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PubMed2003

Chimaerism and erythroid marker expression after microinjection of human acute myeloid leukaemia cells into murine blastocysts.

It has been suggested that the embryonic microenvironment can control the survival and the transformed phenotype of tumour cells. Here, we addressed the hypothesis that the murine embryonic microenvironment can induce the differentiation of human tumour cells. To examine such interactions, we injected human leukaemic cells into preimplantation murine blastocysts at embryonic day 3.5 of gestation (E3.5). Microinjection of human KG-1 myeloid leukaemia cells and primary human acute myeloid leukaemia (AML) cells led to the generation of chimaeric embryos and adults. We observed that in E12.5 murine embryos, KG-1 cells were preferentially detected in yolk sac and peripheral blood, while primary AML cells mainly seeded the aorta gonad mesonephros region of chimaeric embryos. Analysis of the donor contribution in 15 different adult tissues showed that progeny of primary AML cells seeded to various haematopoietic and nonhaematopoietic tissues. Chimaeric embryos and adults showed no apparent tumour formation. Furthermore, analysis of chimaeric E12.5 embryos revealed that the progeny of human KG-1 cells activated erythroid-specific human globin and glycophorin A expression. In summary, our data indicate that human AML cells activate markers of erythroid differentiation after injection into early murine embryos.

Animals↗

PubMed2003

Microfluidic valve with cored glass microneedle for microinjection.

In this paper, a new microinjection device was constructed by fusing a glass microneedle and a PDMS-based microvalve. The microneedle was fabricated via traditional micropipette pulling. The PDMS-based microvalve regulates the fluid flow in the microchannel and microneedle. The 'ON/OFF' operation of the valve was controlled by manually supplied pneumatic pressure. The valve membrane utilized a two level geometry to improve control at low flow rates. The relation between pressure and flow was measured and the results showed that very small volumes of fluid (>1 nl) could be controlled. The valve operation was investigated by monitoring the tip of the needle and pneumatic pressure simultaneously and it demonstrated very stable 'ON/OFF' operation to the pressure change.

Dimethylpolysiloxanes↗

PubMed1987

Microinjected glutathione S-transferase Yb subunits translocate to the cell nucleus.

We have previously shown that a 30 kDa DNA-binding protein isolated from rat cell nuclei exhibits the chemical and immunological properties of glutathione S-transferase Yb subunits [Bennett, Spector & Yeoman (1986) J. Cell Biol. 102, 600-609]. It was of interest, therefore, to determine whether Yb subunits isolated from rat liver nuclei would return to nuclear fractions upon reintroduction to cell cytoplasms via red-blood-cell-mediated fusion. Labelled Yb subunits were associated with nuclear fractions 60 min after cell fusion. The microinjected protein remained associated with the nuclei for 18 h and was not extractable with low-salt washes. In addition, injected Yb subunits were found to equally distribute between extractable (56%) and residual (44%) nuclear fractions. These experiments demonstrate that glutathione S-transferase Yb subunits isolated from nuclei rapidly translocate to nuclei upon reintroduction into cell cytoplasms.

Animals↗

PubMed1989

Effects of inhibitors on aldolase breakdown after its microinjection into HeLa cells.

1. The regulation of protein breakdown as well as the generation of intermediates in the pathway from intact protein to amino acids was investigated by using 3H-labelled N-ethylmaleimide-modified aldolase (NEM-aldolase) as an indicator protein after its microinjection into HeLa cells. 2. NEM-aldolase degradation to acid-soluble products proceeded at a slower rate than that of endogenously labelled total cell protein, and was inhibited to a greater extent by 3-methyladenine, leupeptin and NH4Cl. The combination of leupeptin plus NH4Cl was particularly effective, decreasing the NEM-aldolase breakdown rate by 90%. 3. Measurements of the loss of radioactivity from the aldolase band located from fluorograms after SDS/polyacrylamide-gel electrophoresis showed that NEM-aldolase breakdown was much more rapid when measured by this method. The effects of insulin, 3-methyladenine, leupeptin and NH4Cl on this breakdown were also substantial. 4. Substantial amounts of peptide intermediates in the breakdown pathway of NEM-aldolase accumulated in cells. The production of small intermediates (less than 30 kDa) accounted for approx. 40% of the NEM-aldolase degraded in control cultures. Addition of NH4Cl increased the proportion of these intermediates. Large intermediates, between 31 and 38 kDa, were particularly evident in the presence of the cysteine proteinase inhibitor leupeptin, but almost no small intermediates were detected. 5. The results are best explained by the degradation of NEM-aldolase being predominantly a lysosomal process, with cysteine proteinases involved in early proteolytic steps and other proteinases that have acid pH optima required for the complete catabolism of small intermediates.

Adenine↗

PubMed1994

Receptor-mediated Mn2+ influx in rat hepatocytes: comparison of cells loaded with Fura-2 ester and cells microinjected with Fura-2 salt.

In single Fura-2 ester-loaded hepatocytes, stimulation by vasopressin, but not emptying of the agonist-sensitive Ca2+ store by 2,5-di-(t-butyl)hydroquinone, resulted in an increase in the rate of Fura-2 fluorescence-quenching by Mn2+. Similarly, in cells microinjected with Fura-2 salt, vasopressin stimulated Mn2+ entry while 2,5-di-(t-butyl)hydroquinone or thapsigargin did not. The pattern of Fura-2 quenching by Mn2+ only correlated with the movement of Mn2+ across the plasma membrane.

Animals↗

PubMed1999

Dissociation between force and [Ca2+]i during extra systoles in guinea-pig ventricular muscle microinjected with fura-2.

Thin trabeculae were dissected from the right ventricle of guinea-pig heart and stimulated to contract isometrically at 0.5 Hz (26 degrees C). Rapid and transient changes of force were obtained by inducing three extra systoles (ES1-3) at 450-ms intervals. The two regular contractions (P1-2) following (ES1-3) were potentiated. Fura-2 salt was microinjected into the preparation to monitor intracellular calcium ([Ca2+]i). Three distinct phases of [Ca2+]i were seen: (1) a rapid rising phase to about 200 nmol L(-1), (2) a slower rising phase to a peak at 400 nmol L(-1), and (3) a slow decline to about 50 nmol L(-1). During ES1, there was a discrepancy between force, which decreased, and peak [Ca2+]i, which increased to 600 nmol L(-1). It is likely that the increased [Ca2+]i during the extra systoles reflects increased sarcolemmal calcium inflow, causing post-extra-systolic potentiation. Ryanodine (1-2 microM) was added to inhibit the intracellular calcium release and thus reduce the intracellular [Ca2+]i gradients following excitation. Ryanodine inhibited phase 1 of [Ca2+]i and abolished post-extra-systolic potentiation. There was a close relationship between dF/dt and [Ca2+]i with ryanodine during control and ES1-3. It is likely that fura-2 reports a spatially averaged [Ca2+]i and that phase 1 of the signal therefore apparently underestimates activator calcium in the close vicinity of the contractile elements.

Animals↗

PubMed2000

[Ca2+]i following extrasystoles in guinea-pig trabeculae microinjected with fluo-3 - a comparison with frog skeletal muscle fibres.

Force production of cardiac muscle is highly dependent on the interval between the excitations. The aim was to investigate relations between intracellular calcium ([Ca2+]i) and force when a stimulus protocol, with three extrasystoles (ESs) at various intervals, was used. The relation between [Ca2+]i and force was compared with that in frog skeletal muscle fibre. Fluo-3 was microinjected into thin cardiac trabeculae to monitor [Ca2+]i. During steady-state [Ca2+]i consisted of a rapid rise (phase 1) that lasted until peak dF/dt (rate of force development) and was followed by a slower rise (phase 2) that coincided with the action potential and had a peak after peak force. The decline in [Ca2+]i outlasted the duration of the contraction. As the ES intervals were prolonged, there was a gradual restitution of force and of the amplitude and rate of rise of phase 1 [Ca2+]i. Peak dF/dt was linearly related to the amplitude of phase 1 [Ca2+]i during restitution and potentiation of force. Skeletal muscle fibres were loaded with fluo-3-AM. From [Ca2+]i the amount of calcium bound to troponin ([Ca-T]) as a function of time was estimated. Force production of the skeletal muscle fibre could be predicted from [Ca-T] when the signal was delayed (time constant 36 ms). This finding indicates that the recorded [Ca2+]i in skeletal muscle represents activator calcium. In cardiac muscle probably only phase 1 [Ca2+]i represents activator calcium. Phase 2 [Ca2+]i probably represents calcium entry during the action potential and does not activate the contractile system to any significant extent.

Action Potentials↗

PubMed2000

Transinfection of Wolbachia in the mediterranean flour moth, Ephestia kuehniella, by embryonic microinjection.

Wolbachia are maternally transmitted intracellular bacteria found in many arthropod species. They cause a reproductive incompatibility called cytoplasmic incompatibility (CI) in several hosts, including the Mediterranean flour moth, Ephestia kuehniella. Two strains of E. kuehniella, one from Yokohama city and the other from Tsuchiura city, express different levels of CI: the Yokohama strain expresses CI at a higher level than the Tsuchiura strain. In order to determine whether the difference of CI levels depends on Wolbachia or the host, we performed transinfection experiments in E. kuehniella by means of embryonic microinjection, and successfully transferred Wolbachia carried by the Yokohama strain into the Tsuchiura strain, from which the original Wolbachia had been removed by tetracycline treatment. The resulting transinfected strain expressed CI at a level near that of the Yokohama strain, suggesting that, in these strains of E. kuehniella, the level of CI is determined by Wolbachia rather than by the host.

Animals↗

PubMed1999

Carbachol microinjection into the caudal peribrachial area induces long-term enhancement of PGO wave activity but not REM sleep.

This study presents new findings of carbachol-induced long-term ponto-geniculo-occipital (PGO) enhancement lasting five days, but without REM sleep enhancement. A quantitative analysis of the number and types of bilateral PGO wave events during slow wave sleep with PGO activity (SP) and REM was performed in each of four cats over a period of six days following a single unilateral microinjection of carbachol nanospheres into the caudolateral peribrachial area. The results demonstrate increases in the summed total of all PGO wave events to continue for five days postcarbachol reaching a peak sixfold increase on day three in SP and REM. The tendency of PGO waves to occur in clusters of greater than three waves increased sevenfold on day three in SP and fourfold during REM. These findings indicate a dissociation of long-term PGO enhancement from long-term REM enhancement, and suggest that even a sixfold increase in PGO activity alone is not, in itself, sufficient to produce the cholinergic orchestration of REM sleep enhancement.

Animals↗

PubMed1997

Developmental fate of single embryonic stem cells microinjected into 8-cell-stage mouse embryos.

Embryonic stem (ES) cells are pluripotent and capable of differentiating into somatic as well as germ cell lineages when conjoined with blastomeres of early mouse embryos. However, the developmental potential of single ES cells has not been fully investigated. We injected single murine ES cells (A3-1 cell line) of 129 origin into 8-cell mouse embryos (B6xBDF1) and examined the patterns of distribution of ES-cell-derived cells in the blastocysts as well as in the fully grown chimeric mice. The ES cells underwent 1-2 cycles of mitosis between the 8-cell and the blastocyst stage when they were introduced as single cells, whereas those introduced as groups of 2-5 cells did not proliferate during the same period of development. The ES cells and their daughter cells were predominantly incorporated into the ICM. From the 63 8-cell embryos which received single ES cells microinjected into the perivitelline space, 24 newborns were obtained, and 4 (2 fertile males, 1 sterile female and 1 hermaphrodite) of them (16.6%) were chimeric. The test breeding studies revealed that all the progeny of the two chimeric males were derived from spermatozoa of 129 genotype. The relative contribution of the host-derived and the ES-cell-derived cells in different tissues of the chimeric mice was assessed by PCR analyses of the microsatellite polymorphism of genomic DNA extracted from the tissues. In two male germ line chimeras, the testes, the kidneys and the dorsal skeletal muscles exhibited exceptionally high 129 contents. Our results demonstrated that single ES cells which maintain totipotency or pluripotency of high degree are present in a colony of ES cells, and that single ES cells conjoined with the blastomeres of 8-cell-stage embryos may colonize, if the circumstances allow, almost exclusively the germ cells and concomitantly the urogenital cell lineages. Possible correlation between the allocation of the germ line and the urogenital lineages is discussed.

Animals↗

PubMed2001

Activation of the maternally preset program of apoptosis by microinjection of 5-aza-2'-deoxycytidine and 5-methyl-2'-deoxycytidine-5'-triphosphate in Xenopus laevis embryos.

The present study examines the effects on embryogenesis of microinjecting Xenopus laevis fertilized eggs with 5-aza-2'-deoxycytidine (5-Aza-CdR), which induces hypomethylation of DNA, and 5-methyl-2'- deoxycytidine-5'-triphosphate (5-methyl-dCTP), which induces hypermethylation of DNA. Embryos injected with either one of these analogs cleaved normally until the mid-blastula stage, but underwent massive cell dissociation and stopped development at the early gastrula stage. Dissociated cells that appeared here were positive by terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick end-labeling and contained fragmented nuclei with condensed chromatin. The DNA from these cells formed a "ladder" on electrophoresis. Furthermore, the induction of cell dissociation by 5-Aza-CdR and 5-methyl-dCTP was postponed by 2-3 h by co-injection of Bcl-2 mRNA and the normal metabolite (CdR and dCTP, respectively). Using a specific antibody against 5-methyl-cytosine, we confirmed that 5-Aza-CdR induces hypomethylation, whereas 5-methyl-dCTP induces hypermethylation in X. laevis embryos before the onset of cell dissociation. Incorporation of radioactive precursors revealed that synthesis of DNA, and also RNA, is inhibited significantly in both 5-Aza-CdR-injected and 5-methyl-dCTP-injected embryos. These results show that 5-Aza-CdR and 5-methyl-dCTP are incorporated into DNA and induce apoptosis, probably through alteration of DNA methylation coupled with inhibition of DNA replication and/or transcription.

Animals↗

PubMed2002

Behavioral, morphologic, and electroencephalographic evaluation of seizures induced by intrahippocampal microinjection of pilocarpine.

PURPOSE: We studied, by means of video-EEG and neo-Timm histochemistry, the behavioral, electrophysiologic, and structural characteristics of seizures induced by intrahippocampal microinjection of pilocarpine (HIP-PILO), a selective model of temporal lobe epilepsy (TLE). METHODS: We investigated the behavioral and electrophysiologic (hippocampus and amygdala EEG) evaluation of status epilepticus (SE) induced by HIP-PILO and the consequent spontaneous recurrent seizures (SRSs). We evaluated hippocampal structural rearrangements after SE by means of neo-Timm staining. RESULTS: HIP-PILO induced SE in 17 (71%) of 24 animals. Although three animals displayed spontaneous remission of SE (not used in analysis) before the established SE duration (90 min), none of those undergoing SE died. Of SE animals, 10 (71%) of 14 had SRSs. All animals with SE had clear-cut mossy fiber sprouting (MFS) in the inner molecular layer of the dentate gyrus and epileptiform activity in hippocampus and amygdala. CONCLUSIONS: HIP-PILO rats displayed SE, SRS, MFS, and limbic epileptiform activity, without animal loss after SE. Thus, our data support this as a selective and efficient model of TLE.

Animals↗

PubMed1999

Assessment and validation of a microinjection method for kinetic analysis of [Ca2+]i in individual cells undergoing apoptosis.

Normal and malignant epithelial cells are induced to undergo apoptosis by a large variety of mechanistically diverse agents. Regardless of inducing agent, apoptosis characteristically occurs asynchronously within a population of epithelial cells over a period of 12-96 h and is associated with permeability and enzymatic perturbations. Pre-loading of cells with acetoxymethyl esters (AM) derivatives of fluorescent Ca2+ indicators (i.e. fura-2, indo-1, fluo-3) by passive diffusion allows longitudinal kinetic analysis of acute [Ca2+] changes subsequent to exposure to apoptosis inducing agents. Using prostate cancer cell lines, however, it is demonstrated that dye leakage and compartmentalization into organelles limit such passive loading to longitudinal [Ca2+]i measurements of < 2 h. Post-loading of cells exposed to the apoptosis inducing agent for several hours is also inaccurate owing to decreased loading efficiency and de-esterification of the probes resulting in increased production of fluorescent Ca(2+)-insensitive dye species. To accurately measure kinetics of [Ca2+]i changes longitudinally in individual cells undergoing apoptosis, cells were microinjected with fura dextran and maintained in a physiologic environment. [Ca2+] and morphological changes characteristic of apoptosis were then followed simultaneously in individual cells over several days following exposure to the apoptosis inducing agent.

Animals↗

PubMedDate not supplied

Inhibition of nociceptive withdrawal reflex by microinjection of interleukin 2 into rat locus coeruleus.

This study was to examine the effects of microinjection of human recombinant interleukin 2 (IL-2) into locus coeruleus (LC) on spinal nociception. Following application of IL-2 (0.1 microl, 10 pM) into LC, the percentage of inhibition of nociceptive C responses of reflex at 3, 9, 15, 21 and 27 min after injection were 88.2 +/-9.4%, 84.0 +/- 11.8%, 89.7 +/- 10.5%, 57.1 +/- 8.7% and 26.3 +/- 12.2%, respectively. Also, the expression of Fos protein in superficial dorsal horn was reduced by 73.01 +/- 13.58% of control (P<0.0001). Naloxone (10 microg, i.p.) completely blocked the IL-2-induced inhibition of C responses. The results clearly show that IL-2 receptors present in LC mediate descending inhibition of the spinal nociception, which may couple with the activation of opioid receptors on LC neurons.

Animals↗

PubMed1988

Influence of microinjection of corticosterone into ventromedial hypothalamus on hepatic acetate metabolism in rabbits.

Corticosterone was injected into the ventromedial hypothalamus (VMH) of rabbits, and changes in hepatic acetate metabolism were studied. The microinjection of corticosterone with seasame oil into the VMH of intact rabbits increased the rates of 14C transfer from 14C-1-acetate into CO2, glucose, ketone bodies, triglyceride, free cholesterol, free fatty acids and phospholipids but decreased those of 14C transfer into cholesterol ester. However, corticosterone injected into the VMH of rabbits with VMH lesions or the parietal cortex of intact rabbits was without effect on the hepatic acetate metabolism. From these results it might be suggested that the VMH is an integral part of the corticosterone-sensitive brain regulator system in the hepatic acetate metabolism.

Acetates↗

PubMed1989

Production of transgenic merino sheep by microinjection of ovine metallothionein-ovine growth hormone fusion genes.

Seven transgenic Merino sheep have been produced by the technique of pronuclear microinjection. Two different Sheep Metallothionein-1a-Sheep Growth Hormone fusion genes were used. Four of the transgenic sheep, all of which contained the gene MTsGH5, did not express the transgene. The remaining three sheep carrying the second fusion gene, MTsGH9, expressed the gene at high levels in a variety of tissues and had elevated blood levels of sheep growth hormone.

Animals↗

PubMed1994

Early sperm-egg interaction after sperm microinjection.

The early events of sperm-egg interaction occurring 1-3 h after multiple sperm injection into the perivitelline space (PVS) and after direct injection into the ooplasm of human oocytes are reported. The sperm acrosome reaction occurred in the PVS but was not detected within the ooplasm. Sperm in the PVS were incorporated into the ooplasm in the usual manner described in vitro, after completion of the acrosome reaction, and a block to polyspermy was evident at the oolemma. However, sperm incorporation into the ooplasm was not clearly defined and requires further investigation. Sperm were also incorporated into oolemma-bound vacuoles within the ooplasm and breaches in the ooplasm were seen after intracytoplasmic injection. Both normal and abnormal sperm were found in the PVS and ooplasm, even though sperm from donors with normal semen parameters were used. The effect of freezing in liquid nitrogen on demembranation of sperm for microinjection is also reported.

Acrosome↗

PubMed2000

High-frequency intrachromosomal gene conversion induced by triplex-forming oligonucleotides microinjected into mouse cells.

To test the ability of triple helix-forming oligonucleotides (TFOs) to promote recombination within chromosomal sites in mammalian cells, a mouse LTK(-) cell line was established carrying two mutant copies of the herpes simplex virus thymidine kinase (TK) gene as direct repeats in a single chromosomal locus. Recombination between these repeats can produce a functional TK gene and occurs at a spontaneous frequency of 4 x 10(-6) under standard culture conditions. When cells were microinjected with TFOs designed to bind to a 30-bp polypurine site situated between the two TK genes, recombination was observed at frequencies in the range of 1%, 2,500-fold above the background. Recombination was induced efficiently by injection of both psoralen-conjugated TFOs (followed by long-wave UVA light; 1. 2%) and unconjugated TFOs alone (1.0%). Control oligomers of scrambled sequence but identical base composition were ineffective, and no TFO-induced recombination was seen in a control LTK(-) cell line carrying an otherwise identical dual TK gene construct lacking the 30-bp polypurine target site. TFOs transfected with cationic lipids also induced recombinants in a highly sequence-specific manner but were less effective, with induced recombination frequencies of 6- to 7-fold over background. Examination of the TFO-induced recombinants by genomic Southern blotting revealed gene conversion events in which both TK genes were retained, but either the upstream (57%) or the downstream gene (43%) was corrected to wild type. These results suggest that, with efficient intracellular delivery, TFOs may be effective tools to promote site-specific recombination and targeted modification of chromosomal loci.

Animals↗