Xenopus oocyte expression system

The molecular analysis of the Sh K channel transcription unit indicated that at least ten different channel subunits may be expressed in Drosophila cells. This observation raised a number of important questions only some of them can be addressed here. Each subunit has been expressed in the Xenopus oocyte expression system [11,12,29,39]. These experiments indicate that each subunit expresses distinct... 

Heterologous expression systems hERG current (whole cell patch clamp) Mammalian cell expression (CHO, HEK-293, mouse L-ceUs, COS-7) and Xenopus oocytes expression system Witchel et al. 77 Zou et al. 78 Cavero et al. 79 Martin et al. 80 McDonald et al.81... 

Pros and Cons of the Xenopus Oocyte Expression System.326... 

One limitation of the Xenopus oocyte expression system is the presence of endogenous channels. Although these are few in number, they can interfere with the analysis and interpretation of small currents mediated by exogenously expressed ion channels. Such interference can be identified by carrying out control studies of un-injected or water-injected oocytes in parallel with experiments on oocytes expressing the receptor of interest. 

Lovinger DM, Merritt A, Reyes D (1994) Involvement of N- and non-N-type calcium channels in synaptic transmission at corticostriatal synapses. Neurosci 62(1) 31 10 McAllister SD, Griffin G, Satin LS, Abood ME (1999) Cannabinoid receptors can activate and inhibit G protein-coupled inwardly rectifying potassium channels in a xenopus oocyte expression system. J Pharmacol Exp Ther 291(2) 618-26... 

Pillai G, Brown NA, McAllister G, Milligan G, Seabrook GR (1998) Human D2 and D4 dopamine receptors couple through betagamma G-protein subunits to inwardly rectifying K+ channels (GIRK1) in a Xenopus oocyte expression system selective antagonism by L-741,626 and L-745,870 respectively. Neuropharmacology 57 983-987. 

The Xenopus oocyte expression system can be used for characterization of products of specific mRNAs transcribed in vitro from cDNA isolates. Expression cloning of novel cDNAs, whose function can be assayed following expression, can also be performed using oocytes. In the latter case, as shown in Eig. 4.3, total mRNA is fractionated by size, and expression in oocytes is used to identify mRNA fractions capable of producing the protein of interest. The enriched mRNA fractions are used for cDNA preparation. The cDNA of interest is identified by its ability to select (by hybridization) its mRNA from the total mRNA pool. The selected mRNA is assayed again in oocytes, as shown in Fig. 4.3. Repeated rounds of this procedure with further enrichment of the desired cDNA from the pool leads to the isolation of the cDNA. Alternatively, in vitro transcription of the cDNA pool can also be used to generate mRNA for injection and assay. Component cDNAs of the pool that... 

Although the mechanisms that regulate CBi receptor synthesis, posttransla-tional modification, degradation, and internalization remain largely unknown, studies are beginning to address this area. In hippocampal neuronal cultures and in a Xenopus oocyte expression system, CBi receptor desensitization has been shown to require G protein-coupled receptor kinase (GRK) and -arrestin (Jin et al. 1999 Kouznetsova et al. 2002). Internalization of CBi receptors following agonist treatment in CBi receptor-transfected cells (Hsieh et al. 1999) and hippocampal cultures (Coutts et al. 2001) has been shown to lead to internalization of CBl receptors. 

The Xenopus oocyte expression system can be used to study macroscopic junctional conductances, but not to make single-channel measurements. Dual voltage clamp of coupled cells under conditions of reduced junctional conductance can reveal single-channel transitions (41, 46), but the problems of accessibility and series hemichannels remain. 

Rdl orthologs have since been cloned from many other insect species and, when heterologously expressed in the Xenopus oocyte expression system or in cell lines, give membrane receptors that behave like insect GABA receptors. Rdl... 

DmGluCla, but not Rdl, exists in the Drosophila nervous system and binds aver-mectin but not nodulisporic acid, both of which are allosteric activators of GluCls. Another population, assembled from both DmGluCla and Rdl subunits and binding both avermectin and nodulisporic acid, also exists in the Drosophila nervous system, but coexpression of DmGluCla and Rdl in the Xenopus oocyte expression system does not yield functional heteromultimers, indicating that other subunits may be needed [54]. 

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