The Roboocyte: Automated cDNA/mRNA Injection and Subsequent TEVC Recording on Xenopus Oocytes in 96-Well Microtiter Plates

Schnizler, Katrin; Küster, Mike; Methfessel, Christoph; Fejtl, Michael

doi:10.3109/10606820308253

Cited by 36 publications

(17 citation statements)

References 13 publications

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“…For in-cell NMR measurements in oocytes, we used a robotic injection device that ensured the highest consistency in cellular sample deposition, both in terms of localization and quantity, and facilitated the most reproducible injection routines (19). Oocytes were placed in 96-well plates and manipulated in a high-throughput fashion, within a minimum amount of time (Ͻ2 min per plate) and with no error-prone manual interference.…”

Section: Resultsmentioning

confidence: 99%

Quantitative NMR analysis of the protein G B1 domain in Xenopus laevis egg extracts and intact oocytes

Selenko¹,

Serber

Gadea

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

216

227

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We introduce a eukaryotic cellular system, the Xenopus laevis oocyte, for in-cell NMR analyses of biomolecules at high resolution and delineate the experimental reference conditions for successful implementations of in vivo NMR measurements in this cell type. This approach enables quantitative NMR experiments at defined intracellular concentrations of exogenous proteins, which is exemplified by the description of in-cell NMR properties of the protein G B1 domain (GB1). Additional experiments in Xenopus egg extracts and artificially crowded in vitro solutions suggest that for this biologically inert protein domain, intracellular viscosity and macromolecular crowding dictate its in vivo behavior. These contributions appear particularly pronounced for protein regions with high degrees of internal mobility in the pure state. We also evaluate the experimental limitations of this method and discuss potential applications toward the in situ structural characterization of eukaryotic cellular activities.in-cell ͉ high-resolution ͉ liquid-state S tructural investigations of biomolecules are typically confined to artificial and isolated in vitro experimental setups. To study proteins in their native environment, i.e., within cells, recent attempts have aimed at the development of in vivo techniques for structural biology (1). X-ray crystallography and cryo-electron microscopy are intrinsically restricted from in vivo approaches because of their requirement for crystalline or vitrified specimens. NMR spectroscopy, the only other method for structural investigations at the atomic level, allows for the direct and selective observation of NMR-active nuclei within any NMR-inactive environment and can thus be used to structurally investigate labeled proteins inside living cells (2, 3). To date, all applications of in-cell NMR spectroscopy have been conducted in bacterial cells. Highresolution in vivo NMR experiments have been reported for the structural and functional characterization of cellular proteins (4-8), protein dynamics (9), and protein-protein interactions (10). Here, we have used intact Xenopus laevis oocytes to develop the first eukaryotic cellular system for intracellular NMR analyses of biomolecules at high resolution and at close-to-physiological levels of intracellular sample concentrations.Xenopus oocytes have long served as important laboratory tools in cellular and developmental biology (11-13). Their large size renders them suitable for microinjection and permits the precise cytoplasmic deposition of defined amounts of labeled, NMR-active proteins into an otherwise unlabeled, native cellular environment. This ensures quantitative sample delivery and a higher degree of reproducibility of in vivo measurements than is generally achievable by prokaryotic in-cell NMR methods, which overexpress labeled protein and conduct measurements within the same cells. Moreover, because labeled recombinant proteins are produced in Escherichia coli and conventionally purified before injection into oocytes, any background labeli...

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Section: Resultsmentioning

confidence: 99%

Quantitative NMR analysis of the protein G B1 domain in Xenopus laevis egg extracts and intact oocytes

Selenko¹,

Serber

Gadea

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

216

227

View full text Add to dashboard Cite

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“…Voltage clamp was performed with a standard voltage-clamp amplifier (Gene-Clamp 500 amplifier; Molecular Devices, Sunnyvale, CA). For automated recording, the position of the cells and the recording head stage, the amplifier, the solution exchange, and the data acquisition were under full computer control using a software package developed by D. Bertrand (Department of Neuroscience, Centre Medical Universitaire, Geneva, Switzerland) as described by Schnizler et al (2003). All the recordings reported here were obtained at a membrane potential of Ϫ80 mV.…”

Section: Electrophysiological Assaysmentioning

confidence: 99%

“…Preparation of Xenopus oocytes, injection of receptor cDNA, and electrophysiological measurements of receptor activity were performed as described previously (Methfessel et al, 1986;Schnizler et al, 2003). Pieces of ovary were excised from anesthetized adult female Xenopus laevis.…”

Section: Electrophysiological Assaysmentioning

confidence: 99%

The Novel α7 Nicotinic Acetylcholine Receptor AgonistN-[(3R)-1-Azabicyclo[2.2.2]oct-3-yl]-7-[2-(methoxy)phenyl]-1-benzofuran-2-carboxamide Improves Working and Recognition Memory in Rodents

Boess¹,

Vry²,

Erb³

et al. 2007

J Pharmacol Exp Ther

Self Cite

136

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The relative contribution of ␣4␤2, ␣7 and other nicotinic acetylcholine receptor (nAChR) subtypes to the memory enhancing versus the addictive effects of nicotine is the subject of ongoing debate. In the present study, we characterized the pharmacological and behavioral properties of the ␣7 nAChR agonist. ABBF bound to ␣7 nAChR in rat brain membranes (K i ϭ 62 nM) and to recombinant human 5-hydroxytryptamine (5-HT) 3 receptors (K i ϭ 60 nM). ABBF was a potent agonist at the recombinant rat and human ␣7 nAChR expressed in Xenopus oocytes, but it did not show agonist activity at other nAChR subtypes. ABBF acted as an antagonist of the 5-HT 3 receptor and ␣3␤4, ␣4␤2, and muscle nAChRs (at higher concentrations). ABBF improved social recognition memory in rats (0.3-1 mg/kg p.o.). This improvement was blocked by intracerebroventricular administration of the ␣7 nAChR antagonist methyllycaconitine at 10 g, indicating that it is mediated by ␣7 nAChR agonism. In addition, ABBF improved working memory of aged rats in a water maze repeated acquisition paradigm (1 mg/kg p.o.) and object recognition memory in mice (0.3-1 mg/kg p.o.). Rats trained to discriminate nicotine (0.4 mg/kg s.c.) from vehicle did not generalize to ABBF (0.3-30 mg/kg p.o.), suggesting that the nicotine cue is not mediated by the ␣7 nAChR and that selective ␣7 nAChR agonists may not share the abuse liability of nicotine. Our results support the hypothesis that ␣7 nAChR agonists may provide a novel therapeutic strategy for the treatment of cognitive deficits with low abuse potential.Nicotine enhances cognitive functions, such as attention, learning, consolidation, and retention, in both animals and humans, through activation of brain nicotinic acetylcholine receptors (nAChRs) (Levin et al., 1999(Levin et al., , 2006. These ligandgated ion channels are homopentamers formed by five identical subunits (␣7 nAChR) or heteropentamers of multiple ␣ and ␤ subunits. Various isoforms of these subunits have been identified (␣2-␣10; ␤2-␤4; for reviews, see Paterson and Nordberg, 2000;Gotti et al., 2006). The most common nAChRs found in the brain are the ␣7 subtype with a low affinity for nicotine and the ␣4␤2 subtype with a high affinity for nicotine. Evidence from neuroanatomical, electrophysiological, and behavioral studies support a role for both of these receptor subtypes in processes of learning and memory.

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“…Electrophysiological characterization of Slc16a6a was performed at Ecocyte Biosciences using a Robocyte automated two-elec-trode voltage clamp recorder (Schnitzler et al 2003). Xenopus oocytes were harvested and injected with either empty pGEMT vector or pGEMT containing slc16a6a cDNA preceded by a Kozak translation initiation sequence (59-GCCACCACC-39).…”

Section: Electrophysiologymentioning

confidence: 99%

A monocarboxylate transporter required for hepatocyte secretion of ketone bodies during fasting

Hugo¹,

Cruz-García²,

Karanth³

et al. 2012

Genes Dev.

121

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To find new genes that influence liver lipid mass, we performed a genetic screen for zebrafish mutants with hepatic steatosis, a pathological accumulation of fat. The red moon (rmn) mutant develops hepatic steatosis as maternally deposited yolk is depleted. Conversely, hepatic steatosis is suppressed in rmn mutants by adequate nutrition. Adult rmn mutants show increased liver neutral lipids and induction of hepatic lipid biosynthetic genes when fasted. Positional cloning of the rmn locus reveals a loss-of-function mutation in slc16a6a (solute carrier family 16a, member 6a), a gene that we show encodes a transporter of the major ketone body b-hydroxybutyrate. Restoring wild-type zebrafish slc16a6a expression or introducing human SLC16A6 in rmn mutant livers rescues the mutant phenotype. Radiotracer analysis confirms that loss of Slc16a6a function causes diversion of livertrapped ketogenic precursors into triacylglycerol. Underscoring the importance of Slc16a6a to normal fasting physiology, previously fed rmn mutants are more sensitive to death by starvation than are wild-type larvae. Our unbiased, forward genetic approach has found a heretofore unrecognized critical step in fasting energy metabolism: hepatic ketone body transport. Since b-hydroxybutyrate is both a major fuel and a signaling molecule in fasting, the discovery of this transporter provides a new direction for modulating circulating levels of ketone bodies in metabolic diseases.

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The Roboocyte: Automated cDNA/mRNA Injection and Subsequent TEVC Recording on Xenopus Oocytes in 96-Well Microtiter Plates

Cited by 36 publications

References 13 publications

Quantitative NMR analysis of the protein G B1 domain in Xenopus laevis egg extracts and intact oocytes

Quantitative NMR analysis of the protein G B1 domain in Xenopus laevis egg extracts and intact oocytes

The Novel α7 Nicotinic Acetylcholine Receptor AgonistN-[(3R)-1-Azabicyclo[2.2.2]oct-3-yl]-7-[2-(methoxy)phenyl]-1-benzofuran-2-carboxamide Improves Working and Recognition Memory in Rodents

A monocarboxylate transporter required for hepatocyte secretion of ketone bodies during fasting

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