Chapter 20
Expression Cloning with Xenopus Oocytes
DANIEL MARKOVICH, ANDREAS WERNER AND HEINI MURER
lntroduction
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 celllines. 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.
Correspondence to Daniel Markovich, U niversity of Queensland, Department ofPhysiology and Pharmacology, Brisbane, 4072, Australia (phone +61-7-3365-1400; fax +61-73365-1766; e-mail danielm@plpk.uq.edu.au), Andreas Werner, Max-Planck-Institut für
molekulare Physiologie, Rheinlanddamm 201, Dortmund, 44139, Germany, Heini
Murer, Universität Zürich, Physiologisches Institut, Winterthurerstrasse 190, Zürich,
8057, Switzerland
Expression Cloning with Xenopus Oocytes
DANIEL MARKOVICH, ANDREAS WERNER AND HEINI MURER
lntroduction
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 celllines. 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.
Correspondence to Daniel Markovich, U niversity of Queensland, Department ofPhysiology and Pharmacology, Brisbane, 4072, Australia (phone +61-7-3365-1400; fax +61-73365-1766; e-mail danielm@plpk.uq.edu.au), Andreas Werner, Max-Planck-Institut für
molekulare Physiologie, Rheinlanddamm 201, Dortmund, 44139, Germany, Heini
Murer, Universität Zürich, Physiologisches Institut, Winterthurerstrasse 190, Zürich,
8057, Switzerland