Xenopus oocytes protocol

In vitro transcription from cloned cDNA sequences is required to produce mRNA that can be injected into Xenopus oocytes for expression of the encoded protein and is achieved by standard procedures. While oocyte isolation and injection generally follows the protocols provided below, in vitro transcription into mRNA may be modified in accordance to the instructions provided with... 

A variety of extract preparations have been used to support protein and snRNP import in vitro (Adam et ai, 1991 Moore and Blobel, 1992 Newmeyer and Wilson, 1991 Marshallsay and Luhrmann, 1994 Palacios et al, 1996). The most commonly used sources are Xenopus oocytes and eggs and rabbit reticulocyte lysate. The biochemical fractionation of these extracts led to the identification of the essential soluble protein factors for nuclear protein import (see Section I). Detailed protocols for the surgical removal of ovary from Xenopus and the induction of egg laying have been provided in a previous volume in this series (Kay and Peng, 1991). Here we present specific modifications of the basic protocols to give optimal nuclear protein and snRNP import in vitro. 

As with other in vitro systems, different mRNAs exhibit differing translational efficiencies in the Xenopus extract. Where comparisons have been possible, relative translational yields in the extract reflect those seen on microinjection into Xenopus oocytes. Natural mRNAs normally translate best, while the performance of most synthetic mRNAs is much enhanced when transcribed from the vector pSP64T (Krieg and Melton, 1984), where the cDNA is flanked by sequences from the Xenopus P-globin cDNA. A reliable protocol for the synthesis of capped transcripts in vitro is given in Matthews and Colman (1991). 

In the following, a short overview of the expression cloning method with Xenopus laevis oocytes is given. Protocols for standard procedures are provided in the Appendix, while those methods that are subject to frequent modification are described in general and the reader is referred to the corresponding handbooks for further details. 

As mature Xenopus laevis females carry 30,000 oocytes and a typical experiment consumes not more than 1,000 oocytes, it is not necessary to kill a frog for each experiment. Instead, pieces of the ovary are removed by laparotomy and the incision is sutured. The same animal can serve as a donor of fresh oocytes after a convalescence of 2-3 weeks, and 10-15 oocyte preparations can be made from each frog without affecting its health. Hence, not more than 20-30 animals, in most cases 10-15, are maintained at the same time. For details see also Protocol 1 in the Appendix. 

Numerous protocols describing the care and maintenance of Xenopus laevis frogs and the isolation of oocytes from their ovaries have been published (Cohnan, 1984 Goldin, 1992 Quick and Lester, 1994 Stiihmer and Parekh, 1995 Theodoulou and Miller, 1995). The following methods are based primarily on the procedures described by Yao and colleagues (2000). 

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