Expression Cloning Strategies for Glycosylphosphatidylinositol-Anchor Biosynthesis Enzymes and Regulators

This chapter discusses the expression cloning strategies for glycosylphosphatidylinositol (GPI)-anchor biosynthesis enzymes and regulators. The structure and biosynthetic pathway of the GPI anchor suggest the involvement of about 10 genes in its biosynthesis. Some genes are represented by mammalian mutant cell lines that are deficient in GPI-anchor biosynthesis. Cloning the genes is important for understanding molecular mechanisms of the biosynthesis and its regulation. It is also important for understanding the pathogenesis of paroxysmal nocturnal hemoglobinuria, a GPI-anchor deficiency. Both stable and transient expression cloning methods that rely on phenotypic complementation of mutant cells are useful to clone genes for GPI-anchor biosynthesis. A similar transient expression cloning method with cotransfection is also used to clone a gene for complementation class H. The approach is applicable for both rodent and human mutant cells if cotransfection to those cells is efficient enough to produce a sufficient number of transfectants that represent the entire library. If the transfection efficiency is too low for cotransfection, a classic approach in which large T-expressing mutant cells are first established and used as recipients for a cDNA library are used. It is used to clone a gene for complementation class B.