Exogenous protein expression in Xenopus oocytes: basic procedures.
The oocytes of the South African clawed frog Xenopus laevis have been widely used as a reliable system for the expression and characterization of different types of proteins, including ion channels and membrane receptors. The large size and resilience of these oocytes make them easy to handle and to microinject with different molecules such as natural mRNAs, cRNAs, and antibodies. A variety of methods can then be used to monitor the expression of the proteins encoded by the microinjected mRNA/cRNA, and to perform a functional characterization of the heterologous polypeptides. In this chapter, after describing the equipment required to maintain X. laevis in the laboratory and to set up a microinjection system, we provide detailed procedures for oocyte isolation, micropipet and cRNA preparation, and oocyte microinjection. A method for the labeling of oocyte-synthesized proteins and for the immunological detection of the heterologous polypeptides is also described.
Bossi
Elena
E
Laboratory of Cellular and Molecular Physiology, Department of Structural and Functional Biology, University of Insubria, Varese, Italy.
Fabbrini
Maria Serena
MS
Ceriotti
Aldo
A
D000818:Animals; D008845:Microinjections; D008967:Molecular Biology / Q000379:methods*; D009865:Oocytes / Q000378:metabolism*; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D014982:Xenopus laevis
D011506:Proteins; D012333:RNA, Messenger