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PubMed1983

Transformation by iontophoretic microinjection of DNA: multiple integrations without tandem insertions.

DNA transformations of mouse tissue culture cells and mouse embryos were carried out by iontophoretic microinjection of DNA. Iontophoresis involves the use of an externally applied electric current to expel DNA molecules from the injection micropipette into the impaled cell (microelectrophoresis). Restriction analysis of transformants obtained by using this procedure demonstrated that this method gave strikingly different results from those previously obtained by others with transformants generated by pressure injection. First, tandem insertions were never observed in any of our transformants. Second, despite the lack of tandem insertions, we have obtained transformants which have integrated many copies of the injected DNA sequences, probably via a comparably large number of independent integration events. And third, in one transformant, an injected BamHI restriction fragment was found to have been integrated in its entirety; this included the preservation of the terminal BamHI recognition sequence. Based on these observations, we discuss the potential usefulness of iontophoretic DNA microinjections in DNA transformation studies that are focused either on the analysis of the regulation of gene expression or on the targeting of DNA sequences into the eucaryotic cell genome.

Animals↗

PubMed1983

Regulation of adenovirus transcription by an E1a gene in microinjected Xenopus laevis oocytes.

The regulation of adenovirus type 5 gene expression by the E1a gene product was examined in microinjected Xenopus laevis oocytes. Chimeric genes were constructed which included the promoter region of early adenovirus type 5 gene 3 and the structural sequence which codes for the bacterial enzyme chloramphenicol-3-O-acetyltransferase (CAT). A plasmid containing this chimeric gene as well as plasmids containing the E1a gene were coinjected into oocyte nuclei. The presence of the E1a gene was shown to increase CAT activity by up to 8.5-fold over basal levels. Synthesis of the functional product from the E1a gene requires the removal of intron sequences by RNA splicing. The E1a gene and a derivative that precisely lacks the intron were equally effective in increasing CAT activity, suggesting that splicing of the primary E1a transcript is efficiently accomplished in the oocyte nucleus. This was confirmed by directly examining the E1a mRNAs by the S1 mapping procedure. A protein extract from adenovirus type 5-infected HeLa cells enriched for the E1a protein may supplant the E1a plasmid in enhancing CAT activity. Synthesis of the CAT enzyme after gene injection is invariant in oocytes from the same frog, but oocytes from different frogs show a high degree of variability in their ability to synthesize the CAT enzyme. Microinjected X. laevis oocytes appear to be an extremely useful system to study the effects of protein elements on transcription.

Acetyltransferases↗

PubMed1984

Role of the p53 protein in cell proliferation as studied by microinjection of monoclonal antibodies.

Two monoclonal antibodies against the p53 protein, PAb 122 and 200-47, were microinjected into mammalian cells as a probe to determine the role of the p53 protein in cell proliferation. PAb 122 recognizes the p53 proteins of mouse and human cells but not of hamster cells, whereas 200-47 recognizes the p53 proteins of mouse and hamster cells but not of human cells. The ability of these antibodies to inhibit serum-stimulated DNA synthesis of cells in culture correlates with their ability to recognize the species-specific antigenic determinants. More important, however, is the observation that microinjected PAb 122 inhibits the transition of Swiss 3T3 cells from G0 to S phase, but has no effect on the progression of these cells from mitosis to the S phase.

Acridine Orange↗

PubMed1985

Promoter domains required for expression of plasmid-borne copies of the herpes simplex virus thymidine kinase gene in virus-infected mouse fibroblasts and microinjected frog oocytes.

A transient expression assay was used to measure the relative template activities of mutated tk genes in mouse L cells induced in trans by herpes simplex virus (HSV). In this assay, expression of the wild-type HSV type 1 tk gene is induced at least 200-fold by the superinfecting virus. Genetic lesions that were assayed include 5' deletions, clustered base substitutions, single base substitutions, intrapromoter inversions, and intrapromoter recombinants with the HSV type 2 tk gene. Roughly half of the mutations that were tested were found to weaken tk expression efficiency, and the remaining mutations did not alter expression. The spatial distribution of mutations that reduce expression efficiency in trans-induced mouse fibroblasts facilitated the construction of a map of promoter domains. The most gene-proximal promoter domain is located between 16 and 32 base pairs (bp) upstream of the tk mRNA cap site and contains a TATA homology. Two more distally located promoter domains were mapped to discrete locations upstream from the TATA homology. One of these distal domains is located between 47 and 79 bp upstream from the mRNA cap site, and the other is located between 84 and 105 bp upstream from the tk gene. The boundaries of these three promoter domains, with one exception, coincided with the set of domains delineated previously in a frog oocyte microinjection assay. The concordant behavior of tk promoter mutants in microinjected frog oocytes and trans-induced mouse fibroblasts leads us to propose that recognition and activation of the HSV tk promoter is mediated by cellular transcription factors that are common to frogs and mice.

Animals↗

PubMed1986

Sequences involved in accurate and efficient transcription of human c-myc genes microinjected into frog oocytes.

By microinjecting a series of deletion mutant constructs into Xenopus laevis oocytes, transcriptional control regions, two promoters, of the human c-myc gene were defined. In the case of the first promoter, sequences between -60 and -37 relative to the transcription start site contained an element essential for promoter activity. In the case of the second promoter, sequences between -66 and -56 relative to the initiation site appeared to be involved in accurate and efficient transcription. In both cases, the region identified as the essential promoter element contained GGGCGG or GGCGGG,GC box-like sequences, suggesting that c-myc gene promoter activity may be controlled by transcription factor Sp1 binding in the microinjected oocytes.

Animals↗

PubMed1986

Upstream domains of the Xenopus laevis rDNA promoter are revealed in microinjected oocytes.

The DNA sequences involved in promoting transcription of the Xenopus laevis rRNA genes were determined by microinjecting a series of deletion mutants into oocyte nuclei. A very small promoter region is sufficient to direct efficient transcription when templates are microinjected at high rDNA concentration, since 5'delta- 9 and 3'delta +6 templates are fully active. However, as the concentration of injected template is decreased, an increasing requirement for upstream domains, extending to nucleotide approximately -170, is observed. The major downstream border of the required region does not change. This apparently expanding 5' promoter border results from the fact that, as the rDNA concentration is decreased, transcription from templates lacking the upstream promoter domain falls off much more sharply than does transcription from a complete promoter. In fact, the deleted promoters are virtually inactive below a threshold rDNA concentration. It is indeed the rDNA concentration that is important, for coinjected vector DNA does not increase the level of transcription obtained from low concentrations of the 5' deletions. From these data we conclude that polymerase I transcription factors can recognize and initiate transcription from a small core promoter domain, but that sequences extending upstream to nucleotide approximately -170 increase the efficiency of initiation. A model is presented that could account for these results.

Animals↗

PubMed1987

Induction of neurite formation in PC12 cells by microinjection of proto-oncogenic Ha-ras protein preincubated with guanosine-5'-O-(3-thiotriphosphate).

Rat pheochromocytoma (PC12) cells differentiate to neuronal cells in response to nerve growth factor. It has been shown that microinjection of oncogenic but not proto-oncogenic p21 protein induces morphological differentiation in PC12 cells (D. Bar-Sagi and J. R. Feramisco, Cell 42:841-848, 1985). In this paper we describe a recombinant human proto-oncogenic Ha-ras protein which can effectively induce neurite extension of PC12 cells when microinjected as a complex with guanosine-5'-O-(3-thiotriphosphate). The protein was found to be less effective when complexed with GTP. On the other hand, an oncogenic ras protein coinjected with guanosine-5'-O-(2-thiodiphosphate) was entirely inactive. These results indicate that the binary p21-GTP complex, but not the p21-GDP complex, is effective in inducing differentiation in PC12 cells, irrespective of the oncogenic or the proto-oncogenic protein.

Adrenal Gland Neoplasms↗

PubMed1988

Microinjection of fos-specific antibodies blocks DNA synthesis in fibroblast cells.

Transcription of the protooncogene c-fos is increased greater than 10-fold within minutes of treatment of fibroblasts with serum or purified growth factors. Recent experiments with mouse 3T3 cell lines containing inducible fos antisense RNA constructs have shown that induced fos antisense RNA transcripts cause either a marked inhibition of growth in continuously proliferating cells or, conversely, a minimal effect except during the transition from a quiescent (G0) state into the cell cycle. Since intracellular production of large amounts of antisense RNA does not completely block gene expression, we microinjected affinity-purified antibodies raised against fos to determine whether and when during the cell cycle c-fos expression was required for cell proliferation. Using this independent method, we found that microinjected fos antibodies efficiently blocked serum-stimulated DNA synthesis when injected up to 6 to 8 h after serum stimulation of quiescent REF-52 fibroblasts. Furthermore, when fos antibodies were injected into asynchronously growing cells, a consistently greater number of cells was prevented from synthesizing DNA than when cells were injected with nonspecific immunoglobulins. Thus, whereas the activity of c-fos may be necessary for transition of fibroblasts from G0 to G1 of the cell cycle, its function is also required during the early G1 portion of the cell cycle to allow subsequent DNA synthesis.

Animals↗

PubMed1996

An improved microinjection protocol for the transfer of Wolbachia pipientis between infected and uninfected strains of the flour beetle Tribolium confusum.

The lethality of halocarbon and other oils to hatching larvae of the flour beetle Tribolium confusum limits existing microinjection protocols, because postinjection survivorship is only 5.6% of the eggs injected. We report the development of an oil-free protocol that improves survivorship fivefold. We used this protocol to transfect the cytoplasmic endosymbiont Wolbachia pipientis from infected donor eggs to uninfected host eggs and observed reproductive incompatibility in 40% of the surviving, injected eggs. Compared with mock injected controls (35.9% survival) or microinjection of uninfected cytoplasm (32.3% survival), injection of Wolbachia-infected cytoplasm into host eggs significantly reduced egg-to-adult survival (25.1%).

Animals↗

PubMedDate not supplied

Microinjection into crane-fly spermatocytes.

We were successful in microinjecting fluorescently labelled material into crane-fly spermatocytes. In our experiments, we obtained four results. (i) In most attempts, the membrane stretched around the micropipette and prevented entry of fluorescent material, even when the micropipette appeared to be pushed completely through the cell. This confirms suppositions from earlier micromanipulation experiments that the elastic membrane prevents the micropipette needle from entering the cell. (ii) In some attempts, cells lysed upon contact with the micropipette. (iii) In other attempts, we successfully injected fluorescent material into cells. (iv) Fluorescent material left the cells after injection, often passing into adjacent cells. Although our success rate is low, microinjection into crane-fly spermatocytes is indeed possible.

Animals↗

PubMed2004

Bradykinin microinjection in the paratrigeminal nucleus triggers neuronal discharge in the rat rostroventrolateral reticular nucleus.

A small collection of neurons in the dorsal lateral medulla, the paratrigeminal nucleus (Pa5), projects directly to the rostroventrolateral reticular nucleus (RVL). Bradykinin (BK) microinjections in the Pa5 produce marked pressor responses. Also, the Pa5 is believed to be a component of the neuronal substrates of the somatosensory response and the baroreflex arc. Considering the developing interest in the functional physiology of the Pa5, the present study was designed to characterize RVL neuronal activity in response to BK microinjections in the Pa5 as well as to phenylephrine-induced blood pressure increases in freely behaving rats. Of the 46 discriminated RVL neurons, 82% responded with a 180% mean increase in firing rate after BK application to the paratrigeminal nucleus, before the onset of the blood pressure increase. Thirty (79%) of the RVL BK-excited neurons were baroreceptor-inhibited units that responded with a 30% decrease in firing rate in response to a phenylephrine-produced increase of blood pressure. Twenty-seven (71%) units of the latter population displayed cardiac-cycle-locked rhythmic activity. The findings demonstrate a BK-stimulated functional connection between the Pa5 and RVL that may represent the neural pathway in the BK-mediated pressor response. This pathway may be relevant to baroreflex mechanisms since it relates to cardiovascular pressure-sensitive neurons.

Animals↗

PubMed2006

Microinjection of exogenous somatostatin in the dorsal vagal complex inhibits pancreatic secretion via somatostatin receptor-2 in rats.

Previous studies have suggested that somatostatin inhibits pancreatic secretion at a central vagal site, and the dorsal vagal complex (DVC) is involved in central feedback inhibition of the exocrine pancreas. The aim of this study was to investigate the effect of exogenous somatostatin in the DVC on pancreatic secretion and the somatostatin receptor subtype(s) responsible for the effect. The effects of somatostatin microinjected into the DVC on pancreatic secretion stimulated by cholecystokinin octapeptide (CCK-8) or 2-deoxy-d-glucose (2-DG) were examined in anesthetized rats. To investigate the somatostatin inhibitory action site, a somatostatin receptor antagonist [SRA; cyclo(7-aminoheptanoyl-Phe-d-Trp-Lys-Thr)] was microinjected into the DVC before intravenous infusion of somatostatin and CCK-8/2-DG. The effects of injection of a somatostatin receptor-2 agonist (seglitide) and combined injection of somatostatin and a somatostatin receptor-2 antagonist (CYN 154806) in the DVC on the pancreatic secretion were also investigated. Somatostatin injected into the DVC significantly inhibited pancreatic secretion evoked by CCK-8 or 2-DG in a dose-dependent manner. SRA injected into the DVC completely reversed the inhibitory effect of intravenous administration of somatostatin. Seglitide injected into the DVC also inhibited CCK-8/2-DG-induced pancreatic protein secretion. However, combined injection of somatostatin and CYN 154806 did not affect the CCK-8/2-DG-induced pancreatic secretion. Somatostatin in the DVC inhibits pancreatic secretion via somatostatin receptor-2, and the DVC is the action site of somatostatin for its inhibitory effect.

Animals↗

PubMed2003

Cardiovascular responses to substance P in the nucleus tractus solitarii: microinjection study in conscious rats.

The cardiovascular effects of substance P (SP) microinjections in the nucleus tractus solitarii (NTS) were evaluated in conscious rats. We chose this model because it is an effective way to access some of the cardiovascular effects of neurotransmitters in the NTS without the inconvenience of blunting pathways with anesthetic agents or removing forebrain projections by decerebration. The cardiovascular responses to SP injections were also evaluated after chronic nodose ganglionectomy. We found that, in conscious rats, SP microinjections into the NTS induced hypertension and tachycardia. Unilateral and bilateral SP injections into the NTS caused a slow increase in blood pressure and heart rate that peaked 1.5-5 min after injection and lasted for 20-30 min. Nodose ganglionectomy increased the duration of the pressor and tachycardic effects of SP and enhanced the pressor response. These data show that SP in the NTS is involved in pressor pathways. The supersensitivity to SP seen after nodose ganglionectomy suggests that vagal afferent projections are involved in those pressor pathways activated by SP in the NTS.

Animals↗

PubMed2001

Hemodynamic effects elicited by microinjection of glutamatergic agonists into NTS of conscious rats.

In this study, we characterized the arterial pressure, heart rate, and regional vascular conductance responses elicited by unilateral microinjection of ionotropic glutamatergic agonists N-methyl-D-aspartic acid (NMDA and non-NMDA) into the nucleus of tractus solitarius (NTS) of conscious rats. Microinjections of NMDA and S-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) caused changes in mean arterial pressure (MAP). Lower doses elicited decreases in MAP, whereas higher doses elicited biphasic responses (decreases followed by increases). Both agonists induced bradycardia and elicited dose-dependent vasoconstriction in the renal, mesenteric, and hindquarter beds. AMPA elicited delayed vasodilation in the hindquarter bed but NMDA did not. Bradycardia and initial hypotension produced by each agonist were abolished by systemic administration of the muscarinic antagonist methylatropine. However, methylatropine did not affect either the vasoconstriction or the vasodilatation. The contrasting hemodynamic effects produced by NMDA and AMPA could be caused by activation of differential subsets of NTS neurons. Preferential activation of one subset could produce the NMDA-related responses, whereas activation of another subset would elicit AMPA-related responses.

Animals↗

PubMed1988

Cardiovascular responses to microinjection of ANF into dorsal medulla of rats.

Atrial natriuretic factor (ANF) has been suggested as a putative neurotransmitter in central pathways involved in the control of the cardiovascular system. To investigate this possibility, 50 nl of 10(-7) M ANF were microinjected into discrete sites in the nucleus of the tractus solitarius (NTS) where baro- and chemoreceptor afferents terminate. Injections into 36 of a total of 66 sites in the NTS of paralyzed artificially ventilated Wistar rats under urethan anesthesia were found to produce a significant decline in heart rate [HR; -9.2 +/- 2.9 (SE) beats/min, P less than 0.05] and mean arterial pressure [MAP; -11.1 +/- 1.2 (SE) mmHg, P less than 0.01]. Similar responses were also present in anesthetized animals breathing spontaneously. Microinjection of an inactive peptide analogue or of saline did not produce cardiovascular changes. It was also found that ANF injection into the cuneate nucleus (20 of 38 sites) and the spinal trigeminal complex (28 of 42 sites) produced a decrease in MAP and HR that were of the same magnitude as those seen in the NTS. Injections of ANF into the medial longitudinal fasciculus (n = 22), hypoglossal nucleus (n = 9), area postrema (n = 16), and dorsal motor nucleus of the vagus (n = 11) did not change HR or MAP. These results suggest that ANF may serve as a neurotransmitter involved in cardiovascular reflexes mediated by specific nuclei in the dorsal medulla.

Afferent Pathways↗

PubMed1994

Mechanisms of pressor and bradycardic responses to L-glutamate microinjected into the NTS of conscious rats.

Microinjection of increasing doses of L-glutamate (L-Glu, 0.03-5.0 nmol/100 nl) into the nucleus tractus solitarii (NTS) produced a dose-related pressor and bradycardic response. Prazosin virtually abolished the pressor response but produced no changes in the bradycardic response to L-Glu, indicating that bradycardia is not reflex in origin. The bradycardic response was blocked by atropine. In three different groups of rats, excitatory amino acid receptors in the NTS were blocked by increasing doses of kynurenic acid (0.5, 2.0, and 10.0 nmol/100 nl) and the pressor and bradycardic responses to L-Glu (1 nmol/100 nl) were reduced in a dose-related pattern. Reflex bradycardia induced by an increase in pressure caused by phenylephrine (iv) was also blocked by kynurenic acid. These data show that microinjection of L-Glu into the NTS of conscious rats produced pressor and bradycardic responses, which are due to the activation of two independent autonomic pathways. The data also indicate that the activation of both pathways is mediated by excitatory amino acid receptors. Considering that reflex bradycardia was also blocked by kynurenic acid, we suggest that L-Glu and excitatory amino acid receptors are part of the parasympathetic limb of the baroreceptor reflex. The pressor response to L-Glu is also mediated by excitatory amino acid receptors, but its physiological meaning is still unclear.

Animals↗

PubMed1995

Effects on breathing of medullary bicuculline microinjections in immature opossums.

The role of medullary gamma-aminobutyric acid A (GABAA)-receptor activation in the expression of breathing was studied in suckling opossums (Didelphis virginiana) from 4 to 8 wk of age. Animals were anesthetized with a thiobarbiturate, and the ventral medulla was exposed so that drugs could be microinjected into the medulla with a two-barrel glass micropipette. The GABAA antagonist bicuculline at approximately 1-3 pmol total dose was injected in volumes < 1 nl; injections of saline with pontamine sky blue dye were used as controls and to mark location. Breathing pattern was assessed using diaphragm electromyography (EMG). Effective sites for bicuculline microinjection were obtained in the ventrolateral medulla, with both lateral reticular and inferior olive nuclei present as rostrocaudal markers. Responses among 18 tested animals included increased duration of breaths (Ttot) and duration of inspiration (Ti). Peak amplitude of the diaphragm EMG and peak amplitude of the diaphragm/Ti were not consistently affected. Transient apnea, lasting at least 8 s, occurred in five of the animals. Control solutions did not elicit respiratory responses. These results suggest that neural circuitry in the medulla, using the neurotransmitter GABA, can have an early role in the modulation of breathing pattern.

Animals↗

PubMed1998

Microinjection of a cyclooxygenase inhibitor into the anteroventral preoptic region attenuates LPS fever.

Considerable evidence supports the role of prostaglandins in fever production, but the neuroanatomic sites of prostaglandin synthesis that produce fever remain unknown. With the use of a novel microinjection technique, we injected the cyclooxygenase inhibitor ketorolac into the preoptic area (POA) to determine which preoptic regions produce the prostaglandins required for fever. Initial experiments demonstrated that intravenous ketorolac blocked the fever normally produced by lipopolysaccharide (LPS) 5 micrograms/kg i.v. Microinjection of ketorolac into the POA had no effect on body temperature, and injection of artificial cerebrospinal fluid into the POA did not alter LPS fever. Injection of ketorolac into the anteroventral POA markedly decreased the fever produced by LPS, compared with injections into more rostral, caudal, or dorsal locations. These observations indicate that prostaglandin synthesis in the anteroventral preoptic region is necessary for the production of fever.

Analgesics, Non-Narcotic↗