CRNA-injected oocytes

The chlorambucil-bile acid conjugate inhibited the uptake of Na" "-dependent taurochorate into both the liver and the intestinal membranes in a concentration-dependent manner [24]. Kullak-Ublick et al. [25] demonstrated that a chlorambucil-taurocholic acid conjugate was transported by the bile acid transporter in cRNA-injected oocytes. Furthermore, the liver-selectivity of HMG-CoA reductase inhibitors conjugated to bile acids have been investigated [26,27]. The Na -dependent uptake of taurocholate into rat hepatocytes and into rat ileal brush border membrane vesicles was found to be inhibited by the bile acid prodrugs of HMG-CoA reductase inhibitors in a concentration-dependent manner. Bile acid-prodrugs, which... 

High levels of protein expression are generally achieved with cRNA injection. This technique requires the in vitro synthesis of the appropriate cRNA from the template cDNA (see later). Although this approach can be time-consuming and costly, it remains the most common technique used to ensure the robust expression of receptors and ion channels in the oocyte membrane. In our laboratory, we routinely use the cRNA injection technique to promote high expression levels of LGIC receptors (GABAa receptor subtypes, nACh and 5-HT3 receptors). 

Schnizler et al., 2003) permits the automation of both cRNA injection and two-electrode voltage clamp recordings from multiple oocytes in standard 96-well plates. Although the cost and maintenance of such an apparatus is likely to be restricted to industrial research environments, other automated oocyte perfusion systems have been described that would be suitable for smaller scale laboratories (e.g., Joshi et al., 2004). 

The heterologous expression of proteins, including ligand-gated ion channels, is most commonly achieved by injection of cDNA or cRNA encoding the protein of interest into the oocyte. 

RNA transcript combinations are prepared for injection by adding a small volume (0.5-1.0 pL) of the cRNA(s) encoding each required subunit to a 1.5 mL microfuge tube. Individual subunit cRNAs are combined and pulse spun for a few seconds and kept on ice until ready for injection. Preparation of a suitable pipette for microinjection of transcripts is imperative to ensure proper injection with minimum damage to the oocyte membrane. 

The Xenopus oocyte can reliably express LGIC receptors. In our laboratory, we have seen robust expression of the Torpedo nAChR, 5-HT3 receptors and various GABAa receptor subtypes in oocytes. Injection of cRNA transcripts is a convenient and reproducible way to achieve the expression levels needed for functional analysis of receptor subtypes. We have found that functional characterization with this system complements biochemical experiments conducted on native receptors or those that have been expressed in mammahan cells. A combination of these approaches is essential for furthering our understanding of structure-function relationships in these receptors. 

Molecular-biological technologies offer an elegant way out. If there is cDNA for a and a, you let a living cell do the hybridization (e.g., Xenopus-oocytes). cRNAs from each subunit are injected into the cell. As a result, the cell synthesizes the a und a subunits and combines them to form the oligomer species. If a-cRNA and a-cRNA are translated with the same efficiency, the amounts of a-cRNA and a-cRNA control the amounts fa and fa from a and a. 

Complementary RNAs (cRNAs) are synthesized from corresponding complementary DNAs (cDNAs) coding for the receptor gene of interest such as the glutamate receptor and are individually injected into Xenopus laeves oocytes individually or in desired receptor subunit combinations of 16-20 ng per subunit using a microinjector and prepared for electrophysiological recording. 

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