Expression Plasmids

The cDNA encoding for the full-length wild-type T7 RNAP has been previously cloned in the pMR-78 vector (29) and is presented in Fig. 1. The expression of the insert is under the control of the Ptac promoter, which is inducible by IPTG. This vector has a specific bla gene that confers ampicillin resistance for colony selection and growth. It is designed to express an N-terminal his-tagged T7 RNAP for purification with a Ni2+ iminodiacetic acid affinity column. The cDNAs used should contain a T7...

Imaging Techniques

We describe the basic principles of epifluorescence microscopy, video rate and linescan confocal laser scanning microscopy (CLSM), and TIRFM with respect to imaging intracellular Ca2+ signals in Xenopus oocytes. The most commonly used Ca2+ indicators are comprised of a chromophore conjugated to a Ca2+ chelator (BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N' N'-tetracetic acid))) backbone (7,8). Ratiometric Ca2+ indicators demonstrate a spectral shift in either their excitation (e.g., Fura-2) or...

Oocyte Nuclear Injection

Steps for preparing Xenopus oocytes for microinjection. For detail, see Subheading 3.2. Fig. 3. Steps for preparing Xenopus oocytes for microinjection. For detail, see Subheading 3.2. 7. Rinse three times in LSB for 5 min each. If necessary, oocytes can be stored overnight at 4 C, and the protocol can be continued the next day. 8. Sequentially dehydrate the agar-embedded oocytes in an ascending series of ethanol and embed in Epon (Fluka) as follows dehydrate samples in 50, 70, and 90...

Microtransplantation of Neurotransmitter Receptors From Cells to Xenopus Oocyte Membranes

New Procedure for Ion Channel Studies Ricardo Miledi, Eleonora Palma, and Fabrizio Eusebi The Xenopus oocyte is largely used as a cell expression system for studying both structure and function of transmitter receptors and ion channels. Messenger RNA extracted from the brain and injected into oocytes leads to the synthesis and membrane incorporation of many types of functional ion channels. A new method was developed further to transplant neurotransmitter receptors from human brain or cultured...

Electrophysiology

Two-Electrode Voltage Clamp 3.4.1.1. Wiring the Rig 1. The basic wiring of the essential components of an oocyte rig can be very simple. The leads to the microelectrodes are connected to their respective connections in the amplifier. 2. The analog output (referred to as the scaled output in the Geneclamp 500 and as Vout in the Warner OC50) is connected to the analog in port of an A D converter. 3. The voltage command port of the amplifier must also be connected to the analog output of...

Oocyte Expression With Injection of Purified T7 RNA Polymerase

Xavier Altafaj, Nathalie Joux, Michel Ronjat, and Michel De Waard The Xenopus oocyte is a widely used system for protein expression. Investigators have had the choice between two different techniques injection into the cytoplasm of in vitro transcribed complementary RNA (cRNA) or injection into the nucleus of complementary DNA (cDNA). We report on a third expression technique that is based on the combined injection of cDNA and purified T7 RNA polymerase directly into the cytoplasm of oocytes....

Chromatin Immunoprecipitation for Studying Transcriptional Regulation in Xenopus Oocytes and Tadpoles

David Stewart, Akihiro Tomita, Yun-Bo Shi, and Jiemin Wong Understanding the accurate temporal and spatial regulation of gene expression during development requires knowledge of the spectrum of transcription factors and cofactors involved and their functional interplay with chromatin. Chromatin immunoprecipitation (ChIP) has become a powerful technique that allows us to do so. A typical ChIP assay involves (1) treating cells or tissues with formaldehyde to rapidly crosslink chromatin-associated...

Info

TR recruits corepressors SMRT and N-CoR for repression in the absence of T3 and coactivators SRC-1 and p300 for activation in the presence of T3. (A) A simple illustration of the structure of the 4XUAS-pTRPA reporter. Groups of oocytes were injected with or without Gal4-TR Gal4(DBD)-TR(LBD) (200 ng L, 18.4 nL oocyte) and the single-stranded 4XUAS-pTRPA reporter (100 ng L, 18.4 nL oocyte). The oocytes were incubated at 18 C overnight with or without T3 (50 nM) and processed for...

Using a Xenopus Oocyte Protein Refolding Assay

Heikkila, Angelo Kaldis, and Rashid Abdulle Heat shock proteins (Hsps) are molecular chaperones that aid in the folding and translocation of protein under normal conditions and protect cellular proteins during stressful situations. A family of Hsps, the small Hsps, can maintain denatured target proteins in a folding-competent state such that they can be refolded and regain biological activity in the presence of other molecular chaperones. Previous assays have employed cellular lysates...

Oocyte Preparation Maintenance and Injection

Sonication Centrifugation Supernatant

Stage V and VI oocytes are prepared according to the procedure described by De Waard and Campbell (33), which is further described next 1. Maintain mature X. laevis female frogs under a cycle of 12 h light 12 h dark in water tanks at 16 C. 2. Choose a healthy frog, place it into a small and secure container, add water for partial body immersion, and add 0.03 (w v) of 3-aminobenzoic acid ethyl ester to the water to anesthetize the animal. Partial body immersion is important to avoid drowning of...

Assay of Apoptosis Using Fractionated Interphase Egg Extracts

Reconstitution Extract Cytosolic and Heavy Membrane Fractions 1. Add ATP-regenerating mix (1 10 dilution of 0.2 Mphosphocreatine and 1 100 of 0.5 mg mL creatine kinase and 0.2 M ATP ) to 170 L of cytosolic extract. 2. Add 14 L freshly prepared heavy membrane fraction enriched in mitochondria (Subheading 3.3.2.). 3. Incubate at room temperature. Withdraw samples at 30-min intervals to assess cytochrome c release and caspase activity (Subheadings 3.2.2.-3.2.3. see Note 10). 3.4.2....

John M Walker Series Editor

323 Arabidopsis Protocols, Second Edition, edited by Julio Salinas and Jose J. Sanchez-Serrano, 2006 322 Xenopus Protocols Cell Biology and Signal Transduction, edited by X. John Liu, 2006 321 Microfluidic Techniques Reviews and Protocols, edited by Shelley D. Minteer, 2006 320 Cytochrome P450 Protocols, Second Edition, edited by Ian R. Phillips and Elizabeth A. Shephard, 2006 319 Cell Imaging Techniques, Methods and Protocols, edited by Douglas J. Taatjes and Brooke T. Mossman, 2006 318 Plant...

Oocyte Isolation and Enucleation X Shawn Liu and X Johne

Xenopus laevis oocytes are popular cells in experimental biology. Fully grown oocytes are large ( 1.3-mm diameter) with an enormous nucleus ( 300- im diameter). Oocytes are generally isolated by either manual dissection (manual defolliculation) or enzymatic (mainly with collagenase preparations) digestion of the extracellular connective tissues. In this chapter, we describe both procedures, which are routinely used in our laboratory. However, manual defolliculation does not actually remove the...

Introduction to Nucleocytoplasmic Transport

Molecules and Mechanisms Reiner Peters Nucleocytoplasmic transport, the exchange of matter between nucleus and cytoplasm, plays a fundamental role in human and other eukaryotic cells, affecting almost every aspect of health and disease. The only gate for the transport of small and large molecules as well as supramolecular complexes between nucleus and cytoplasm is the nuclear pore complex (NPC). The NPC is not a normal membrane transport protein (transporter). Composed of 500 to 1000 peptide...

Cytoplasmic mRNA Polyadenylation and Translation Assays Maria Piqu Jos Manuel Lpez and Ral Mndez

Vertebrate development is directed by maternally inherited messenger RNAs that are synthesized during the very long period of oogenesis. These dormant mRNAs usually contain short poly(A) tails and are stored as mRNA ribonucleoproteins that preclude riboso-mal recruitment. In Xenopus laevis oocytes treated with the meiosis-inducing hormone progesterone, their poly(A) tails are elongated, and the mRNAs are mobilized into polysomes. This cytoplasmic polyadenylation is directed by cis-acting...

Materials

Unless stated otherwise, all reagents may be purchased from Sigma Chemicals (St. Louis, MO www.sigmaaldrich.com). 1. Frogs X. tropicalis may be purchased from any of a number of commercial suppliers, including Xenopus One (Dexter, MI www.xenopusone.com), NASCO (Fort Atkinson, WI www.nascofa.com), or Xenopus Express (Plant City, FL www.xenopus.com). A number of Web resources describe husbandry of these animals in great detail (see www.xenbase.org for details and links). 2. Masui's oocyte medium...

Xenopus Egg Extracts

In vitro-assembled nuclei can be reconstituted using cytoplasmic extracts of the eggs of female X. laevis, combined with chromatin from the demembranated sperm of male X. laevis. The reconstituted nuclei are capable of nuclear import as well as a complete round of DNA replication. Female frogs are induced to lay unfertilized eggs (Subheading 3.1.1.). These eggs are then fractionated to obtain cytosol and membrane fractions (Subheading 3.1.2.). The cytosol fraction is a concentrated solution of...

Ubiquitin Mediated Protein Degradation in Xenopus Egg Extracts

Anna Castro, Suzanne Vigneron, Cyril Bernis, Jean-Claude Labbe, and Thierry Lorca Events controlling cell division are governed by the degradation of different regulatory proteins by the ubiquitin-dependent pathway. In this pathway, the attachment of a polyubiquitin chain to a substrate by an ubiquitin-ligase targets this substrate for degradation. Xenopus egg extracts present many advantages for the study of the cell cycle, including the availability of a large quantity of material...

Notes

The protease inhibitor PMSF is dissolved in ethanol and stored at 4 C. Trace amounts are added to LSB immediately before use. Fig. 9. Visualization of the reversible calcium-mediated opening and closing of the nuclear baskets by time-lapse AFM. (A,B) The same NPCs with closed baskets (i.e., -Ca2+) (A) could be opened after addition of 0.1 mM Ca2+ (B). The opposite is true in C and D the same NPCs with open baskets (i.e., +0.1 mM Ca2+) (C) could be closed after addition of 1 mM EGTA (i.e.,...

Assessment of Apoptosis in Fractionated Extract

Subcutaneous Lymph Sac Frog

Cytosolic and Mitochondria Heavy Membrane Fractions 2. Mitochondria heavy membrane fraction. 2.5.2. Assessment of Cytochrome c-Induced Apoptosis 5. Purified cytochrome c (from equine heart Sigma) make stock solution in water at 1 mg mL and store at -80 C for up to 1 mo. 2.6. Immunodepletion, Addition of Recombinant Proteins, and Antibody Neutralization 2. Preimmune serum or immunoglobulin G (IgG). 4. Phosphate-buffered saline PBS 8 g NaCl, 0.2 g KCl, 0.1 g CaCl2, 0.1 g MgCl2-6 H2O, 1.15 g...

Biosensor Detection of Cytoplasmic Analytes

Preparation of Detector Cell Cells to be used as IP3 detectors are prepared as follows (5-8,17) 1. Plate BHK21 cells 24 to 48 h prior to experiments in an open cell chamber made by attaching a silicon O ring to a no. 1 glass cover slip. 2. Coat the cover slip with collagen to improve cell adherence to the glass by pipeting 200 L collagen solution onto the cover slip followed by air drying. If desired, the coated cover slip can be sterilized by a 70 ethanol wash. 3. Maintain the cells in...

B

Emitted from a mercury or xenon lamp source. The wavelength is established by placing an excitation filter in the light path. An image of the light source, depicted here as the filament in a lightbulb (Fig. 1A), is placed at the back aperture of the objective by a lens. From this focal plane, diverging light emitted from each point on the filament is collected by the objective lens and projected through the imaging plane as parallel rays. A significant advantage of the conventional optical...

Steroidogenesis

Sex steroids appear to be secreted primarily by the ovaries of female frogs (30,31). During breeding season, serum estradiol levels increase, which stimulates vitellogenin production by the liver. The direct effects of estradiol on amphibian ovarian follicle development are still not well understood. Sex steroid production reaches a maximum during ovulation, when gonadotropins secreted from the pituitary stimulate both estrogen and androgen production however, the exact amounts of the various...

The Physiology of the Xenopus laevis Ovary Melissa A Rasar and Stephen R Hammes

Xenopus laevis has been used for many decades to study oocyte development and maturation. The Xenopus oocytes' large size, relative abundance, and clearly defined progression of physical characteristics from oogonia to eggs make them ideal for studying oogenesis. In addition, the ability of steroids to trigger Xenopus oocyte maturation in vitro has resulted in their extensive use for the study of the complexities of meiosis. Interestingly, steroid-induced maturation of Xenopus oocytes occurs...

Studying Fertilization in Cell Free Extracts

Focusing on Membrane Lipid Raft Functions and Proteomics Ken-ichi Sato, Ken-ichi Yoshino, Alexander A. Tokmakov, Tetsushi Iwasaki, Kazuyoshi Yonezawa, and Yasuo Fukami Xenopus oocytes, eggs, and embryos serve as an ideal model system to study several aspects of animal development (e.g., gametogenesis, fertilization, embryogenesis, and organogenesis). In particular, the Xenopus system has been extensively employed not only as a living cell system but also as a cell-free or reconstitutional...

Use of Xenopus laevis Oocyte Nuclei and Nuclear Envelopes in Nucleocytoplasmic Transport Studies

In this chapter, two techniques for the analysis of transport through the nuclear pore complex are described. In the first technique, nuclei isolated manually from Xenopus laevis oocytes are used to measure the import kinetics of fluorescent substrates by confocal fluorescence microscopy. In the second technique, referred to as optical single transporter recording (OSTR), isolated Xenopus oocyte nuclei, perforated nuclei, or isolated nuclear envelopes are tightly bound to planar transparent...

Contributors

Rashid Abdulle Department of Biology, University of Waterloo, Waterloo, Ontario, Canada Ueli Aebi M. E. M ller Institute for Structural Biology, Biozentrum, University of Basel, Basel, Switzerland Nancy L. Allbritton Department of Physiology and Biophysics, University of California Irvine, Irvine, CA Genevi ve Almouzni Research Section, Institut Curie, UMR218 du Centre National de la Recherche Scientifique (CNRS), Paris, France Xavier Altafaj Canaux Calciques, Fonctions et Pathologies, CEA, D...

Defolliculation of Xenopus tropicalis Oocytes

Ovarian oocytes are wrapped in several layers of follicle cells and theca, which protects them and provides them with nourishment during oogenesis. As with X. laevis, these layers may be removed by manually using forceps or enzymatically using a protease solution (24). However, the smaller size of X. tropicalis oocytes makes manual defolliculation a challenge (although not insurmountable) even to an accomplished X. laevis defolliculator. In either case, first tease the ovarian fragments apart...

Mm

Fig. 1. (A) Simultaneous brightfield fluorescence images of an excised oocyte patch. The solid white overlay in brightfield is our interpretation of the location of the fluorescent excised patch the dotted white line is the pipet (our interpretation) in brightfield. In the boxed area, the pipet orifice presumably veered out of the confocal plane. Membrane was stained with a cys-teine-specific rhodamine reagent. Modified from ref. 45. (B) A very large oocyte patch and traces showing VG sodium...

Enucleation for Western Blot Analysis

Although the aforementioned enucleation protocol is suitable for obtaining healed enucleated oocytes for functional studies, it is not recommended in studies in which the nucleus and enucleated oocyte are to be analyzed simultaneously. This is because the nucleus extruded into the half-strength OR2 will swell and become very fragile. The alternative enucleation method described in the following paragraph was originally developed by Gall et al. (12). The advantages of this protocol are as...

Chromatin Assembly of DNA Templates Microinjected Into Xenopus Oocytes

Dani le Roche, Genevi ve Almouzni, and Jean-Pierre Quivy The packaging of deoxyribonucleic acid (DNA) into chromatin within the eukaryotic nucleus can affect processes such as DNA replication, transcription, recombination, and repair. Therefore, studies aimed at understanding at the molecular level how these processes are operating have to take into account the chromatin context. We present a method to assemble DNA into chromatin by nuclear microinjection into Xenopus oocytes. This method...

Hemisecting Xenopus Oocytes and Eggs

The large size of Xenopus oocytes and eggs hampers the penetration of antibodies, particularly after fixation with aldehydes. To aid antibody penetration, we routinely Fig. 2. Triple-fluorescence labeling of mitotic and meiotic spindles in Xenopus oocytes and embryos. Oocytes (A-C) and blastula-stage embryos (D F) were fixed in methanol as described and stained with rabbit anti-XKCMl and Alexa 488-conjugated goat antirabbit immunoglobulin G (A and D), rat anti-a-tubulin (YL1 2) and Alexa 546...

MNase assay

Analysis of chromatin assembly. Left Autoradiography of a supercoiling gel showing a time-course of chromatin assembly coupled to DNA synthesis. DNA was extracted and purified after 15 min, 30 min, 1 h, 2 h, and 4 h following injection. The positions of the super-coiled form (form I) and the nicked and closed relaxed forms (forms II and Ir, respectively) are indicated on the right. The intermediates reflecting partial duplexes for which complete complementary DNA synthesis is not...

Defolliculation by Collagenase Treatment

There are two obvious advantages in isolating oocytes by collagenase treatment. The first one is to obtain a large quantity of oocytes without stressing your eyes and hands. The second one is that the oocytes obtained in such a fashion are truly defolliculated (similar to that shown in Figs. 2C,D). There are drawbacks as well. Collagenase treatment adversely affects the metabolic rate of oocytes. The most noticeable is the marked depression in endogenous protein synthesis (4). A common remedy...

Low Speed Interphase Extract

Described next are the steps involved in preparing an LSE from eggs that have been harvested and washed as described in the Subheading 3.2.3. Preparation of LSE is an intermediate step in the preparation of HSE (Subheading 3.4.) and NPE (Subheading 3.5.). For HSE, we first prepare LSE from the eggs of 6 frogs for NPE, we prepare LSE from the eggs of 15 frogs. 1. Transfer the washed eggs to Falcon 2059 tubes. Allow the eggs to settle and aspirate the supernatant. Pack the eggs by gentle...

Manual Defolliculation With Sandpaper Stripping

It is customary in our lab for a new recruit to spend the first 2 to 3 wk to learn and practice manual defolliculation. We follow the description provided in Smith et al. (7). Place a piece of ovarian tissue containing 50 to 100 oocytes in a 6-cm dish filled with Ca2+-free OR2. Under a dissecting microscope, place two pairs of sharp forceps Fig. 2. Manually defolliculated oocytes before and after sandpaper stripping. (A) The surface of a manually defolliculated oocyte appears rough under a...

Using the Xenopus Egg Extract Reconstitution System Paula Deming and Sally Kornbluth

It was first shown by Newmeyer and colleagues in the 1990s that the molecular events of apoptosis could be reconstituted in vitro using Xenopus egg extracts. When the egg extract is allowed to incubate at room temperature for an extended time, the biochemical events of apoptosis are activated spontaneously. The features of apoptosis in the Xenopus reconstitution system mimic those that occur in mammalian cells Cytochrome c is released from the mitochondria, caspases are activated, cellular...

Exploring RNA Virus Replication in Xenopus Oocytes Andrea V Gamarnik and Raul Andino

Microinjection of poliovirus RNA in Xenopus oocytes initiates a complete and authentic viral replication cycle that yields newly synthesized infectious virus. This system can be used to study the molecular mechanism of the different steps involved in virus replication. Interestingly, viral replication only occurs if poliovirus RNA is coinjected with factors present in HeLa extracts. We have determined that two HeLa cell factors are required for viral replication in oocytes, one involved in...

CDNA Libraries and EST Sequences

The NIH Xenopus Initiative includes cDNA libraries that are part of the IMAGE (Integrated Molecular Analysis of Genomes and Their Expression) Consortium gene collection. The collection currently contains 52 X. laevis libraries and 12 X. tropicalis libraries (Table 1). Details of each library can be found at the IMAGE Web site (http image.llnl.gov ) and at NCBI's UniGene library browsers (http www.ncbi.nlm.nih.gov UniGene lbrowse2.cgi TAXID 8355 and http www.ncbi.nlm.nih.gov UniGene lbrowse2.cgi...

Preparation and Microinjection of Xenopus Oocytes

Because detailed protocols for preparing and microinjecting Xenopus oocytes are listed elsewhere in this book, we only give a brief summary covering the essential steps. The subheadings outline terminal and survival surgery removal of epithelial layers, follicular cells, and the vitelline membrane and oocyte microinjection. 3.1.1. Terminal and Survival Surgery 1. Place a female X. laevis frog (Fig. 1A) in a 0.17 solution of MS-222 (made up in water) until sufficiently anesthetized. 2. If all of...

And Pesticides

Steven David Buckingham, Luanda Pym, and David Barry Sattelle The Xenopus laevis oocyte offers one of the most convenient expression systems for assaying the actions of candidate ligands on cloned ionotropic neurotransmitter receptors also known as ligand-gated ion channels LGICs . Their large size makes injection of complementary ribonucleic acid or complementary deoxyribonucleic acid and electro-physiological recording very easy. Furthermore, Xenopus oocytes translate messages very...

DNA Replication in NPE

Sperm chromatin or plasmid DNA can be used as a template for efficient DNA replication when incubated sequentially in HSE and NPE Fig. 1A . Incubation of the DNA template with HSE leads to pre-RC formation. Subsequent addition of NPE stimulates replication initiation by providing high levels of several replication factors see Subheading 1. , while also limiting DNA replication to a single round because of the presence of inhibitors that block the formation of pre-RCs 6 . Complete DNA...

By Mitotic Xenopus Egg Extract in Semi Intact MDCK Cells

Fumi Kano, Katsuya Takenaka, and Masayuki Murata Semi-intact cells are cells with plasma membranes that have been permeabilized by bacterial pore-forming toxins or surfactants. The addition of mitotic Xenopus egg extract to semi-intact cells can reconstitute a number of intracellular events that occur specifically at the onset of mitosis. In this chapter, we describe methods for reconstituting the disassembly of the Golgi apparatus by introducing mitotic Xenopus egg extract into semi-intact...

And Chromosome Condensation in Xenopus Egg Extracts

Methods are presented for preparing cytoplasmic extracts from Xenopus laevis eggs and their utilization to reconstitute and monitor events of the cell cycle in vitro. Addition of sperm nuclei to crude extracts and cycling of the reaction through interphase and back into metaphase promotes formation of bipolar spindles capable of segregating their duplicated chromosomes. Reactions can be spun down onto cover slips for immunofluorescence analysis. High-speed extracts support mitotic chromosome...

Chromosomal DNA Replication in a Soluble Cell Free System Derived From Xenopus Eggs

Walter Cytoplasmic egg extracts from the frog Xenopus laevis represent a powerful cell-free system to study eukaryotic chromosomal DNA replication. In the classical approach, sperm chromatin is added to unfractionated egg cytoplasm, leading to the assembly of transport-competent nuclei that undergo a single, complete round of DNA replication. The need for nuclei in this system has been circumvented. Sperm chromatin or plasmid DNA is first incubated with...

Uni Gene Clusters

Computational methods have been developed that predict transcriptional units from EST sequences. NCBI's UniGene project used an experimental algorithm to group hundreds of thousands of Xenopus dbEST sequences into a set of about 20,000 gene-oriented clusters see The analyses included Xenopus messenger RNAs mRNAs and draft or high-throughput cDNA HTC sequences HTC available from GenBank. UniGene predicted that 360,000 of the X. laevis sequences available in September 2004 represented about...

Functional Studies in Xenopus Egg Extracts

Although the X. laevis extract system is biochemically and cytologically accessible, it is not practical for genetic analyses of cell cycle events. In contrast, extracts may be biochemically manipulated through a number of techniques that mimic some powerful tools available in genetic model systems. As Xenopus genome databases expand to facilitate proteomic analyses, the extract system is also developing as a tool to identify novel factors through biochemical and functional assays. Null...

Performing an Analysis

Incubation of detector cells with Mag-fura-2 is performed initially. Fluorescent loading of cells in individual chambers is done serially every 30 to 60 min to have freshly loaded cells prepared for each run. While cells are incubating with Mag-fura-2, an isolated oocyte is injected with a-32P ATP and allowed to recover. The detector cells are permeabilized and washed on the stage of the microscope. On completion of the permeabilization step, a cell is chosen to act as the detector cell, and...

Localized Sampling of Oocyte Cytoplasm

Surgically remove stages V and VI oocytes from X. laevis frogs. 2. Isolate individual oocytes after collagenase digestion 13 . 3. Maintain oocytes at 18 C in ND96 until use. 4. Make a 0.5-mm V-shaped depression within a shallow 1-2 mm reservoir created in a block of electrically nonconductive material such as delrin. The shallow reservoir should be contiguous with a deeper reservoir of 1 to 2 mL total volume Fig. 2 . 5. In preparation for sampling of oocyte cytoplasm into a capillary, place...

For Subcellular Biochemical Assays

Sims, Veronica Luzzi, and Nancy L. Allbritton The Xenopus oocyte is a widely used model cell for studies of signal transduction mechanisms. Advances in microanalytical methods have made it feasible to perform rapid, localized collection of cytoplasm from individual Xenopus oocytes. Analytes contained in the cytoplasmic sample are separated by electrophoresis in a capillary and simultaneously transferred to a detection region. The development of bioengineered cells as sensitive...