In addition to developing methods for in vivo and in vitro micronucleus detection (i.e., MicroFlow), Litron has made great progress in the development of a novel endogenous mutation assay.  This new assay is based on the detection of mutation in the Pig-a gene locus.

Several characteristics of the Pig-a gene make it a favorable candidate for a mutation screening assay:

• The product of this gene has a critical role in the production of glycosylphophatidylinositol (GPI) anchors, a structure used to target specific proteins to the cell surface. Disruption of this gene eliminates the expression of these proteins on the cell surface, and this phenotype can be readily identified by flow cytometric analysis. 
  
  
• The Pig-a gene is located on the X-chromosome and thus has only one functional copy in both males and females. Therefore, as opposed to autosomal genes with two alleles, only a single mutation event or “hit” is required to affect the Pig-a gene’s function and produce the characteristic phenotype.

Even with the favorable characteristics described above, a Pig-a mutation remains a rare event. For this reason, many cells need to be interrogated per analysis. The combination of flow cytometry with peripheral blood analysis is ideal for this purpose, since hundreds of thousands of cells may be scored in a short time.

Specifically, this methodology examines red blood cells (RBCs) for aberrant GPI anchor expression. Additionally, by focusing on the youngest RBCs, i.e., reticulocytes, this method has been shown to detect alterations in GPI anchor-mediated surface protein expression in as little as one week following in vivo exposure to chemical mutagens.

Conclusions

There is considerable potential for this assay in pharmaceutical development and chemical safety assessment, among other areas, where a simple in vivo mutation assay could provide valuable toxicological data. The ability of this assay to integrate with standard toxicity studies means more efficient use of resources for required in vivo testing.

Since the Pig-a gene is conserved across mammalian species of toxicological interest, this assay offers the potential to bridge the gap between preclinical studies and human clinical trials. Litron is continuing to develop this promising assay in an effort to improve the ability of researchers and regulatory agencies to provide the public with safe and effective products.

Micronucleus Scoring           |           MicroFlow Kits           |          R & D           |           About Us          |           Home