Enhancer Trapping in Mosquitoes

Lewis Hun, Ph.D. is a postdoctoral research at the University of California, Riverside and is interested in new strategies for controlling mosquitoes and the diseases they transmit

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A recent manuscript published on Genes Genomes Genetics by Reid et al (2018) reports an exciting improvement to piggyBac-based promoter-trap designed in Anopheles stephensi. The development of this new piggyBac-based promoter-trap designs provides further knowledge and exciting venues to examine tissue-specific gene expression with appropriate promoters.

Briefly, the piggyBac promoter-trap element was inserted into the TTAA piggyBac insertion site. The promoter-trap reporters in these vectors consisted of the open reading frame of the fluorescent protein gene AmCyan. The splicing of the promoter-trap element and formation of the tetramer AmCyan protein produce a functional AmCyan reporting fluorescent protein. However, longer protein tags from the endogenous transcript may interfere with the quaternary functional structure of AmCyan.  To solve this problem, the internal ribosomal entry site  (IRES) element was inserted to the splicing promoter trap element, and this allowed the AmCyan protein to express independently of the endogenous peptide tag, which may enhance potential interference of a peptide tag on the quaternary structure formation of AmCyan.

Mechanism of enhancer trapping and gene trapping. a , b Enhancer trap ( ET ) vectors encode a basal promoter ( P b ) upstream of a reporter gene such as fluorescent marker gfp or transcription activator 
Conceptual basis for conducting enhancer trapping. Promoter trapping relies on a similar strategy but reporter genes are configured to only be expressed when inserted into an actively transcribed gene.

In the context of controlling disease transmission, vector control remains the frontline approach. Therefore, it is critical to understand the basic biology of the mosquito vector. This novel piggyBac-based promoter-trap technology provides a new tool to precisely target specific genes in a particular tissue, which will in turn result into a better understanding of how each gene functions. More importantly, this technology provides a unique tool to study specific stages of the mosquito development.  This is valuable information that can be used to target mosquito development such as creating more effective larvicide. Although this technology represents a step forward for the mosquito research community, we must further determine whether this technology may pose any potential fitness disadvantage after integrated into the mosquito genome.

This study also showed that piggyBac is highly active in the genome of An. stephensi, hence it can be used to detect genes using reporter gene expression-based technologies. Besides, it demonstrated that piggyBac-based ‘gene-traps’ are based on reporter gene expression only from an intra-genic insertion of piggyBac. This technology can be used to easily screen for genes expressed in various tissues, at the same time providing the tools to analyze temporal and spatial patterns of gene expression. The insertion mutation resulting from piggyBac integration will be useful for determining the role of the gene in different tissue functions. 

Mechanism of piggyBac transposition. Image credit Skipper et al. Journal of Biomedical Science 2013 20:92 doi:10.1186/1423-0127-20-92

This study also showed that piggyBac is highly active in the genome of An. stephensi, hence it can be used to detect genes using reporter gene expression-based technologies. Besides, it demonstrated that piggyBac-based ‘gene-traps’ are based on reporter gene expression only from an intra-genic insertion of piggyBac. This technology can be used to easily screen for genes expressed in various tissues, at the same time providing the tools to analyze temporal and spatial patterns of gene expression. The insertion mutation resulting from piggyBac integration will be useful for determining the role of the gene in different tissue functions. 

Overall, the contribution of this work to the piggyBac-based promoter-trap technology is substantial and will open new paths to understand the basic biology of this important vector.

Reid W, Pilitt K, Alford R, Cervantes-Medina A, Yu H, Aluvihare C, Harrell R, O’Brochta DA (2018) .An Anopheles stephensi Promoter-Trap: Augmenting Genome Annotation and Functional Genomics. G3 (Bethesda). 2018 Oct 3;8(10):3119-3130. doi: 10.1534/g3.118.200347

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