Functional Genomics & RNAi

FAQ

TargeTron® Design Site FAQs
Adapting TargeTron® to Other Organisms FAQs


TargeTron® Gene Knockout System FAQs

 

  1. Why is this system better than homologous recombination if I have all of the conditions optimized?
    The TargeTron System provides many benefits over homologous recombination:
    1. The TargeTron System gives much higher insertion efficiencies than homologous recombination. With selection, efficiencies are near 100%. Without selection, insertion efficiencies are still ~1-80% (Perutka et. al. 2003. Use of Computer-designed Group II Introns to Disrupt Escherichia Coli DexH/D-box Protein and DNA Helicase Genes. J. Mol. Bio. 336, 421-429).
    2. The TargeTron System has very minimal host specific requirements and can be used over a wide range of bacterial species.
    3. The TargeTron System significantly reduces the amount of time required to generate a knockout. In E. coli, knockouts can be generated in as little as three days in the lab using TargeTron. In Clostridium, it typically takes six months to one year to generate a knockout. Using TargeTron, researchers are able to generate a knockout in Clostridium in only two weeks (Chen et. al. 2005. Construction of an Alpha Toxin Gene Knockout Mutant of Clostridium perfringens Type A by Use of a Mobile Group II Intron. Appl. and Env. Micro. 71, 7542-7547).

  2. Has the TargeTron system been validated in my organism?
    The TargeTron system has been validated in both Gram-positive and Gram-negative bacteria. It has been proven in the following organisms:
    • Staphylococcus aureus
    • Escherichia coli
    • Lactococcus lactis
    • Shigella flexneri
    • Salmonella typhimurium
    • Clostridium perfringens

    Work is being done in additional organisms. Data will be posted to the TargeTron website (www.sigmaaldrich.com/targetron) as it becomes available.


  3. Will the vector included in the kit work in other Gram-negative bacteria, such as Vibrio, without having to do any sub-cloning?
    The system has not yet been validated in Vibrio, but if your organism is normally compatible with E.coli plasmids (T7 promoter and origin of replication), then it is likely that the TargeTron system would work for Vibrio. To use the system in Vibrio, one would most likely have to co-transfect with TargeTron Vector pAR1219 (T2076), which expresses T7 RNA polymerase. Similar experiments were done by Michael Karberg in Shigella and Salmonella (Karberg et. al. 2001. Group II Introns as Controllable Gene Targeting Vectors for Genetic Manipulation of Bacteria. Nature Biotechnology. 19, 1162-1167 1.52 Mb pdf).

  4. Does the TargeTron vector need to replicate to be successful?
    All TargeTron applications to date have used replicating plasmids to express the intron. No data has been generated to show that expression vector replication is required. The group II intron RNA and the Intron Encoded Protein only need to be expressed at sufficient levels to generate the functional RNP’s required to target the gene of interest and to insert the intron to disrupt the gene of interest. The DNA encoding the RNP must be transformed into the host in quantities sufficient for RNP expression. Currently, the p15A ori is used (~10-12 copies per cell) and is successful at expressing sufficient RNP during log-phase growth under plasmid selection using chloramphenicol. For highly competent organisms, it might be possible to express sufficient RNP without vector replication, but this has not been demonstrated.

  5. How do I adapt TargeTron to my organism?
    Please see the Adapting TargeTron for use in Other Organisms FAQs.

  6. How can I generate multiple knockouts using the TargeTron Gene Knockout System?
    Multiple knockouts can be generated without selection by using the TargeTron kit in conjunction with the pACD4 TargeTron Vector Set (no selectable marker) (TV0010). Multiple knockouts can be generated using removable selection by using the TargeTron Vector pACD4K-C-loxP (T2826) with the TargeTron kit.
    For more information, please visit www.sigmaaldrich.com/targetronvectors.

  7. Are there other selectable markers being developed for the TargeTron system?
    No. Vectors without selection and with removable selection are now available. To generate knockouts without using selection, the pACD4 TargeTron Vector Set (TV0010) is recommended. To generate knockouts using removable selection, TargeTron Vector pACD4K-C-loxP (T2826) is recommended.

  8. Can I knockout genes in a plasmid?
    Any DNA sequence may be an insertion site for the TargeTron system.

  9. How much sequence can be brought in using the TargeTron system?
    A few kilobase pairs should be feasible and approximately 2Kb has been inserted to date. It is sequence-dependent due to potential effects of an inserted sequence on intron RNA folding and stability.

  10. Can sequence be knocked-in without group II intron sequence on either side of the insertion?
    In prokaryotic organisms, group II intron sequence will be on either side of the knock-in insertion sequence. Specifically inserting knock-in sequence without group II intron sequence also being inserted has been done using the retrohoming mechanism with eukaryotic exon sequence in a plasmid based system using intron splicing sequence on either side of the knock-in exon sequence. An intron sequence of a eukaryotic gene was then targeted (Sullenger, Molecular Therapy 2005). Group II introns have also been shown to increase homologous recombination of a co- transformed DNA fragment site specifically (Karberg et. al. 2001. Group II Introns as Controllable Gene Targeting Vectors for Genetic Manipulation of Bacteria. Nature Biotechnology. 19, 1162-1167 1.52 Mb pdf).

  11. Do you see polar effects with this knockout strategy?
    You may see polar effects using TargeTron if your gene is part of an operon. Bringing in a native promoter in domain IV of the intron RNA may relieve some of the polar effects. One researcher suggested that cloning your gene of interest out of the operon and into a plasmid would allow the gene to be knocked out in the plasmid without seeing polar effects.

  12. How stable is the knockout after several generations?
    Data in Lactococcus lactis mutants passaged for 80 generations illustrated that 100% of the mutants were stable. (Frazier et. al. 2003. Genetic Manipulation of Lactococcus lactis by Using Targeted Group II Introns: Generation of Stable Insertions without Selection. Appl. and Env. Micro. 69, 1121-1128 897 Kb pdf).

  13. Can the insertion be conditional?
    Yes. If the intron is inserted in the sense orientation, the IEP can be expressed in trans and restore the ORF mRNA to wild-type status (Frazier et. al. 2003. Genetic Manipulation of Lactococcus lactis by Using Targeted Group II Introns: Generation of Stable Insertions without Selection. Appl. and Env. Micro. 69, 1121-1128 897 Kb pdf).

 


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TargeTron® Gene Knockout System