Expression Cloning with Xenopus Oocytes

Abstract

Expression cloning using Xenopus laevis oocytes has been proven to be an excellent tool for the structural/functional identification of proteins of all origins. Due to their great availability, large size and relative ease of handling, X. laevis oocytes are optimal tools for the expression and cloning of proteins, when compared to traditional expression systems, such as Escherichia coli or eukaryotic cell lines. The Xenopus oocyte system, pioneered in 1971 (Gurdon et al. 1971), is able to efficiently transcribe and translate injected genetic information; perform assembly of the foreign protein products; correctly process the nascent polypeptides; and target them to the proper subcellular compartment. Some advantages of the Xenopus oocyte expression system over other functional expression systems (e.g. in somatic cells) are: easy and rapid transfer of genetic information by microinjection; simple handling of single cells after transfer of genetic information; high proportion of cells expressing transferred genetic information and good control of the oocyte environment. In this section, we will describe a general strategy which can be successfully used for expression cloning of proteins using Xenopus oocytes. The manipulation of oocytes, protein assay (in our case transport measurements), cDNA library screening and cloning by expression, will be covered in detail below.