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PubMed2001

Effects of cryopreservation of pronuclear-stage rabbit zygotes on the morphological survival, blastocyst formation, and full-term development after DNA microinjection.

The objectives of this study were to examine the freezing sensitivity of pronuclear-stage rabbit zygotes and to produce transgenic rabbits using the cryopreserved zygotes. Zygotes were cryopreserved either by one of two vitrification protocols or by one of the two conventional freezing protocols. The morphological survival rates of zygotes subjected to two-step freezing in 1.5 M ethylene glycol and 0.1 M sucrose (74%) or to vitrification in 7.2 M ethylene glycol and 1.0 M sucrose (81%) were higher than those subjected to freezing in 1.5 M DMSO (46%) or to vitrification in a mixture of 2.0 M DMSO, 1.0 M acetamide, and 3.0 M propylene glycol (41%). But the in vitro development into blastocysts of zygotes cryopreserved by vitrification (17%) or to a lesser extent by freezing (52%) was impaired, when compared to that of fresh control zygotes (89%). Next, a fusion gene composed from bovine aS1-casein promoter and a human GH structural gene (2.8 kb) was microinjected into the pronucleus of rabbit zygotes frozen-thawed in ethylene glycol and sucrose. Then, the presence of exogenous DNA in the genome of newborn offspring was determined by PCR. The post-injection survival of frozen zygotes (97%) was the same as that of fresh control zygotes (96%). However, of 18 offspring derived from 414 frozen-thawed and DNA-injected zygotes, no transgenic rabbits were produced. Of 52 offspring derived from 403 DNA-injected fresh zygotes, 3 transgenic rabbits were found. Here we report the first rabbit offspring resulting from zygotes cryopreserved at the pronuclear-stage, although the cryopreservation procedure employed must be improved if zygotes are to be used for systematic production of transgenic rabbits.

Animals↗

PubMed1989

Microinjection of cholecystokinin into the rat ventral tegmental area potentiates dopamine-induced hypolocomotion.

Cholecystokinin, (CCK) 1-400 ng, significantly potentiated the hypolocomotion induced by dopamine, when simultaneously microinjected bilaterally into the ventral tegmental area (VTA) of rat brain. Within this dose range, CCK had no effect alone on ambulatory locomotion. Topographical analysis indicated that the modulatory effect of CCK was greatest in the central and caudal regions of the VTA, and absent at sites lateral, dorsal, rostral, and caudal to the VTA. Pharmacological analysis indicated that both unsulfated CCK octapeptide (100 ng) and the C-terminal tetrapeptide of CCK (400 ng) potentiated dopamine-induced hypolocomotion in a manner identical with sulfated CCK octapeptide (100 ng). Proglumide, an antagonist of the peripheral-type CCK receptor, did not block the potentiating actions of CCK, at doses of proglumide up to 500 mg/kg i.p., or 100 micrograms into the ventral tegmental area. L-364,718, an antagonist of the peripheral-type CCK receptor with lesser affinity for the central-type CCK receptor, blocked the potentiating actions of CCK at relatively high doses of L-364,718 (1-10 mg/kg i.p.). These findings suggest that CCK acts as a facilitatory modulator of dopamine at a central-type CCK receptor on the A10 cell bodies.

Animals↗

PubMed1993

Microinjection of an nm23 specific antibody inhibits cell division in rat embryo fibroblasts.

Expression of the nm23/NDP-K gene correlates with reduced metastasis in some tumors and with increased proliferation in both nontransformed and transformed cells in culture. Decreased nm23/NDP-K expression results in mitotic arrest in neuroblasts of developing Drosophila. In order to better understand the biological role(s) of nm23 in non-transformed cells, an nm23-specific antibody was introduced into rat embryo fibroblasts and effects on DNA synthesis and cell cycle progression were analyzed. Microinjection of the nm23 antibody inhibited cell division with no apparent effect on DNA synthesis. Control experiments revealed that the survival of cells injected with the nm23 antibody was similar to that of control antibody injected cells in the absence of cell division. These results suggest that in mammalian fibroblasts, as in Drosophila, nm23 expression may be necessary for progression through the cell cycle.

Animals↗

PubMed1999

Microinjection of Ca2+ store-enriched microsome fractions to dividing newt eggs induces extra-cleavage furrows via inositol 1,4,5-trisphosphate-induced Ca2+ release.

The cleavage signal transferred to the future cleavage cortex during anaphase has been proposed as "cleavage stimulus," but no signal has proved to induce cleavage furrows. The local Ca2+ transient along the cleavage furrow has been reported, but the Ca2+ source has remained unknown. To address these questions, we studied functions of Ca2+ stores in dividing newt eggs and found that microinjection of the Ca2+ store-enriched microsome fraction to the dividing newt egg induced a local extra-cleavage furrow at the injection site in 64-67% of the injected newt eggs while coinjection with inositol 1,4, 5-trisphosphate receptor (IP(3)R) antagonists heparin or anti-type 1-IP(3)R antibody clearly suppressed this induction (5 and 11% in induction rates, respectively). Injection of cerebellar microsomes from the type 1-IP(3)R-deficient mice induced extracleavage furrows albeit at a low rate (19%). Our observations strongly suggest that Ca2+ stores with IP(3)R induce and position a cleavage furrow via IP(3)-induced Ca2+ release (IICR) as Ca(2+)-releasing machinery and putative cleavage stimulus itself.

Anaphase↗

PubMed1994

Transcription of amphibian lampbrush chromosomes is disturbed by microinjection of HSP70 monoclonal antibodies.

To investigate the possible involvement of HSP70 in nuclear transport of proteins associated with the transcription process in amphibian oocyte lampbrush chromosomes, we examined the effect of anti-HSP70 mouse monoclonal antibodies on the transcriptional activity of lampbrush chromosomes. When injected into the oocyte cytoplasm, anti-HSP70 induced disorganization followed by retraction of chromosomal lateral loops. This retraction reflected inhibition of transcription, which was confirmed by our electron microscopic observations. At the same time, while nucleoli were morphologically disorganized, nucleolar transcription persisted. These modifications were completely reversible. In contrast, no modifications were observed when antibodies were directly microinjected into nuclei. Similar observations were registered at the level of lampbrush chromosomes when wheat germ agglutinin was injected into oocyte cytoplasm. All of these results suggest that HSP70 mediates the nuclear transport of proteins involved in the lampbrush chromosome transcriptional process.

Animals↗

PubMed1994

Human regulatory subunit RI beta of cAMP-dependent protein kinases: expression, holoenzyme formation and microinjection into living cells.

The human regulatory subunit RI beta of cAMP-dependent protein kinases was expressed in Escherichia coli as a fusion protein with glutathione S-transferase. Purification was performed by affinity chromatography on glutathione-agarose beads after cleavage with thrombin. The human recombinant RI beta protein migrated at 55 kDa on SDS-PAGE and displayed immunoreactivity with an anti-human RI beta antiserum. Furthermore, the purified recombinant RI beta protein was shown to exist as a dimer that was able to form holoenzyme with the catalytic subunit C alpha. The rate of RI beta 2C alpha 2 holoenzyme formation was faster in the presence than in the absence of MgATP. The kinase activity measured before and after adding cAMP to the holoenzyme showed that the presence of cAMP resulted in holoenzyme dissociation and release of active C alpha-subunit, due to cAMP binding to RI beta. Compared to a RI alpha 2C alpha 2 holoenzyme, the RI beta 2C alpha 2 holoenzyme exhibited a more than twofold higher sensitivity to cAMP. The subcellular localization of RI beta was analyzed in quiescent REF-52 fibroblasts and Wistar rat thyroid (WRT) cells after microinjection of fluorescently labeled proteins into the cytoplasm. A cytoplasmic distribution was observed when free RI beta was injected, whereas free C alpha injected into the cytoplasm appeared in the nucleus. When holoenzymes with labeled RI beta and unlabeled C alpha, or unlabeled RI beta and labeled C alpha, were injected, unstimulated cells showed fluorescence in the cytoplasm of both cell types. REF-52 cells stimulated with 8-bromo-cAMP (8-Br-cAMP) and WRT cells treated with thyrotropin (TSH) showed fluorescence mainly in the cytoplasm when RI beta was the labeled subunit of the in vivo dissociated holoenzyme. In contrast, nuclear fluorescence was evident from the release and translocation of labeled C alpha from the holoenzyme complex after stimulation with 8-Br-cAMP or TSH.

Allosteric Regulation↗

PubMed1995

Sensitive and rapid detection of beta-galactosidase expression in intact cells by microinjection of fluorescent substrate.

Bacterial beta-galactosidase, coded by lacZ, is a widely used reporter for studies of transcriptional activity of eukaryotic promoters at the single cell level. Unfortunately, current detection methods, like X-gal cytochemistry, are slow, have suboptimal sensitivity, and are incompatible with cell survival. By a novel approach based on microinjection into cells of the fluorogenic substrate 5-chloromethylfluorescein di-beta-D-galactopyranoside lacZ gene expression was detected without affecting cell viability or proliferative capacity. The method was far more sensitive than the conventional X-gal cytochemistry in all cell systems tested (primary hepatocytes, fibroblasts, and glioma cells). Results were obtained within seconds to minutes after injection, and cells remained fluorescent for hours.

Animals↗

PubMed1996

Fluorescently labeled phosphatidylinositol transfer protein isoforms (alpha and beta), microinjected into fetal bovine heart endothelial cells, are targeted to distinct intracellular sites.

Upon permeabilization of Swiss mouse 3T3 fibroblasts, an isoform of phosphatidylinositol transfer protein (PI-TP) was preferentially retained, a major part of which was associated with the perinuclear Golgi system (K. J. de Vries, A. Momchilova-Pankova, G. T. Snoek, and K. W. A. Wirtz, Exp. Cell Res. 215, 109-113, 1994). In the present study, the intracellular localization of this isoform (PI-TP beta) and the regular form (PI-TP alpha) was investigated in fetal bovine heart endothelial cells by microinjection of fluorescently labeled analogs followed by confocal laser scanning microscopy. The PI-TP alpha and PI-TP beta used were purified from bovine brain cytosol and covalently labeled with sulfoindocyanine dyes. By this novel method it was found that PI-TP beta was preferentially associated with perinuclear membrane structures whereas PI-TP alpha was predominantly present in the nucleus and in the cytoplasm. This intracellular localization was confirmed by indirect immunofluorescence indicating that the fluorescently labeled PI-TP alpha and PI-TP beta were targeted to the same sites as their endogeneous counterparts.

Animals↗

PubMed1998

Focal microinjection of gamma-acetylenic GABA into the rat entorhinal cortex: behavioral and electroencephalographic abnormalities and preferential neuron loss in layer III.

Neuron loss in layer III of the entorhinal cortex (EC) occurs in patients with temporal lobe epilepsy and in several animal models of the disease and may play a role in the development of spontaneously recurring seizures. This damage can be reproduced in rats by a focal microinjection of the indirect excitotoxin aminooxyacetic acid into the EC (Neurosci. Lett., 147: 185, 1992). We have now examined a similar but approximately 20 times more potent toxin, gamma-acetylenic GABA (GAG), for its ability to produce seizures and neurodegeneration in the rat EC. EEG activity was recorded continuously for 48 h after a focal injection of 4 micrograms GAG into the rat EC. Seizure episodes, spiking, and other irregularities occurred with a latency of 150 min. Behavioral abnormalities were observed in all animals and were always accompanied by EEG seizures. The behavioral changes subsided gradually, but EEG seizures continued up to 24 h after GAG treatment. Nissl and silver-stained tissue sections obtained 2-3 days after the injection of 4 micrograms GAG revealed neuron loss which preferentially affected the medial part of layer III of the EC, and caused a modest lesion in the hilar region of the ventral hippocampus. The neurodegenerative potency of GAG, in contrast to the effects of aminooxyacetic acid, was not influenced by the depth of anesthesia during surgery. A slight increase in the dose of GAG (to 5 micrograms) resulted in more severe behavioral seizures, causing generalized convulsions with salivation and loss of righting posture in 3 of 13 rats. These animals also showed a marked enlargement of the lesioned area, with substantial neuronal loss occurring in layer III of the EC, in the hilus of the dentate gyrus, and occasionally also in homotopic structures of the contralateral hemisphere. Seizure activity and lesions induced by 4 micrograms GAG were prevented by the NMDA receptor antagonist Dizolcipine (MK-801) (4 mg/kg, i.p., 10 min before and 12 h after GAG). These data support the notion of a close correlation between the occurrence of seizures and neuronal loss in layer III of the EC. Taken together, the study suggests that intraentorhinal injections of GAG may provide an advantageous model for the study of epileptogenic and epileptic mechanisms.

4-Aminobutyrate Transaminase↗

PubMed2002

Effects of angiotensin II microinjected into medial amygdala on male sexual behavior in rats.

Research was undertaken to study the role of central angiotensin in the modulation of male sexual behavior, testing the effect of angiotensin II (Ang II) injections into the medial amygdaloid nucleus (MeA). The sexual behavior of adult male Wistar rats was evaluated, 15 min after bilateral intra-amygdaloid microinjection (0.3 microl) of saline and 5 doses of Ang II: 10; 25; 50; 100, and 150 fmol. The effects of the Ang II receptor blockade were also studied. We tested the effect of coinjection of Ang II (50 fmol) with the AT1 antagonist, losartan (20 pmol) and the AT2 antagonist, CGP 42112 (1 pmol). Ang II inhibited sexual behavior and this inhibition was prevented by the coinjection of AT1 antagonist, losartan, or the AT2 antagonist, CGP 42112. Results show that Ang II has a powerful effect on male sexual behavior, which may be mediated by both AT1 and AT2 receptors.

Amygdala↗

PubMed1995

Genetic transformation of an entomopathogenic nematode by microinjection.

We report the first successful transformation of an entomopathogenic nematode. Foreign genes were introduced in Heterorhabditis bacteriophora HP88 by microinjection using vectors carrying the Caenorhabditis elegans genes coding for the roller phenotype and 16-kDa heat shock protein (hsp16) gene. A translational fusion made by inserting lacZ in frame into hsp16 was expressed in the body musculature, hypodermis, and pharyngeal muscles. The transcription of the hsp16/lacZ transgenes resulted in the rapid synthesis of detectable levels of beta-galactosidase. This research opens new avenues for genetic modification of entomopathogenic nematodes.

Animals↗

PubMed1999

Modeling transcriptional regulation using microinjection into Xenopus oocytes.

Transcriptional regulation is a complex process that requires cooperation between specific DNA sequence elements, the DNA-binding proteins that bind to these sequences, the general transcriptional machinery, and chromatin. Oocyte microinjection offers a technique to study the integrated transcription process while still providing the opportunity to experimentally perturb this process. We describe here techniques for manipulating DNA templates and the protein complement of the oocyte to study multiple facets of transcriptional regulation. We present sample results showing that the GAL4-VP16 fusion activator is sensitive to distance in constructs containing only a minimal promoter, but can activate transcription at greater distances when proximal promoter elements are present.

Animals↗

PubMed2002

The effect of cell division on the cellular dynamics of microinjected DNA and dextran.

Gene delivery is a multistep process that is being studied to increase its efficiency, a major hurdle for effective gene therapy. Our study focused on the nuclear entry step by microinjecting a mixture of fluorescent dextran and the pEYFP-Nuc plasmid (encoding a nuclear-targeted, enhanced GFP) into the cytoplasm of nondividing and dividing cells that were selected using non-chemical means. After 10 and 1000 ng/microl of plasmid DNA (pDNA) were cytoplasmically injected, 28% and 50% of the cells that had not divided expressed GFP, respectively, compared with 50% and 90% for the cells that had divided. This result suggested that pDNA can enter the nonmitotic nuclei of mononucleated cells, albeit at a lower efficiency than mitotic nuclei. The ability of pDNA to enter the intact nuclei of nondividing cells is consistent with our previous experience using multinucleated myotubes and digitonin-permeabilized cells in culture and using intravascular naked pDNA delivery in vivo. An explanation for the small effect of cell division was provided by studies using fluorescently labeled molecules and confocal fluorescent microscopy. They showed that the bulk of large dextran, and similarly pDNA, was excluded from re-formed nuclei after mitosis, thereby limiting the effect of cell division on the nuclear entry of pDNA.

Cell Division↗

PubMed2002

Muscimol diffusion after intracerebral microinjections: a reevaluation based on electrophysiological and autoradiographic quantifications.

Intracerebral muscimol injection is widely used to inactivate discrete brain structures during behavioral tasks. However, little effort has been made to quantify the extent of muscimol diffusion. The authors report here electrophysiological and autoradiographic results obtained after muscimol injection (1 microg/microl) either into the nucleus basalis magnocellularis (0.1-0.4 microl) or into the thalamic reticular nucleus (RE, 0.05-0.1 microl). In 52 rats, multiunit recordings were collected either in the RE or in the auditory thalamus during the 2 h following muscimol injection. Decreases in neuronal activity were observed up to 3 mm from the injection site; their time of occurrence was a function of the distance between the injection and recording sites. Because these decreases cannot be explained by physiological effects, they likely reflected muscimol diffusion up to the recording sites. Autoradiographic studies involved 25 rats and different experimental conditions. Optical density (OD) measures indicated that after a survival time of 15 min, a 0.05 microl injection produced a labeled area of 5.25 mm(2) at the injection site and a rostrocaudal labeling of 1.7 mm. Increasing the survival time to 60 min, or increasing the injected volume to 0.1 microl, systematically led to a larger labeled area at the injection site (8-12 mm(2)) and to a larger rostrocaudal diffusion (2.0-2.5 mm). Direct quantifications of radioactivity by a high-resolution radioimager validated the OD measures and even indicated a larger muscimol diffusion (up to 3.25 mm). Thus, these data point out that muscimol diffusion after intracerebral microinjection is larger than usually supposed. The relationships between these results and those obtained in behavioral studies are discussed.

Animals↗

PubMed1993

Deletion analysis of a replication origin of human cytomegalovirus by a novel assay system with a combination of microinjection and polymerase chain reaction.

We have developed a sensitive assay for DNA replication in mammalian cells that enables us to detect replicated DNA fragments of human cytomegalovirus. A 1/100 portion of DNA extracted from 1000 microinjected cells was subjected to polymerase chain reaction after digestion with appropriate restriction enzymes to differentiate replicated from nonreplicated plasmids. A portion of the amplified DNA was electrophoresed to detect replicated DNA. Subfragments of the HindIII fragment A (24 kb, map units 0.37-0.47) of strain Towne, which contains a previously identified origin of DNA replication (oriLyt), were analyzed by the assay system. A 4.3-kb subfragment (AatII-SacI) replicated as efficiently as the HindIII fragment A. Efficient replication ability was lost with a 1.3-kb deletion from the AatII end or a 0.9-kb deletion from the SacI end. These results suggest that the boundaries of oriLyt of Human cytomegalovirus strain Towne lie within the 1.3- and the 0.9-kb regions of the 4.3-kb fragment.

Base Sequence↗

PubMed1994

Nuclear import of microinjected influenza virus ribonucleoproteins.

Influenza virus ribonucleoproteins (vRNPs) devoid of the matrix protein (M1) were isolated and introduced into the cytoplasm of CHO and MDCK cells by microinjection. The injected vRNPs were found to be imported into the nucleus, and the RNA was transcribed. Their uptake into the nucleus was ATP-dependent, inhibited by antibodies to the nuclear pore complex, unaffected by the prior acidification of the vRNPs, and not inhibited by amantadine. The results showed that for productive infection, all the early stages of the viral entry pathway (receptor interaction, endocytosis, acid exposure, and membrane fusion) can be bypassed. Once the vRNPs are stripped of M1 and separated from each other, they are competent for import into the nucleus by constitutive cellular processes. Second, the results showed that while the amantadine block for incoming virus is manifested at the level of nuclear entry of the vRNPs, the actual import event per se is not affected. The results are consistent with a recent hypothesis that amantadine inhibits a step needed to prime the core for uncoating, which takes place before the virus has reached the cytosol.

Amantadine↗

PubMed2006

Chromatin assembly of DNA templates microinjected into Xenopus oocytes.

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 allows in vivo chromatin formation in a nuclear environment. We provide the experimental procedures for oocyte preparation, DNA injection, and analysis of the assembled chromatin.

Animals↗

PubMed1984

Renal handling of cadmium: a study by tubular microinjections.

109Cadmium was microinjected, in different chemical forms, into the proximal or distal cortical tubules of adult rats and recovery in the pelvic urine from the ipsilateral kidney was measured. There was marked uptake of inorganic cadmium along the proximal tubules, and only negligible uptake in the distal nephron. The fractional uptake of the complex cadmium-cysteine in the proximal tubule was inversely related to the cysteine concentration. The chelate cadmium-pentetic acid was completely recovered in the pelvic urine.

Animals↗