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Results for “Microinjection”

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At least 523 records · Page 29Linked to original sources

PubMed1997

Microinjection of anti-alpha-tubulin antibody (DM1A) inhibits progesterone-induced meiotic maturation and deranges the microtubule array in follicle-enclosed oocytes of the frog, Rana pipiens.

Microinjection of anti-alpha-tubulin (Dm1A) inhibited progesterone-induced meiotic maturation in large follicle-enclosed oocytes of the frog, Rana pipiens. DM1A (46 nl; 10 mg/ml) injection significantly increased the ED50 value for progesterone as determined by germinal vesicle dissolution (GVD) bioassay. By contrast, low doses of microinjected DM1A (46 nl; 2.5 mg/ml), anti-actin (clone KJ43A), anti-cytokeratin (C-11), anti-intermediate filament antibody (IFA), generic IgG (46 nl; 20 mg/ml) or sodium azide (46 nl; 1 mg/ml), an antibody preservative, were without inhibitory effect in this bioassay. Microinjected, affinity-purified DM1A (46 nl; 7.5 mg/ml) was also inhibitory, but preabsorption with pure tubulin prior to injection significantly reduced the inhibitory effect. DM1A injection had no effect on centrifugation-induced germinal vesicle migration (GVM). Previous work indicated that drugs (e.g. demecolcine and nocodazole), which destabilise microtubules, enhance both centrifugation-induced GVM and progesterone-induced GVD in Rana oocytes. Taking these results together, it is suggested that DM1A injection may have differential effects on microtubules in this cell. Thus, while the majority of microtubules were apparently depolymerised by DM1A (46 nl; 10 mg/ml) injection, a small subpopulation appeared to be stabilised as bundles. Confocal immunofluorescence microscopy of follicle-enclosed oocytes after DM1A injection revealed a major loss of microtubules throughout the cell; however, apparent sparse bundles of microtubules arranged in an approximately 600 microns shell were associated with the injectate region 24 h post-injection. By contrast, control follicle-enclosed oocytes topically labelled with DM1A post-fixation had extensive microtubule arrays similar to those previously reported in Xenopus oocytes. Intracellular recording after DM1A injection and progesterone treatment yielded an intermediate membrane potential (Vm = -31.8 mV) compared with control (immature) DM1A-injected cells (Vm = -44.7 mV) or potassium balanced salt solution (KBS)-injected cells matured with progesterone (Vm = -13.9 mV). These results suggest that DM1A injection does not completely inhibit electrophysiological changes initiated by progesterone. Working hypotheses are proposed that suggest a role for microtubules in the action of progesterone which normally lifts the prophase I block in the Rana follicle-enclosed oocyte.

Animals↗

PubMed2004

Effect of GnRH injection timing in the production of pronuclear-stage zygotes used for DNA microinjection.

This study was aimed at developing a hormonal treatment protocol in order to optimize the proportion of pronuclear-stage embryos to be used for DNA microinjection in a goat transgenic founder production programme. A total of 46 adult BELE and 47 adult standard goats (1-5 years old) were used as donors and recipients, respectively. They were heat-synchronized using intravaginal sponges containing 60 mg medroxyprogesterone acetate for 10 days with an injection of 125 microg cloprostenol on the morning of the eighth day. Recipients were injected with 400 IU eCG at the time of sponge removal while donors received a total of 133 mg NIH-FSH-P1 (Folltropin-V) given twice daily in decreasing doses over 3 days starting 48 h before sponge removal. Ovulation was induced in donors by injecting 100 microg of GnRH at 24 h (GnRH24) or 36 h (GnRH36) after sponge removal. Embryo recovery was performed by oviduct flushing following a standard mid-ventral laparotomy procedure. The proportion of embryos in the pronuclear stage of development was higher in the GnRH36 group (90% vs 34%, p < 0.01). Embryos were microinjected with a DNA expression cassette followed by transfer to the oviduct of synchronized recipients. A higher, yet not statistically significant, pregnancy rate was found in the recipients transferred with pronuclear-stage embryos compared with those transferred with 2-cell-stage embryos (64% vs 37%, chi-square p = 0.06). One transgenic female founder was produced from the group of recipients transferred with pronuclear-stage microinjected embryos.

Animals↗

PubMed2005

Preimplantation development and viability of in vitro cultured rabbit embryos derived from in vivo fertilized gene-microinjected eggs: apoptosis and ultrastructure analyses.

Microinjection (Mi) of gene constructs into pronuclei of fertilized eggs is a widely used method to generate transgenic animals. However, the efficiency of gene integration and expression is very low because of the low viability of reconstructed embryos resulting from cell fragmentation and cleavage arrest. As a consequence, only a few viable embryos integrate and express transgene. Since cellular fragmentation and cleavage stage arrest in embryos may be associated with apoptosis, we aimed to test the hypothesis that the low viability of Mi-derived eggs is caused by a high rate of apoptosis in embryos, as a result of the detrimental effect of Mi. Pronuclear stage eggs (19-20 hours post-coitum, hpc) were microinjected with several picolitres of DNA construct into the male pronucleus (gene-Mi); the intact eggs (non-Mi) or eggs microinjected with phosphate-buffered saline (PBS-Mi) served as controls. Epidermal growth factor (EGF; 0, 20 and 200 ng/ml) was added to the culture medium and the embryos were cultured up to 94-96 hpc. Apoptosis was detected using the TUNEL assay, and the ultrastructure was analysed using electron microscopy of Durcupan ACM thin sections of the embryo. Gene-Mi embryos had significantly lower (p < 0.05) blastocyst yields and a higher percentage of cleavage-arrested embryos than those in the non-Mi group. In gene-Mi groups, approximately 40% of all cleavage-stage-arrested embryos had fragmented blastomeres. Both gene-Mi- and PBS-Mi-derived blastocysts had a significantly higher TUNEL index (p < 0.001) and lower total cell number (p < 0.05) than the non-Mi embryos. Comparison of the quality of gene-Mi embryos with that of PBS-Mi embryos indicated that the deleterious effect of Mi on the embryo was caused by the Mi procedure itself, rather than DNA. EGF (at 20 ng/ml) had beneficial effects on the quality of gene-Mi-derived embryos, eliminating the influence of the Mi procedure on apoptosis and embryo cell number. Ultrastructural analysis confirmed a higher occurrence of apoptotic signs (nuclear membrane blebbing, areas with electron-dense material, numerous apoptotic bodies) in Mi-derived cleavage-arrested embryos compared with untreated or Mi-derived normal-looking embryos. These findings suggest an association between embryo cleavage arrest and apoptosis in Mi-derived embryos. Inclusion of EGF in the embryo culture medium can eliminate the detrimental effect of Mi on embryo quality.

Animals↗

PubMed2000

Transformation of barley by microinjection into isolated zygote protoplasts.

Barley zygote protoplasts were mechanically isolated, embedded in agarose droplets, and microinjected with a rice actin promoter Act1-gusA-nos gene construct. On average 62% of the cells survived the injection and of these 55% continued development into embryo-like structures and eventually to plants. PCR screening for the presence of a 307-bp fragment in the middle of the gusA gene showed that on average 21% of the derived structures contained this fragment. However, among the hundreds of injected zygotes, derived structures and regenerants we only found significant GUS expression in two cases (embryo-like structures nine days after injection). Two lines of green plants, derived from zygotes microinjected with linearized plasmid (line A147-1) or an isolated Act1-gusA-nos gene cassette (line A166-h) proved to be transgenic. Line A147-1 appeared to contain a single and intact copy of the expression cassette but a PCR based progeny analysis indicated the presence of additional shorter fragments of the cassette. Line A166-h appeared to contain a single fragment of the gusA gene that was transferred to the progeny as a single Mendelian trait. One additional fragment of the gusA gene was identified in this line. The present data show that transformation of barley by microinjection of DNA into isolated zygotes is feasible but also that gene expression rarely is achieved, possibly due to degradation of the introduced DNA.

Actins↗

PubMed2000

Generation of transgenic mice and germline transmission of a mammalian artificial chromosome introduced into embryos by pronuclear microinjection.

We have generated transgenic mice by pronuclear microinjection of a murine satellite DNA-based artificial chromosome (SATAC). As 50% of the founder progeny were SATAC-positive, this demonstrates that SATAC transmission through the germline had occurred. FISH analyses of metaphase chromosomes from mitogen-activated peripheral blood lymphocytes from both the founder and progeny revealed that the SATAC was maintained as a discrete chromosome and that it had not integrated into an endogenous chromosome. To our knowledge, this is the first report of the germline transmission of a genetically engineered mammalian artificial chromosome within transgenic animals generated through pronuclear microinjection. We have also shown that murine SATACs can be similarly introduced into bovine embryos. The use of embryo microinjection to generate transgenic mammals carrying genetically engineered chromosomes provides a novel method by which the unique advantages of chromosome-based gene delivery systems can be exploited.

Animals↗

PubMed2000

Study of the in vitro maturation of mouse oocytes induced by microinjection of maturation promoting factor (MPF).

PURPOSE: Maturation promoting factor (MPF) acts at the resumption of meiosis and nonspecifically throughout the animal species. There exists a considerable body of literature on MPF, but little work has been done to study the induction of maturation of mammalian oocytes by microinjection of extracted MPF. METHODS: Immature (GV-stage) mouse oocytes were microinjected MPF extracted from matured Xenopus eggs in the presence of dbcAMP. RESULTS: The rate of germinal vesicle, breakdown (GVBD) induced at 24 hr after MPF injection was significantly higher (90.5%) than that of the control (2.2%), which was injected with HTF medium containing dbcAMP (P < 0.0001). The rate of extrusion of the first polar body at 24 hr after MPF injection was significantly higher (84.1%) than that of the same control (1.1%) (P < 0.0001). CONCLUSIONS: From these results, it is concluded that the maturation of mammalian oocytes can be induced by the microinjection of MPF extracted from other species.

Animals↗

PubMed1995

Effects of a microinjection of morphine into the amygdala on the acquisition and expression of conditioned fear and hypoalgesia in rats.

A unilateral microinjection of morphine into the amygdala impaired the acquisition of fear and hypoalgesic responses in rats exposed to a heated floor in a hot-plate apparatus. This impairment was dose dependent, receptor specific, and not observed in rats microinjected with morphine into the caudal basolateral amygdala. A microinjection of morphine into the amygdala reduced the expression of fear responses and of naloxone-sensitive hypoalgesic responses, but did not reduce the expression of naloxone-insensitive hypoalgesic responses. The results document an involvement of opioidergic mechanisms in the amygdala in learned danger and of the amygdala in the control of opioid hypoalgesic responses. They also suggest that learned danger can activate antinociceptive mechanisms independently of the amygdala.

Amygdala↗

PubMedDate not supplied

Expression of a microinjected immunoglobulin gene in the spleen of transgenic mice.

Transgenic mice were produced by microinjection of a rearranged, functional immunoglobulin kappa gene into fertilized mouse eggs and implantation of the microinjected embryos into foster mothers. Mice that integrated the injected gene were mated and the DNA, RNA and serum kappa chains of their offspring were analysed. The data from offspring of three different transgenic mice indicate that the microinjected gene is expressed in the spleen, but not the liver of mice which inherited the injected gene.

Animals↗

PubMed1999

Respiratory action of capsaicin microinjected into the nucleus of the solitary tract: involvement of vanilloid and tachykinin receptors.

1. The respiratory response to microinjection of capsaicin into the commissural nucleus of the solitary tract (cNTS) of urethane-anaesthetized rats was investigated in the absence and presence of the competitive vanilloid (capsaicin) antagonist, capsazepine, and selective tachykinin NK1, NK2 and NK3 antagonists (RP 67580, SR 48968 and SR 142801, respectively). 2. Microinjection of capsaicin reduced respiratory frequency but not tidal volume (VT), leading to an overall reduction in minute ventilation (VE). The effect was dose-dependent between 0.5 and 2 nmol capsaicin. Doses greater than 2 nmol produced apnoea. Tachyphylaxis was observed following repeated injection of capsaicin (1 nmol, 30 min apart). 3. Capsazepine (1 nmol) had no effect on frequency or VT when injected alone but completely blocked the respiratory response to capsaicin (1 nmol). 4. RP 67580 (1 but not 5 nmol) alone depressed frequency and VT slightly. Moreover, RP 67580 appeared to potentiate the bradypnoeic effect of capsaicin. In contrast, SR 48968 and SR 142801 (1 and 5 nmol) alone had no significant effect on respiration. However, both agents significantly attenuated the reduction in frequency produced by capsaicin. 5. In conclusion, microinjection of capsaicin into the cNTS decreases overall ventilation, primarily by reducing frequency. The action of capsaicin appears from the data to be mediated by vanilloid receptors since it is blocked by the competitive vanilloid antagonist capsazepine and is subject to tachyphylaxis. However, since NK2 (SR 48968) and NK3 (SR 142801) receptor antagonists block the actions of capsaicin, we propose that capsaicin acts also by releasing tachykinins from central afferent terminals in the cNTS.

Anesthesia↗

PubMed1987

A putative protein-sequestration site involving intermediate filaments for protein degradation by autophagy. Studies with microinjected purified glycolytic enzymes in 3T3-L1 cells.

Several glycolytic enzymes (lactate dehydrogenase, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase) were radiolabelled by [125I]iodination, conjugation with 125I-labelled Bolton & Hunter reagent and reductive [3H]methylation, and their degradative rates after microinjection into 3T3-L1 cells compared with that of the extracellular protein bovine serum albumin. Although the albumin remains largely cytosolic in recipient cells, the glycolytic enzymes rapidly (less than 30 min) become insoluble, as measured by detergent and salt extractions. The microinjected glycolytic enzymes appear to form disulphide-linked aggregates, are found in a cell fraction rich in vimentin-containing intermediate filaments and histones (nuclear-intermediate-filament fraction), and are degraded slowly by a lysosomal mechanism, as judged by the effects of inhibitors (NH4Cl, leupeptin, 3-methyladenine). 125I-labelled bovine serum albumin appears to be degraded rapidly and non-lysosomally. Prolonged treatment (96 h) of cultured cells with leupeptin results in the accumulation of pulse-labelled ([35S]methionine for 24 h) endogenous cell proteins in the detergent-and salt-non-extractable residue, but NH4Cl and 3-methyladenine do not have this effect. The findings are in terms of the interpretation of experiments involving microinjection of proteins to study intracellular protein protein degradation by autophagy.

Autophagy↗

PubMed1994

An approach towards genetically engineered cell fate mapping in maize using the Lc gene as a visible marker: transactivation capacity of Lc vectors in differentiated maize cells and microinjection of Lc vectors into somatic embryos and shoot apical meristems.

To establish a system for genetically engineered cell fate mapping, different vectors carrying the Lc gene, a member of the R gene family, were delivered into embryonic and meristematic cells of maize by the microinjection technique. Vectors in which the Lc cDNA is driven either by a constitutive promoter (CaMV 35S), with or without the Adh1 intron 1 of maize, or a tissue-specific promoter (phosphoenolpyruvate carboxylase, PEPC) as well as self-replicating wheat dwarf virus (WDV) vectors carrying a Lc-expression-cassette, have been tested. The ability of these vectors to transactivate was evaluated in mesophyll-derived protoplasts of the maize genotype appropriate for these microinjection experiments. The expression product of the introduced Lc gene can substitute for mutated R and B loci, resulting in anthocyanin production. Analogous results were obtained by microinjection into organized tissues, where transactivation of anthocyanin biosynthesis resulted in pigmented sectors in somatic embryos (B79) and in the leaves of plants regenerated from the cultivated shoot apical meristems (K55, r-g, b). The tissue-specific appearance of pigmented sectors in leaves, using the mesophyll-specific PEPC promoter suggests the possibility of using this approach for layer-specific cell fate studies. The presence of the introduced plasmids in leaves showing red sectors 20-30 days after injection was proven by PCR analysis.

Base Sequence↗

PubMed2003

Systemic morphine-induced release of serotonin in the rostroventral medulla is not mimicked by morphine microinjection into the periaqueductal gray.

We used in vivo microdialysis in awake rats to test the hypothesis that intravenous morphine increases serotonin (5-HT) release within the rostral ventromedial medulla (RVM). We also injected morphine into various sites along the rostrocaudal extent of the periaqueductal gray (PAG), and examined the extent of its diffusion to the RVM. Intravenous morphine (3.0 mg/kg) produced thermal antinociception and increased RVM dialysate 5-HT, 5-hydroxyindole acetic acid (5-HIAA), and homovanillic acid (HVA) in a naloxone-reversible manner. As neither PAG microinjection of morphine (5 micro g/0.5 micro L) nor RVM administration of fentanyl or d-Ala(2),NMePhe(4),Gly-ol(5)]enkephalin (DAMGO) increased RVM 5-HT, we were unable to determine the precise site of action of morphine. Surprisingly, peak morphine levels in the RVM were higher after microinjection into the caudal PAG as compared to either intravenous injection or microinjection into more rostral sites within the PAG. Naloxone-precipitated withdrawal in morphine-tolerant rats not only increased extracellular 5-HT in the RVM, but also dopamine (DA) and HVA. We conclude that substantial amounts of morphine diffuse from the PAG to the RVM, and speculate that opioid receptor interactions at multiple brain sites mediate the analgesic effects of PAG morphine. Further studies will be required to elucidate the contribution of 5-HT and DA release in the RVM to opioid analgesia and opioid withdrawal.

Analgesics, Opioid↗

PubMed1994

Microinjection: choice of embryo transfer technique.

Subzonal sperm microinjection (SUZI) is indicated in severe oligoasthenozoospermia, in which the total count of motile sperm is inadequate for in vitro fertilization (IVF), and in cases with repeated failure of fertilization. Sperm for microinjection are selected following centrifugation on a Percoll gradient and stimulation with pentoxifylline and 2-deoxyadenosine. Motile sperm (2-10 per egg) are injected into the perivitelline space and fertilized oocytes are then cultured for two days prior to transfer into the Fallopian tube (tubal embryo stage transfer, TEST) or uterus. During 1992, SUZI results showed a total fertilization rate of 30% (19% were 2 pronuclear, 11% were polyspermic), a transfer rate of 55% and pregnancy rates of 15.2% per transfer and 8.3% per cycle. Recent pregnancy data in mild-moderate male factor infertility showed that gamete intrafallopian transfer (GIFT) results were consistently superior to TEST or IVF, suggesting a beneficial effect of the tubal environment on fertilization and early embryonic development. Accordingly, the combination of SUZI followed by the immediate transfer of injected oocytes into the Fallopian tube, the MIFT procedure, was explored. An initial study of 21 consecutive microinjection candidates showed a clinical pregnancy rate of 24% per cycle. Information regarding fertilization and polyspermy rates was available from supernumerary oocytes in 90% of patients. A randomized, controlled trial comparing MIFT with SUZI or TEST in severe male factor infertility is required to confirm the improved pregnancy rate in MIFT cycles.

Embryo Transfer↗

PubMed1980

Replication and expression of thymidine kinase and human globin genes microinjected into mouse fibroblasts.

A mixture of two recombinant plasmids was microinjected into mouse thymidine kinase-negative fibroblasts (L cells). One plasmid contained the thymidine kinase gene of herpes simplex virus type I and the other contained the human beta globin gene. Seven fibroblast colonies arising from injected cells incubated in hypoxanthine/aminopterin/thymidine medium were analyzed. These microinjected cells were shown to: (i) produce functionally active herpes simplex type I thymidine kinase enzyme, (ii) replicate the human beta globin gene, and (iii) produce human beta globin mRNA sequences at low levels. Thus, the genetic defect (lack of thymidine kinase activity) was corrected by the microinjected thymidine kinase gene, and a coinjected human beta globin gene was replicated and weakly expressed.

Animals↗

PubMed1983

Synthetic leader peptide modulates secretion of proteins from microinjected Xenopus oocytes.

To investigate the role of the leader peptide in modulating secretion from living cells, we injected a synthetic peptide into Xenopus oocytes. The peptide consisted of the NH2-terminal leader sequence of mouse immunoglobulin light chain precursor. We found that the leader peptide has two different roles in regulating secretion from the oocytes. First, it competitively inhibits the synthesis of secretory and membrane proteins but not of cytoplasmic proteins. The inhibition occurs both with oocyte proteins and with proteins directed by coinjected myeloma mRNA. The inhibition reaches a maximum 2 hr after injection and decays within 3 hr. It appears to be mediated through the cell membrane, because 125I-labeled leader peptide segregates into the membrane fraction of microinjected oocytes simultaneously with the interference with methionine incorporation. A second role of the microinjected leader peptide is to induce a rapid acceleration in the rate of export of secretory proteins from the oocyte. The maximal enhancement effect is obtained upon injection of 50 ng of leader peptide per oocyte. It is not merely due to the small size, negative charge, or hydrophobicity of the peptide, because enhanced secretion does not occur when glucagon, poly-L-glutamic acid, or Triton X-100 is injected. Furthermore, immunoreaction of the peptide with specific antibodies prior to microinjection prevents the accelerated export. Our observations indicate that in Xenopus oocytes, the leader peptide is involved in both translocation and later step(s) in the secretory pathway.

Animals↗

PubMed1983

Regulation of catabolism of microinjected ribonuclease A requires the amino-terminal 20 amino acids.

RNase A introduced into the cytoplasm of IMR-90 human diploid fibroblasts by erythrocyte-mediated microinjection is degraded with a half-life of approximately equal to 75 hr in the presence of fetal bovine serum. In response to serum deprivation the degradative rate of microinjected RNase A is enhanced 2-fold. RNase S protein (amino acids 21-124) is degraded with a half-life similar to that of RNase A in the presence of serum, but its catabolism is not increased during serum withdrawal. Reconstitution of RNase S protein with RNase S peptide (amino acids 1-20) restored full enzymatic activity to the S protein as well as the ability of fibroblasts to increase its catabolism during serum deprivation. Finally, RNase S peptide microinjected alone shows the full 2-fold increase in degradative rate during serum withdrawal. These results show that recognition of RNase A for enhanced breakdown during serum deprivation is based on some feature of its amino-terminal 20 amino acids. Furthermore, our results indicate that the enhanced protein catabolism during serum deprivation can be highly selective.

Amino Acid Sequence↗

PubMed1983

Immunospecific vesicle targeting facilitates microinjection into lymphocytes.

Antibody-directed targeting of vesicles to cells dramatically enhances polyethylene glycol-mediated fusion and microinjection. Sealed erythrocyte ghosts, containing fluorescent bovine serum albumin, were targeted to murine spleen and thymus cells, and to lymphocyte, monocyte, and fibroblast cell lines. In all cases, targeted cell populations showed substantial levels of microinjection, whereas populations treated with the fusogen in the absence of targeting were not significantly microinjected. To achieve attachment of vesicles to selected cells, the cells were first labeled with biotin-modified antibody then treated with sealed ghosts prepared from avidin-coupled erythrocytes. This procedure should prove useful when the injection of specific cell populations is desired, or with cell types such as lymphocytes that are difficult to fuse, or when the use of limited reagents necessitates high injection efficiencies.

Animals↗

PubMed1984

Transient complementation of xeroderma pigmentosum cells by microinjection of poly(A)+ RNA.

An assay has been developed in which excision repair deficiency of xeroderma pigmentosum cells is transiently complemented, as measured by unscheduled DNA synthesis, by microinjection of cytoplasmic poly(A)+ RNA derived from HeLa cells. Four different complementation groups of xeroderma pigmentosum have been assayed. Groups A and G showed complementation, whereas groups D and F did not. Survival for cells in each of the groups subsequent to microinjection was approximately equal to 75%. Approximately 10-25% of surviving cells from groups A and G were complemented, as judged by near-normal unscheduled DNA synthesis. Fractionation of cytoplasmic poly(A)+ RNA on a 15-30% nondenaturing sucrose gradient and subsequent microinjection of the individual fractions indicate that repair mRNAs that complement xeroderma pigmentosum groups A and G sediment at approximately 11 S and 12 S, respectively. This assay should be of great utility in the cloning and biochemical analysis of DNA repair genes.

Autoradiography↗