Bypassing obstacles of reverse genetics research approaches

30 September 2015
​A fundamental method in genetic research is the generation of large collections (‘libraries’) of mutant cells or organisms, or cells containing sequence variants of a particular gene. Making such libraries is quite straightforward but characterizing the library is a whole different matter. Kristof Vandewalle and Nico Callewaert (VIB Medical Biotechnology Center, UGent) together with colleagues from VIB and Ghent University have described a method that reports both on the sequence variant as well as on the physical location of sequence-tagged biological entities in well-plate archived clone collections.

Can you explain what makes this novel method so relevant for the research community?
Kristof: For many research questions, a clone library becomes truly useful if we know which mutation or sequence variant is present in each clone. Wellknown examples are collections of yeast strains that each contain a knockout mutation in one gene, or Arabidopsis T-DNA insertion line libraries.

The construction of such pre-characterized libraries of mutants can be extremely valuable, especially in difficult-to-engineer organisms, but often requires huge investments in terms of time and money. We have devised a more efficient alternative: an effective and easy-to-implement parallel sequencing-based approach. Characterizing a 96x96 clone Mycobacterium
bovis BCG transposon insertion library.

You worked with a Mycobacterium mutant library. Will this novel method have implications for
tuberculosis research?
At the moment, large efforts are ongoing in the tuberculosis (TB) research community to generate
genome-wide mutant resources for slow-growing mycobacteria. Our research provides a very versatile and extremely cost-effective tool to construct such resources. We have generated the largest resource of mutants in any strain of the M. tuberculosis complex to date.

These single mutants are now available within our lab for testing and can be provided to any researcher upon request. Moreover, with minor modifications to the setup, this straightforward approach is entirely portable to any ordered library of sequence-tagged biological entities or clone collections.

You demonstrated this approach using a Mycobacterium bovis BCG mutant library, instead of
Mycobacterium tuberculosis, why?
Nico: With M. bovis BCG genetically being over 99% identical to M. tuberculosis, it is a highly
suitable and safer alternative for genetic studies. Moreover, although generated almost a century ago, M. bovis BCG is still the only licensed vaccine against tuberculosis. We and others believe that this vaccine strain has retained properties that can modulate theimmune system and thus prevent a fully protective memory immune response.

By inactivating some of these corresponding genes, we hope to obtain a modified strain that is able to mount an appropriate immune response. This has already been described by our lab in the past using a mutant from the collection with a disruption in the SapM locus. Festjens et al, 2011

Nevertheless, further improvements through additional mutations will probably be required to
progress into clinical trials. To this end, genomewide mutant collections in this particular organism
can be highly valuable to identify more rapidly such additional immunomodulatory genes.

Vandewalle et al., Nat Communications 2015

From left to right: Nele Festjens, Kristof Vandewalle, Nico Callewaert and Evelyn Plets