >95% of the cases both clones chosen had the correct restriction maps, saving substantial time and effort in recombinant plasmid preparation.

Likewise, vectors derived from Adenoassociated virus (AAV) carry two inverted repeats that form 40 bp stems. At the ends, two more nine bp stems branch further ending in loop structures. These two stem-loops themselves comprise direct repeats that are particularly susceptible to deletion when grown in standard E. coli hosts. Extreme example of this is pT-ITR-IL2 vector, (Figure 2) which contains ITR1 and ITR2 stem-loops that are also direct repeats of one another.

To test the Clean Genome® strains’ ability to resolveunstable biotherapeutic pDNA production challenges, Scarab Genomics used the Adenoassociated virus pT-ITR-IL2 Vector. It was transformed into in MDS™42 and unreduced parent strain E. coli K-12 MG1655. Clones containing plasmids with the correct restriction patterns for NotI, KpnI and MscI were selected.

To test stability during growth in E. coli, triplicate clones from both hosts were grown in Luria Broth for 24 hr at 37°C with selection. Culture samples were withdrawn for analysis, and one correct culture of each set was then used to start a serial passage by dilutions of 10-6 – 10-7 in fresh broth. Four more similar serial passages were made, isolating plasmid DNA from each stage for analysis.

The restriction digestion patterns for pT-ITR-IL2 grown in MDS™42 did not change with the serial passages, whereas MG1655-grown plasmids were unstable (Figure 3). The KpnI digests (single site) revealed a smaller plasmid fragment, consistent with loss of the “hammerhead” region between the repeats of the plasmid by internal recombination. The MscI digests (sites on both sides of each ITR) likewise show progressive loss of one of the fragments, consistent with the KpnI digests. DNA sequencing confirmed these results.

Experimental Data
Figure 1: Lentivirus Stability in Reduced Genome E. coli vs. Stbl3™.
Figure 2: An Adenovirus-derived pDNA with Extreme Secondary Structure.
Figure 3: Clean Genome® E. coli Stabilizes pDNA Containing Extreme Secondary Structures.

Learn More: Clean Genome® - A Superior Host for Unstable Plasmid DNA.

Learn More: Chacko S. Chakiath, CS & Esposito, D (2007): Improved recombinational stability of lentiviral expression vectors using reduced-genome Escherichia coli. BioTechniques 43:466-470.

Learn More: Pósfai G, et al., (2006) Emergent properties of reduced-genome Escherichia coli. Science 312:1044-6 and supplemental materials.


Figure 1. Lentivirus Stability in Reduced Genome E. coli vs. Stbl3™. The stability of four variants of a Lentiviral expression plasmid in MDS™42 ΔrecA and Stbl3™ (Life Technologies), showing the proportion of transformants containing intact plasmids (Table 2 Chacko S. Chakiath, CS & Esposito, D (2007) BioTechniques 43:466-470 (October 2007)).



Figure 2: An Adenovirus-derived pDNA with Extreme Secondary Structure. pT-ITR-IL2 contains ITR1 and ITR2 stem-loops that are also direct repeats of one another.



Figure 3: Clean Genome® E. coli Stabilizes pDNA Containing Extreme Secondary Structures.