Creation of Hypomorphic Alleles for Functional Gene Studies

Manipulation of gene expression and activity is a standard approach to evaluate gene function in living organisms. However, current genetic tools for manipulation of gene expression are rather tuned towards complete loss of gene function; they are organism specific, show unpredictable alterations in gene activity (dependent on tissue or cell types within the organism) and change spatial-temporal regulation of gene expression. Hypomorphic mutations, partial loss of gene function, are a valuable tool for both genetic analysis of gene function and for synthetic biology applications. I will present a simple and predictable method to generate hypomorphic mutations in model organisms that targets specific step of translation cycle - translation elongation. This method is dependent on addition of polyA tracks, runs of consecutive adenosine nucleotides, to the gene coding sequence of interest. The consequence of this addition is decrease in translation elongation efficiency, and in all tested cell cultures and model organisms, this decreases mRNA stability and protein expression. As such this method can be used in industry for control of biosynthetic pathways in production of useful secondary metabolites, antibiotics or recombinant antibodies; in synthetic biology for introduction of controllable retrosynthetic and fully engineered pathways; as well as in basic research for ultimate control of gene regulatory pathways in the modelling of diseases.