Lipid-assisted microinjection: introducing material into the cytosol and membranes of small cells.

The microinjection of synthetic molecules, proteins, and nucleic acids into the cytosol of living cells is a powerful technique in cell biology. However, the insertion of a glass micropipette into the cell is a potentially damaging event, which presents significant problems, especially for small mammalian cells (spherical diameter = 2-15 micron), especially if they are only loosely adherent. The current technique is therefore limited to cells that are both sufficiently large or robust and firmly attached to a substrate. We describe here a modification of the standard technique that overcomes some of the problems associated with conventional microinjection but that does not involve the insertion of a micropipette deep into the cell cytoplasm. Instead, this method depends on lipid fusion at the micropipette tip to form a continuous but temporary conductance pathway between the interiors of the micropipette and cell. This technique thus also provides a novel method of transferring lipids and lipid-associated molecules to the plasma membrane of cells.     

Biophysical aspects of reconstruction of a single cell by the methods of cell engineering

The problems of a low efficiency of mammalian cloning are discussed with emphasis on the necessity of the expertise of each step of single cell reconstruction, beginning with microsurgical manipulations. The fact of cell content leakage when the cell is held during microsurgery or microinjections with the help of the conventional method using negative pressure in the holding micropipette was demonstrated in experiments on murine embryos. It was shown that the rate of cell content efflux depends on the value of negative pressure generated in the holding micropipette, and is directly proportional to the dimensions of its orifice and the duration of micromanipulations. An alternative method of cell fixation using the capillary forces of the holding micropipette was proposed. The method optimizes the process of cell fixation, reducing the holding effort by two orders of magnitude. As a result, 92% of embryos remain viable after fixation of embryo, as compared with 39% in the conventional technique. In order to diminish the cell damage produced by the tip of a microinstrument, a new technique of fabricating micropipettes was proposed. The improved method of filling the micropipette with viscous liquids, including DNA, which is described in details in the paper, enabled constant (non-stop) microinjection of more than 1000 cells by hand, without any special automatic device.     

Purification and some properties of human erythrocyte calpastatin

By means of a new type of microinjection apparatus, which has a micropipette located in a hole through the optical axis of the condenser lens, we injected interferon (IFN) or 2',5'-oligoadenylate (2-5A) into mouse L cells, and observed their antiviral effects on the multiplication of vesicular stomatitis virus (VSV). After injection, cells were infected with VSV, and labeled with [3H]uridine in the presence of actinomycin D. The proportion of cells infected with VSV which carried radioactive virus-RNA was determined by autoradiography. IFN introduced directly into L cells had no effect on the virus growth. This result supports the idea that IFN molecules exert their effect from outside the cell membrane without penetrating into the cytoplasm. 2-5A, on the other hand, was able to inhibit the growth of VSV effectively when injected into L cells. The antiviral effect was dependent on the dose of 2-5A injected, and moreover the effect was transient, since it disappeared completely after 24-h incubation.     

A method for microinjection into subpleural alveoli of rat lung in situ

A new technique is described for the micropuncture of rat lung in the intact thorax. Under pentobarbital sodium anesthesia a glass micropipette is passed through a small area of the parietal pleura, which has been cleared of overlying intercostal muscle. The micropipette is passed into the lung parenchyma and withdrawn again without collapsing the lung. The current application of this technique is in the microinjection of fluid into subpleural alveoli, which is illustrated using a suspension of colloidal gold. The gold particles are immediately dispersed over the surface of many alveoli, a small proportion spreading laterally as far as 4-6 mm. There is no evidence of alveolar flooding.     

Rotationally oscillating drill (Ros-Drill) for mouse ICSI without using mercury

Intracytoplasmic sperm injection (ICSI) is an important assisted reproductive technology (ART). Due to deployment difficulties and low efficiency of the earlier (conventional) version of ICSI, especially in the mouse, a piezo-assisted ICSI technique had evolved as a popular ART methodology in recent years. An important and remaining problem with this technique, however, is that it requires small amounts of mercury to stabilize the pipette tip when piezoelectric force pulses are applied. To eliminate this problem we developed and tested a completely different and mercury-free technology, called the "Ros-Drill" (rotationally oscillating drill). The technique uses microprocessor-controlled rotational oscillations on a spiked micropipette without mercury or piezo. Preliminary experimental results show that this new microinjection technology gives high survival rate (>70% of the injected oocytes) and fertilization rate (>80% of the survived oocytes), and blastocyst formation rates in early trials (approximately 50% of the survived oocytes). Blastocysts created by Ros-Drill ICSI were transferred into the uteruses of pseudopregnant surrogate mothers and healthy pups were born and weaned. The Ros-Drill ICSI technique is automated and therefore; it requires a very short preliminary training for the specialists, as evidenced in many successful biological trials. These advantages of Ros-Drill ICSI over conventional and piezo-assisted ICSI are clearly demonstrated and it appears to have resolved an important problem in reproductive biology.     

External mechanical control of the timing of bend initiation in sea urchin sperm flagella

The movement parameters of a sea urchin sperm flagellum can be manipulated mechanically by applying various modes of periodic vibrations to the sperm head held by suction in the tip of a micropipette. The beat frequency of the flagellum readily synchronizes with the frequency of the externally imposed lateral vibration, and the plane of flagellar bending waves adapts itself to the plane of the pipette vibration (Gibbons et al., J. Cell Biol. 101:270a, 1985; Nature 325: 351-352, 1987). In this study, we observed the particular effects of external asymmetric forces on flagellar beating parameters by vibrating the micropipette holding the sperm head in a transverse sawtooth-like motion composed of a rapid effective stroke and a slower recovery stroke, while keeping the vibration frequency constant. The results demonstrate that the timing of bend initiation within the flagellar beat cycle can be controlled mechanically by changing the time point within the vibration cycle at which the micropipette changes its direction of motion. A switch in the sidedness of the asymmetric movement of the micropipette produces dramatic changes in the profiles of bend growth in the basal 5 microns of the flagellum but has almost no effect on the asymmetry or other parameters of bending in the mid- and distal regions of the flagellum. Our results suggest that elastic strain within the basal region of the flagellar structure may play a more significant role in the process of bend initiation than has been realized heretofore.     

Improved technique for studying ion channels expressed in Xenopus oocytes, including fast superfusion.

The study of whole-cell currents from ion channels expressed in Xenopus oocytes with conventional two-electrode voltage clamp has two major limitations. First, the large diameter and spherical geometry of oocytes prevent extremely fast solution changes. Second, the internal medium is not controlled, which limits the experimental versatility of the oocyte expression system. For example, because the internal medium is not controlled, endogenous calcium-activated chloride conductances can contaminate currents measured with channels that are permeable to calcium. We describe a new technique that combines vaseline-gap voltage clamp for oocytes with a fast superfusion system. The vaseline-gap procedure is simplified by having the micropipette that monitors voltage serve a dual role as a perfusion micropipette that controls the internal solution. In addition, the technique provides fast external solution changes that are complete in 30-50 ms. We applied the approach to measure the calcium permeability of a muscle and a neuronal nicotinic acetylcholine receptor. Very fast agonist induced currents were measured without contamination by the secondary activation of calcium-dependent chloride channels.     

Cannabinoid and GABA modulation of sympathetic nerve activity and blood pressure in the dorsal periaqueductal gray of the rat

Sympathoexcitation and increased blood pressure evoked by central networks integrating defensive behavior are fundamental to the acute stress response. A balance between excitatory glutamatergic and inhibitory GABAergic neurotransmission in the dorsal periaqueductal gray (dPAG) results in a tonic level of activity in the alerting system. Neuromodulators such as endocannabinoids have been shown to influence the sympathoexcitatory and pressor components of acute stress in the dPAG, exemplified by the defense response as a model, but the mechanism of integration remains unknown. The present study examines the role of GABA and its interaction with endocannabinoids in modulating sympathetic nerve activity and blood pressure related to the defense response. Microinjection of the broad-spectrum excitatory amino acid dl-homocysteic acid (DLH) identified sites of the defense pathway in the dPAG from which an increase in renal sympathetic nerve activity and blood pressure could be evoked, and subsequent microinjections were made at the same site through a multibarrelled micropipette. Blockade of GABAA receptors or microinjection of the cannabinoid 1 receptor agonist anandamide elicited a renal sympathoexcitation and pressor response. Prior microinjection of the GABAA receptor antagonist gabazine attenuated the sympathoexcitation and pressor response associated with anandamide microinjection. In contrast, the sympathetic response to DLH was enhanced by GABAA receptor blockade. These data demonstrate that sympathoexcitatory neurons in the dPAG are under tonic inhibition by GABA and that endocannabinoids modulate this GABAergic neurotransmission to help regulate components of the defense response.     

Microinjection in the chick blastoderm. An improved method to study the extracellular matrix in the living organism

A microinjection technique for the chick blastoderm is described. With a micropipette attached to a de Fonbrune micromanipulator, 25-45 nl of a reagent was injected into the entophyllic crescent of a chick blastoderm explanted in vitro according to New [7]. This procedure offers the advantage of eliminating the concentration variability which was observed after subblastodisc injection, and in contrast to the in ovo techniques, it allows one to stage the blastoderms properly. To check its applicability, testicular hyaluronidase was injected. On the basis of morphological and histochemical observations we ascertained that the experimental procedure itself did not interfere with the results. This method may provide a reliable experimental procedure with which to study the interactions between several macromolecules and the tissues during morphogenesis.     

Photodynamic assistance increases the efficiency of the process of microinjection in animal cells

A microinjection process is described that involves the photodynamically-assisted puncture of an animal cell membrane by means of a brief and partial degeneration resulting from cell membrane damage. The ratio of dye-injected rat pheochromocytoma cells that became detached three and six days after photodynamically-assisted microinjection was 9%; however, it was 83% when a conventional mechanical microinjection process was used.     

Increase in Plasmodesmatal Permeability during Cell-to-Cell Spread of Tobacco Rattle Virus from Individually Inoculated Cells

A microinjection technique was devised for inoculation of single Nicotiana clevelandii leaf trichome cells with virus particles. By removing inoculated trichomes at various times after microinjection, it was shown that at least 4 hr were required for tobacco rattle virus (TRV; tobravirus group) to move out of primarily inoculated cells. Effects of the early stages of TRV infection on plasmodesmatal permeability were examined by microinjection of fluorochrome-labeled molecules. Fluorescein-labeled insulin A chain (Mr, 2921) and fluorescein-labeled dextran (Mr, 4400) were observed to pass out of individual N. clevelandii trichome cells that had been inoculated with TRV by microinjection 5 hr previously. By contrast, Lucifer Yellow CH-labeled dextran (Mr, 10,000) was restricted to the inoculated cell. None of these fluorescent probes were able to move out of uninoculated cells or out of cells that had been inoculated with TRV only 2 hr previously. The movement of macromolecules through plasmodesmata, therefore, coincided with and probably resulted from cell-to-cell movement of TRV. The results are discussed with reference to the interaction of viruses and plasmodesmata and mechanisms of intercellular virus movement.     

Mediation of vagal cardioinhibitory responses by glutamatergic receptors in the caudal medulla of turtles

Our previous studies showed that electrical stimulation of the nuclei ambiguous (NA) or dorsomotor nuclei of the vagus (DMV) complex in the brain stem of spontaneously breathing pond turtles (Cyclemys fiavomarginata), anesthetized with chloralose (4 mg/100 g) and urethane (40 mg/100 g), produced a marked slowing or even cessation of the heart rate, and resulted in an immediate fall of blood pressure. Results of the present study further demonstrated that the cardioinhibitory responses could also be elicited by microinjection of monosodium glutamate (0.2-20 nl, 50 mM) into the NA/DMV complex in turtles. A two-barrel glass micropipette held in a manipulator was connected to a pneumatic pressure pump for microinjection. The glutamate-induced cardioinhibitory responses could be significantly reduced in a dose-dependent manner by pretreatment with AP-5 (a NMDA receptor antagonist, at 1-8 nmole) or CNQX (a non-NMDA receptor antagonist; at 0.1-0.8 nmole) 20 min before glutamate administration. Histochemical verification by injecting horseradish peroxidase into the cervical vagus nerves revealed that retrogradely labeled glutamatergic neurons in the NA/DMV complex were observed. These results suggest that glutamatergic receptors in the caudal medulla may mediate vagal cardioinhibitory responses in the turtle.     

氯化钠密度梯度离心法制备用于显微注射的外源DNA片段

DNA显微注射是生产转基因动物最可靠和最常使用的一种方法,外源DNA的纯度对显微注射的成功起着至关重要的作用。本文介绍用氯化钠密度梯度离心的方法制备用于显微注射的外源DNA片段。与传统的琼脂糖凝胶回收的方法相比较,用此方法制备的外源DNA片段对小鼠受精卵进行显微注射后,受卵体母鼠的胚胎存活率,以及子代小鼠的外源基因整合率均有明显的提高。这一方法可为进一步提高转基因动物的成功率,提供方法学上的参考。 Abstract:DNA microinjection is the most popular and reliable method of producing transgenic animals.The purity of foreign DNA plays an important role for the success of microinjection.In this study,we introduced the use of sodium chloride step gradients in fractionating foreign DNA fragment for microinjection.The data demonstrated that,compared with the conventional agarose gel extraction method,NaCl purification scheme of toreign DNA could improve the treated embryo survival and foreign DNA intergration rate markedly.     

侧脑室微量注射大麻素受体拮抗剂对orexin-A诱导大鼠能量代谢改变的影响及潜在机制研究

目的:探讨大麻素1型受体(CB1)抑制剂利莫那班对下丘脑外侧区(LHA)微量注射orexin-A诱导的小鼠能量代谢及相关行为变化改变的影响。方法:通过侧脑室微量注射(icv)利莫那班,同时LHA微量注射orexin-A,测量小鼠能量代谢、自主运动的变化,杏仁核(CeA)内多巴胺释放能力以及小鼠摄食量的变化。结果:侧脑室微量注射利莫那班可减弱因LHA微量注射orexin-A引起的小鼠能量代谢变化,降低小鼠自主运动,并且减弱小鼠CeA内多巴胺释放能力。注射(icv)利莫那班未改变LHA微量注射orexin-A所诱导的摄食量增多。此外,LHA双侧注射利莫那班可阻断LHA内注射orexin-A对运动活性的促进作用,但不影响小鼠的摄食量。结论:大麻素受体涉及orexin-A诱导的小鼠中脑边缘系统多巴胺系统活化的调控,对能量代谢及自主运动也有影响,但对食物摄入的调节无明显影响。     

Ionic currents in single isolated bullfrog atrial cells

Enzymatic dispersion has been used to yield single cells from segments of bullfrog atrium. Previous data (Hume and Giles, 1981) have shown that these individual cells are quiescent and have normal resting potentials and action potentials. The minimum DC space constant is approximately 920 microns. The major goals of the present study were: (a) to develop and refine techniques for making quantitative measurements of the transmembrane ionic currents, and (b) to identify the individual components of ionic current which generate different phases of the action potential. Initial voltage-clamp experiments made using a conventional two-microelectrode technique revealed a small tetrodotoxin (TTX)-insensitive inward current. The small size of this current (2.5-3.0 X 10(-10)A) and the technical difficulty of the two-microelectrode experiments prompted the development of a one-microelectrode voltage-clamp technique which requires impalements using a low-resistance (0.5-2 M omega) micropipette. Voltage-clamp experiments using this new technique in isolated single atrial cells reveal five distinct ionic currents: (a) a conventional transient Na+ current, (b) a TTX-resistant transient inward current, carried mainly by Ca++, (c) a component of persistent inward current, (d) a slowly developing outward K+ current, and (e) an inwardly rectifying time-independent background current. The single suction micropipette technique appears well-suited for use in the quantitative study of ionic currents in these cardiac cells, and in other small cells having similar electrophysiological properties.     

A simple, rapid, aseptic method for filling micropipettes by centrifugation

A centrifugation method for sterilizing, storing, and filling micropipettes is described. Each micropipette is held in a centrifuge tube by a rubber stopper which clamps the butt end of the micropipette. The pipettes are sterilized and aseptically stored. A pipette is filled by injecting solution into the butt end of the micropipette. The micropipette is returned to a suspended position in the centrifuge tube and the liquid is rapidly forced into the micropipette tip by centrifugation. The technique is simpler and more rapid than presently used centrifugation methods.     

Influence of microfilament and microtubule inhibitors applied by immersion and microinjection on circulation streaming in the staminal hairs ofTradescantia blossfeldiana

Summary Reaction of cytoplasmic streaming inTradescantia staminal hairs to microfilament and microtubule specific inhibitors, applied either by conventional immersion or by microinjection, indicates that both the actin-myosin and the microtubule system may be involved in driving the particle stream. Cytoplasmic streaming was stopped at relatively high drug concentrations when the cells were immersed in the inhibitor solution. Microinjection of defined concentrations of inhibitor into single, selected cells were effective at concentrations at least two orders of magnitude lower. Further reduction of inhibitor concentrations, however, enhanced streaming up to 100%. When a mixture of cytochalasin D and oryzalin were injected at concentrations that had previously been shown to enhance particle movement, very efficient inhibition occurred and streaming rapidly stopped. Adjacent cells on both sides of the injected cell were also affected: within a few minutes of the injection of microfilament inhibitors the basal cell reacted, followed slightly later by the apical one; microtubule inhibitors caused a reaction in the apical cell earlier than in the basal cell. The results are discussed with respect to the involvement of actin and myosin microfilaments, as well as microtubules, as force generating systems of particle movement.Abbreviations CB cytochalasin B - CD cytochalasin D - Cys cysteine - DMSO dimethylsulfoxid - DTT dithiothreitol - MI microinjection - NBD 7-nitrobenz-2-oxa-1,3-diazole - NEM N-ethylmaleimide Nocodazole methyl [5-(2-thienylcarbonyl)-1 H-benzimidazol-2-yl]carbamate     

Dictyostelium Chemotactic Response to Spatial and Temporal Gradients. Theories of the Limits of Chemotactic Sensitivity and of Pseudochemotaxis

Four aspects of ameboid cell chemotaxis are discussed: 1) Ameboid cells (Dictyostelium discoideum, leukocytes) might orient to chemotaxin gradients by sensing a spatial gradient or a temporal change in the concentration. Using a moving micropipette source of cAMP, we show the D discoideum cells can orient toward a gradient in which the concentration is everywhere decreasing with time–implying a spatial mechanism. 2) The number of molecules N that must be released by a source to orient a cell is limited by the natural concentration “noise” due to diffusion. N is shown to be simply related to the cell size and the distance from the source. 3) We show that previous diffusion equations for cell population movement have not taken the speed variations (klinokinesis) into account properly, and we present a new result that does. 4) We briefly discuss reaction-diffusion models of cell orientation.     

The use of recombinase proteins to generate transgenic large animals

The endogenous properties of recombinase proteins allow them to associate with and bind DNA to catalyze homologous recombination. These endogenous properties of cellular recombination enzymes may be useful to the field of transgenesis. The production of transgenic animals, in particular livestock, is an inefficient process by both conventional pronuclear microinjection techniques and nuclear transfer. Furthermore, the use of pronuclear microinjection is currently limited to the random addition of genes and does not allow for the replacement of an endogenous gene with a more desired one. The functions of cellular recombination enzymes have been exploited to develop a technique that is compatible with pronuclear microinjection and may make the process of generating transgenic livestock more efficient while also enabling the targeting of homologous chromosomal genes. In our hands, transgenic animals generated by the pronuclear microinjection of various recombinase protein-coated DNA fragments led to a higher than expected birth rate as well as transgene integration frequency. Most founder animals generated were likely mosaic, indicating that integration occurred after cell division. The presence of multiple related genes makes detection of any recombination event difficult. Overall, this technique is a straightforward, rapid, and efficient procedure that can be applied to any segment of DNA and any microinjection apparatus, and is less labor intensive than nuclear transfer.     

Chemotactically-induced redistribution of CD43 as related to polarity and locomotion of human polymorphonuclear leucocytes

Leukocyte motility involves pseudopods extension at the leading edge and uropod contraction at the cell rear. Previous studies have shown that the glycoprotein CD43 redistributes to the uropod, when the cells develop polarity and locomotion. The present study addresses the question whether the accumulation of specific membrane molecules, such as CD43 at the contracted uropod precedes or follows development of polarity and locomotion. PMNs were labeled with fluorescent anti-CD43 antibodies and guided to polarize in the direction of a chemoattractant-containing micropipette or, once polarized, they were forced to reverse polarity and movement direction by placing the micropipette behind the uropod. This chemotactically-induced reversal of polarity was used as an efficient tool to analyse the sequence of events. CD43, but not another abundant surface glycoprotein CD45, was concentrated at the uropod. This documents that CD43 redistribution is a selective phenomenon. During reversal of polarity and of locomotion direction, the geometric center of the cell clearly changed direction earlier than the center of anti-CD43 fluorescence intensity. Thus, CD43 redistribution to the new uropod follows rather than precedes reversal of polarity, suggesting that CD43 redistribution is a consequence rather than a prerequisite for polarity and locomotion. PMNs making a U-turn maintained the pre-existing polarity and CD43 remained concentrated at the uropod, even when the front was moving in the opposite direction. Our data show that anterior pseudopod formation, rather than capping of CD43 at the uropod or the position of the uropod determines the direction of locomotion.