Disrupting gene expression is essential for gene function exploration. Creative Biolabs provides ingenious gene targeting system referred to as gene conditional expression. This approach enables the identification of a set of genes that are essential for the fungal pathogen. We also provide gene conditional expression service for bacteria.

Our creative functional genomics approach tailor made for diploid organism allows the conditional knockout of genes. As long as the genomic sequence is available, gene essentiality can be identified and assessed. Our platform demonstrates both the reproducibility and reliability for determining gene essentiality and is extremely suitable for systematic and large-scale essential gene identification and validation in diploid pathogenic microorganisms.

This innovative approach is used through a combination of target gene replacement of one allele and conditional gene expression of the other. One allele is disrupted by PCR-based gene replacement; the second allele is transactive controlled by tetracycline (Tet) promoter which is put upstream from the coding sequence of the targeted gene. Thus, one gene copy is deleted by replacement, and the other is placed under the control of a regulatable promoter. This Tet-regulatable promoter system can achieve conditional repression of a target gene by providing tetracycline. Thus, the addition of Tet in the medium allows the identification of essential genes of the pathogen by the growth phenotype of the corresponding strain. By comparing growth phenotypes of constructed conditionally expressed isolates with wild-type strains, essential genes can be direct demonstrated, and potential targets are identified.

Figure 1. The gene replacement and conditional expression method of target validation (Roemer <em>et al.</em> 2003). Figure 1. The gene replacement and conditional expression method of target validation (Roemer et al. 2003).

After identification, the target set, combined with comparative genomics, allows rational target prioritization. After essential genes identified by our conditional expression method, searching for orthologous genes in the human and other fungi genomes can further aid drug discovery. Those genes absence in humans but conserved in pathogenic fungus may represent broad spectrum targets. The specific genes with no human or other fungi counterpart may be suitable as narrow spectrum targets.

In addition to identifying target genes, constructing a comprehensive gene conditional expression mutant strain collection has many applications which can significantly accelerate functional annotation. The other useful applications for conditional mutant strains may include:

  • Serve as sensitized whole cell assays suitable for high throughput drug screening a wide spectrum of preferred targets regardless of whether the functional or biochemical information is available.
  • Involved in a wide range of cellular processes, including growth assay, biofilm formation and in vivo screens to identify virulent-related factors.
  • Apply to transcriptional profiling for essential drug targets.

For more detailed information, please feel free to contact us or directly sent us an inquiry.

Reference

  1. Roemer T, Jiang B, Davison J, et al. (2003). “Large-scale essential gene identification in Candida albicans and applications to antifungal drug discovery”. Molecular Microbiology 50(1): 167-181. doi:10.1046/j.1365-2958.2003.03697.x.


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