mRNA-injected Oocytes Research Articles

Xenopus Oocytes as immunological vectors to produce monoclonal antibodies to rat brain antigens

A novel approach was developed to raise a panel of monoclonal antibodies (mAb) against brain antigens using Xenopus oocytes as immunological vectors. Xenopus oocytes were injected to express proteins encoded by brain-derived mRNA extracted from rat cerebral cortex. A crude membrane preparation from mRNA-injected oocytes was then used to immunize mice previously rendered immunotolerant to native oocyte membranes. mAb reacting with cryostat cut sections from rat brain were selected and further characterized by immunohistological and immunobiochemical techniques. Several mAb recognized brain specific antigens, including some that were cell type specific and others that revealed a regional binding pattern. A particular group of antibodies recognized an epitope localized exclusively to the cerebellar pinceau terminals. Although some of the hybridomas found in this panel may be products of natural autoreactive lymphocytes, the presence of a specific immune response to mRNA expression products is discussed. These results indicate that mRNA injected oocytes are useful tools to raise mAb to study the molecular diversity of the nervous system.

Expression of ion channels and receptors in Xenopus oocytes using vaccinia virus

The cytoplasmic injection of mRNA synthesized in vitro into Xenopus oocytes is widely used for heterologous expression of ion channels and neurotransmitter receptors. We report two new methods for expression of ion channels and receptors in oocytes using vaccinia virus (VV). 1) A recombinant VV carrying the Shaker H4 K+ channel cDNA driven by the VV P7.5 early promoter was injected into oocytes. 2) A recombinant VV containing the bacteriophage T7 RNA polymerase driven by the P7.5 promoter was coinjected along with plasmids containing a T7 promoter and cDNAs for channels and receptors. The functionally expressed proteins include a) voltage-gated ion channels: the Shaker H4 K+ channel and the rat brain IIA Na+ channel, b) a ligand-gated ion channel: the mouse muscle nicotinic acetylcholine receptor (AChR), and c) a G protein-coupled receptor: the rat brain 5HT1C receptor. After virus/cDNA injection into oocytes, these channels and receptors generally showed characteristics and expression levels similar to those observed in mRNA-injected oocytes. However, the AChR expressed at lower levels in virus/cDNA-injected oocytes than in mRNA-injected oocytes. Because our methods bypass mRNA synthesis, they are more rapid and convenient than the mRNA injection method. Potential applications to structure-function studies and expression cloning are discussed.

Comparison of a Na +/ d-glucose contransporter from rat intestine expressed in oocytes of Xenopus laevis with the endogenous cotransporter

Epithelial Na +/ d-glucose cotransport was incorporated into the plasma membrane of Xenopus oocytes after micro-injection of poly(A) +-mRNA from rat intestine tissue and was detected by measurements of uptake of [ 14C]AMG (methyl α- d-glucopyranoside). In mRNA-injected oocytes, the rate of AMG uptake exceeds the rate of endogenous Na +/AMG cotransport by a factor of up to 30. It is demonstrated that the additionally expressed transport differs qualitatively from the endogenous transport with respect to several parameters which is a prerequisite for the demonstration of expression of a foreign transporter: (1) The expressed system is more sensitive to external glucose or AMG and to the specific inhibitor phlorizin, (2) it is less sensitive to external Na + and to changes in membrane potential, and (3) it is susceptible to inhibition by monoclonal antibodies, known to bind specifically to Na +/glucose contransporters and to modulate the cotransport in kidney and intestine. The use of the antibodies allows one to distinguish between endogenous Na +/AMG cotransport and foreign contransport expressed by injection of foreign mRNA. The expression of the foreign transport leads to transport rates that are high enough to detect the electrical current generated by the Na +/glucose cotransport. This allows future characterization of the cotransport system under voltage-clamp conditions by analyzing membrane current.

Functional expression of the intestinal peptide-proton co-transporter in Xenopus laevis oocytes.

The expression of the intestinal peptide-proton cotransporter was examined in Xenopus laevis oocytes by microinjection of poly(A)+ mRNA prepared from rabbit intestinal mucosal cells. The concomitant expression of the glucose-sodium co-transporter was used as the control for the effectiveness of the expression technique. There was significant endogenous activity of Gly-Sar uptake in water-injected oocytes, but the uptake activity increased nearly 3-fold in poly(A)+ mRNA-injected oocytes. The expression of the peptide transporter was time-dependent. There was no detectable expression on day 1 after injection. The expression became noticeable on day 2 and increased with time, reaching a maximum on day 4. There was no further change on days 5 and 6. The endogenous uptake rate measured in water-injected oocytes, on the contrary, showed a slight decrease during this time. The expressed peptide transporter retained its substrate specificity, having affinity for the dipeptides, Gly-Sar and Gly-Pro, and no or little affinity for the free amino acids, Gly and Sar. The expressed peptide transporter also showed a dependence on a transmembrane H+ gradient for maximal activity. These data demonstrate that the mammalian intestinal peptide-proton co-transporter can be successfully expressed in Xenopus laevis oocytes. This expression system can provide an effective assay procedure to clone the gene encoding the transporter.

Possible heterogeneity of metabotropic glutamate receptors in duced in Xenopus oocytes by rat brain mRNA

Pharmacological properties of metabotropic glutamate receptors were studied in Xenopus oocytes injected with rat brain mRNA. trans-1-Amino-cyclopentyl-1,3-dicarboxylic acid ( t-ACPD), a conformationally restricted analog of glutamate, induced oscillatory inward currents in mRNA-injected oocytes. These t-ACPD responses showed several characteristics identical to those of the other metabotropic responses including the metabotropic glutamate responses stimulated by quisqualate. d,l-2-Amino-3-phosphonopropionate ( d,l-AP3) effectively suppressed the t-ACPD and ibotenate responses. However, quisqualate responses were not affected substantially by d,l-AP3. These findings suggest that the metabotropic glutamate receptors in the oocytes may be classified into at least two subtypes according to their pharmacological properties: one preferentially activated by quisqualate and insensitive to AP3, and the other activated by t-ACPD and ibotenate and antagonized by AP3.

Neurotensin and neuromedin N elevate the cytosolic calcium concentration via transiently appearing neurotensin binding sites in cultured rat cortex cells

Through assessment of the changes in the intracellular free-calcium concentration ([Ca 2+] i), which was measured using the calcium sensitive dye, fura-2, the character of the neurotensin (NT) binding sites which appeared transiently during the early ontogenetic stage in the rat cerebral cortex was analyzed in primary cultures of cerebral cortex cells from neonatal rats. NT (1–1000 nM) elevated [Ca 2+] i of the cells even when extracellular calcium was chelated with 1 mM ethylene glycol-bis(β-aminoethylether)- N, N, N′, N′-tetraacetic acid (EGTA). These findings suggest that the transiently appearing NT-binding sites in the cortex are receptors for NT and that some of the NT-induced increase in [Ca 2+] i is due to mobilization from the intracellular calcium store. Further application of NT after 10 min washing caused an increase in [Ca 2+] i again. This is in contrast to the findings for cortical slices from adult rats and mRNA-injected oocytes; desensitization due to NT was of long duration and further application of NT failed to activate the neurons which had responded the first time to NT. These facts suggest that the character of the NT-binding sites in the cerebral cortex differs between neonatal and adult rats. In addition, we showed that neuromedin N had a similar property to NT as to mobilization of [Ca 2+] i and acted only on NT-responsive cells, suggesting the interaction between NT and neuromedin N at the postsynaptic level via the same receptor.

Dopamine transporter: expression in Xenopus oocytes

Xenopus oocytes can express biologically relevant transport activity after injection of mRNAs encoding several carrier molecules. mRNA from PC12 cells, as well as transcripts from a rat ventral midbrain library, can be expressed in these oocytes and allow them to display pharmalogically specific dopamine uptake. mRNA-injected oocytes incubated with tritiated dopamine contain tritiated dopamine and metabolites; lower amounts of radiolabeled dopamine and more radiolabeled metabolites are found in oocytes co-incubated with cocaine or in water-injected oocytes. Tritiated dopamine uptake into mRNA-injected oocytes is time, sodium, and temperature dependent. It is blocked by cocaine and mazindol, but not by haloperidol. It is not found after injection of mRNA from other brain regions. A size-selected rat midbrain library constructed in the plasma vector pCDM8 yields mRNA transcripts whose injection into oocytes causes cocaine-blockable [ 3H]dopamine uptake. These findings provide an assay for purification of the dopamine transporter cDNA by sib selection techniques.

Expression of the Na(+)-K(+)-2Cl- cotransporter in Xenopus oocytes

The Na(+)-K(+)-2Cl- cotransporter (NKCCT) is important in mediating net salt transport in a variety of cells. As a first step in obtaining structural information on this transport system, we attempted to express it in Xenopus laevis oocytes by injection of poly(A)+ mRNA from rat kidney. Four days after injection, batches of oocytes were tested for 86Rb uptake in presence and absence of 10 microM bumetanide to distinguish that fraction of influx mediated by NKCCT. In the absence of bumetanide, the oocytes formed a bimodal distribution with respect to 86Rb uptake, with some oocytes accumulating significantly more 86Rb than others. In the presence of bumetanide, or when Na was replaced with choline, that group of oocytes taking up more 86Rb did not appear. Sham-injected oocytes did not accumulate sufficient 86Rb to be distinguishable above background. Taken together, these data suggest that some of the mRNA-injected oocytes expressed the rat renal NKCCT. Crude poly(A)+ mRNA was separated by acid-urea agarose gel electrophoresis, and fractions were injected into oocytes. One fraction corresponding to messages of approximately 7 kilobases in length induced a bumetanide-sensitive 86Rb influx resembling that seen with total mRNA. Poly(A)+ mRNA fractionated on sucrose density gradients gave similar results. It is concluded that the rat kidney NKCCT has been expressed in Xenopus oocytes from a high molecular weight fraction of poly(A)+ mRNA.

Glycine-insensitive NMDA-sensitive receptor expressed in Xenopus oocytes by guinea pig cerebellar mRNA

The electrophysiological and pharmacological properties of N-methyl-D-aspartate (NMDA)-sensitive receptors expressed in Xenopus oocytes by injection of total poly(A)+RNAs (mRNAs) from the cerebellum and cerebrum of guinea pigs were compared. The inward current induced by NMDA under voltage-clamp in cerebellar mRNA-injected oocytes was depressed in a voltage-dependent fashion by Mg2+ to show a negative slope conductance and selectively antagonized by D-2-amino-5-phosphonovalerate (D-APV) and phencyclidine (PCP). Glycine (0.01-10 microM) did not potentiate NMDA-induced currents in cerebellar mRNA-injected oocytes, while it potentiated NMDA-induced currents in cerebral mRNA-injected oocytes in a dose-dependent fashion. 6-Cyano-7-nitroquinoxaline-2,3-dione and 7-chlorokynure-nate suppressed the NMDA response but significantly less potently in cerebellar mRNA-injected oocytes than in cerebral mRNA-injected oocytes. These results suggest that the NMDA-sensitive receptor expressed in Xenopus oocytes by guinea pig cerebellar mRNA resembles the cerebral NMDA receptor in its high sensitivities to Mg2+, PCP, and D-APV, but it is distinct from the cerebral NMDA receptor in responsiveness to glycine.

Selective activation of oscillatory currents by trans-ACPD in rat brain mRNA-injected Xenopus oocytes and their blockade by NMDA

Comparison of trans-1-amino-1,3-cyclopentane-dicarboxylate with quisqualate in their action on oscillatory current

Expression of striatal D1 dopamine receptors coupled to inositol phosphate production and Ca2+ mobilization in Xenopus oocytes.

Expression of central nervous system receptors for dopamine was examined by injection of poly(A)+ RNA (mRNA) from rat striatum into oocytes from Xenopus laevis. Electrophysiological measurements in mRNA-injected oocytes indicated that addition of 100 microM dopamine induced an inward current (40-100 nA) that was consistent with the activation of endogenous Ca2(+)-dependent Cl- channels. This current was also elicited by addition of the selective D1 agonist SKF 38393 but not by the selective D2 agonist quinpirole. Prior addition of the dopaminergic antagonist cis-piflutixol completely abolished dopamine-induced currents but had no effect on currents produced by serotonin. Using 45Ca2+ efflux assays, addition of 100 microM dopamine to injected oocytes stimulated efflux 2- to 3-fold. This increase was mimicked by SKF 38393 and was blocked by the D1-selective antagonist (+)SCH 23390 but not by the D2-selective antagonist domperidone. No increase in 45Ca2+ efflux was seen with 100 microM quinpirole. Size fractionation of striatal mRNA yielded a single peak (2.5-3.0 kilobases) of D1 receptor-mediated 45Ca2+ efflux activity in injected oocytes. In addition, dopamine stimulation of oocytes injected with peak fractions and prelabeled with myo-[3H]inositol caused a 3-fold increase in [3H]inositol 1,4,5-triphosphate [( 3H]InsP3) formation. No effect on [3H]InsP3 production or 45Ca2+ efflux was observed, however, in injected oocytes incubated with 1 mM N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate. Thus, in addition to D1 receptors that stimulate adenylyl cyclase, rat striatum contains D1 receptors that can couple to InsP3 formation and mobilization of intracellular Ca2+.

Expression of biologically active human platelet-derived growth factor (PDGF) B-receptor in [formula omitted] oocytes

Platelet-derived growth factor (PDGF) consists of three different isoforms, PDGF-AA, PDGF-AB and PDGF-BB, which bind to at least two types of receptors: the B-receptor, to which only PDGF-BB binds, and the A B receptor, to which all three isoforms bind. Microinjection of synthetic mRNA in Xenopus laevis oocytes was used to obtain cell-surface expression of the human PDGF B-receptor. The production of receptor molecules of correct size (190 kd) was demonstrated by specific immunoprecipitation; the binding properties of the membrane- associated PDGF B-receptor were investigated with highly purified recombinant [ 125I] labeled human PDGF-BB and -AA. Unlike Swiss mouse 3T3 cells, which possess both B- and A B -receptors and, therefore, bind both isoforms with high affinity, the mRNA-injected oocytes bound only the BB isoform. Mock-injected oocytes showed no specific binding.

Postnatal Development of Voltage-Dependent Calcium Channels in the Mouse Brain Disclosed by the <i>Xenopus </i>Oocyte Assay

We assessed the ontogeny of murine voltage-dependent calcium channels by extracting mRNA from brains of mice at different postnatal ages and injecting the mRNA into oocytes of the frog, Xenopus laevis. Voltage-dependent Ca2(+)-activated Cl- channels were measured to assess the presence of Ca2(+) channels. When compared with water-injected oocytes (controls), an increase in Ca2(+) channels was not detected until postnatal day 7. The number of Ca2+ channels peaked between 9 and 18 days and began to decline by 35 days. Bath application of barium, serotonin and the Ca2+ channel antagonist, verapamil, to mRNA-injected oocytes confirmed the presence of Ca2+ channels.

Functional expression of the human HepG2 and rat adipocyte glucose transporters in Xenopus oocytes: Comparison of kinetic parameters

Facilitated glucose transport is a ubiquitous characteristic of animal cells carried out by a family of membrane glycoproteins. Two members of this gene family are the well characterized human erythrocyte protein that has been cloned from the HepG2 cell line and the insulin-sensitive transporter that has been cloned from adipocytes and muscle tissue. In the present study the HepG2 and adipocyte glucose transporters were functionally expressed in Xenopus oocytes after injection of synthetic mRNAs produced by transcription in vitro from cloned cDNAs. Both 2-deoxyglucose uptake and 3-O-methylglucose transport were increased several-fold over basal levels in mRNA-injected oocytes. Increased uptake of 2-deoxyglucose was inhibited completely in the presence of cytochalasin B, and 3-O-methylglucose transport was blocked by D-glucose but not by L-glucose. The half-saturation constant and turnover number for 3-O-methylglucose transport at 22 degrees C via the HepG2 transporter were estimated to be 21 mM and 2.2 x 10(3) s-1 under equilibrium exchange conditions. The half-saturation constant for 3-O-methylglucose transport via the adipocyte transporter under the same conditions was estimated to be 1.8 mM. These data prove the functional identity of the cloned HepG2 and adipocyte cDNAs and indicate that the HepG2 and adipocyte transporters display similar kinetic behavior when expressed in the frog oocyte membrane as compared with their native membrane environments. Thus, the difference in the equilibrium exchange kinetic parameters for glucose transport in the erythrocyte and the adipocyte is a result of the expression of two distinct glucose transporter proteins.

Selective expression of an amiloride-inhibitable Na+ conductance from mRNA of respiratory epithelium in Xenopus laevis oocytes

Poly(A)+RNA was prepared from primary cultures of human nasal polyp epithelia and from native bovine tracheal epithelia. Six to fifty nanograms mRNA were injected into prophase-arrested immature Xenopus laevis oocytes. One to four days later the oocytes were probed with electrophysiological techniques for induction of novel ion conductances. Oocytes injected with mRNA had lower membrane potentials (Vm) and resistances (Rm) than controls. By use of step changes in extracellular Na+ concentration and applying amiloride, a Na+ conductance could be identified in mRNA-injected oocytes, which was inhibited by submicromolar concentrations of amiloride with the same kinetics (Ki = 1.3 X 10(-7) mol/l) as in the original tissue. After complete inhibition of this conductance by 10(-5) mol/l amiloride, Vm and Rm approached the respective values of controls. The data indicate that oocytes express functional epithelial Na+ channels but apparently no other epithelial ion conductances from injected mRNA of respiratory epithelium.

Functional expression of the amiloride-sensitive sodium channel in Xenopus oocytes.

Expression of the amiloride-sensitive sodium channel was examined in Xenopus oocytes that were microinjected with A6 cell mRNA. Amiloride-inhibitable 22Na flux could be measured in intact oocytes 2-3 days after injection with 25 ng of poly(A)+ RNA isolated from aldosterone-treated A6 cells. The rate of 22Na uptake was approximately 15-fold greater in oocytes microinjected with 25 ng of poly(A)+ RNA than in water-injected control oocytes. An increase in 22Na uptake by mRNA-injected oocytes occurred whether the mRNA was isolated from A6 cells grown on a porous or nonporous support. In the presence of 4 mM NaCl, amiloride caused dose-dependent inhibition of 22Na uptake in mRNA-injected oocytes, which was half-maximal at 6 x 10(-8) M. Both 1 microM amiloride and 0.1 microM benzamil inhibited 22Na uptake in mRNA-injected oocytes by greater than 95%, whereas less than 50% inhibition occurred with 1 microM 5-(N-ethyl-N-isopropyl)amiloride. When A6 cell mRNA was size fractionated by sucrose density-gradient centrifugation, amiloride-sensitive 22Na uptake was expressed predominantly by oocytes injected with mRNA from two contiguous fractions.

Neurooncogenes.

Using monospecific antibodies raised against the amino terminal domain of viral-yes protein, we precipitated a 58 kD protein-tyrosine kinase from the brain of Xenopus laevis frog. By a number of criteria, including pattern of expression in various tissues and one-dimensional peptide mapping, we concluded that pp58 is very likely the authentic amphibian yes protein because it is more similar to the chicken yes protein than to any other known tyrosine kinases. The pp58c-yes is expressed in adult brain at elevated levels. In contrast, its level of expression in follicular and denuded oocytes is 30-50 times lower. Because of the low endogenous expression of the oocyte-associated yes kinase, we were able to transiently express and analyze pp62c-yes by injecting oocytes with 27S size fraction of poly(A)+ mRNA isolated from chicken cerebella. The pp62c-yes expressed in oocytes from the exogenous message comigrated on SDS polyacrylamide gel with pp62c-yes from cerebellum and was indistinguishable from it by one-dimensional peptide mapping. The pp62c-yes expressed in oocytes was enzymatically active. A number of phosphotyrosine-containing proteins were detected specifically in the c-yes mRNA-injected oocytes. The usefulness of the Xenopus oocyte expression system to study the functional aspects of c-yes protein and other tyrosine kinases was evaluated.

Properties of the kainate channel in rat brain mRNA injected Xenopus oocytes: ionic selectivity and blockage

The properties of kainate receptor/channels were studied in Xenopus oocytes injected with mRNA that was isolated from adult rat striatum and cerebellum and partially purified by sucrose gradient fractionation. Kainate (3-1000 microM) induced a smooth inward current that was competitively inhibited by gamma-D-glutamyl-aminomethanesulfonate (GAMS, 300 microM). In striatal mRNA-injected oocytes, the kainate current displayed nearly linear voltage-dependence and mean reversal potential (Er) of -6.1 +/- 0.5 mV. In cerebellar mRNA-injected oocytes; Er was nearly identical (-5.1 +/- 1.2 mV) but there was marked inward rectification of the kainate current. Ion replacement studies reveal that the kainate channel is selective for cations over anions, but relatively non-selective among small monovalent cations. Large monovalent cations such as tetrabutylammonium are impermeant and induce a non-competitive block of kainate current that is strongly voltage-dependent. Divalent cations are relatively impermeant in the kainate channel and Cd++ and other polyvalent metals were shown to block kainate current by a mechanism that is only weakly voltage-dependent. A model of the kainate channel is proposed based upon these observations.

Low-concentration N-acetylaspartylglutamate suppresses the climbing fiber response of Purkinje cells in Guinea pig cerebellar slices and the responses to excitatory amino acids of Xenopus laevis oocytes injected with cerebellar mRNA

Whether N-acetylaspartylglutamate (NAAG) at micromolar concentrations shows a modulatory action on the synaptic transmission mediated by excitatory amino acids was investigated using Guinea pig cerebellar slices and the Xenopus laevis oocytes injected with mRNA from Guinea pig cerebellum. The climbing fiber response consisted of an excitatory postsynaptic potential (EPSP) and a plateau potential intracellularly recorded from Purkinje cell dendrite was depressed by 30 μM NAAG; the EPSP was decreased by about 21% in amplitude and the plateau potential was depressed by about 42% in duration. The depolarizations induced by l-aspartate, l-glutamate, N-methyl->d-aspartate and quisqualate in mRNA-injected Xenopus oocytes were non-selectively antagonized by 0.5 μM – 5μM NAAG, the mean % blockade by 5 μM NAAG being about 38%. Higher concentrations (100 μM) of NAAG alone depolarized mRNA-injected oocytes. On the other hand, the high-affinity uptake of l-[ 3H]aspartate by cerebellar slices was enhanced by 33% on average by 10 μM NAAG. These results suggest the possibility that micromolar concentrations of NAAG may attenuate the synaptic transmission mediated by glutamate receptors not only by blocking postsynaptic receptors but also by facilitating the high-affinity re-uptake of transmitter amino acids.

Fate of glyoxysomal malate dehydrogenase from watermelon after heterologous in-vivo translation in xenopus oocytes

Total poly A +-mRNA from watermelon cotyledons was translated in Xenopus laevis oocytes. Watermelon glyoxysomal malate dehydrogenase was found as its higher molecular weight precursor (pre-gMDH) and accumulates over at least 48 hours of translation. Organelle separation and immunocytochemistry located the watermelon pre-gMDH in the cytosol of the oocyte. The heterologous translation product from oocytes can be imported into isolated glyoxysomes from endosperm of castor bean. This import was correct in terms of protection against proteolysis and cleavage of the presequence within the limits of accuracy. We conclude that watermelon pre-gMDH accumulates in mRNA-injected oocytes as an import competent cytosolic precursor.