Integration of Controlled Intracellular Pressure Microinjection, lontophoresis, and Membrane Potential Measurement

Abstract: A novel device for intracellular microinjection was designed to integrate controlled pressure microinjection and electrical microinjection (iontophoresis) with membrane poten tial recording and a limited capacity for turgor measurement. The validity of the device was verified by microinjection of a mixture of the fluorescent probes, Texas Red sulfonyl chloride and 10 kDa-LYCH-dextran conjugate, into epidermal cells of var iegated Coleus blumei leaves. Continuous monitoring of the fluorochrome movement by confocal laser scanning microscopy evidenced that the novel device succeeded in differential micro-injection of the fluorescent probes by pressure and iontophor esis. The multifunctionality of the microinjection system was further demonstrated by showing that both microinjection methods functioned in parallel with cellular membrane poten tial measurements in Vicia faba stem tissue. Advantages and prospective applications of the integrated microinjectionjmem-brane potential measurement system are briefly discussed.     

Generation of Recombinant Chinese Hamster Ovary Cell Lines by Microinjection

Microinjection is a gene transfer technique enabling partial control of plasmid delivery into the nucleus or cytoplasm of cultured animal cells. Here this method was used to establish various recombinant mammalian cell lines. The injection volume was estimated by fluorescence quantification of injected fluorescein isothyocynate (FITC)-dextran. The DNA concentration and injection pressure were then optimized for microinjection into the nucleus or cytoplasm using a reporter plasmid encoding the green fluorescent protein (GFP). Nuclear microinjection was more sensitive to changes in these two parameters than was cytoplasmic microinjection. Under optimal conditions, 80–90% of the cells were GFP-positive 1 day after microinjection into the nucleus or the cytoplasm. Recombinant cell lines were recovered following microinjection or calcium phosphate transfection and analyzed for the level and stability of recombinant protein production. In general, the efficiency of recovery of recombinant cell lines and the stability of reporter protein expression over time were higher following microinjection as compared to CaPi transfection. The results demonstrate the feasibility of using microinjection as a method to generate recombinant cell lines. Revisions requested 27 October 2005; Revisions received 12 December 2005     

Improved developmental potential of rabbit oocytes fertilized by sperm microinjection into the perivitelline space enlarged by hypertonic media

The objectives of the present study were: 1) to develop a simple and more efficient technique for sperm microinjection than is currently available, using the rabbit as a model, and 2) to evaluate the development of rabbit oocytes fertilized by single or multiple sperm microinjection. Hyperosmotic sucrose in phosphate-buffered saline (SPBS) was employed to dehydrate oocytes to increase the perivitelline space for sperm microinjection and prevent possible injury to the vitellus. In the first experiment, 58% (n = 29) oocytes treated with 0.5 M SPBS developed to morulae following multiple sperm microinjection compared, respectively, to 47% (n = 34) and 60% (n = 15) for control IVF with or without sucrose exposure (P greater than 0.05). Blastocyst development from microinjected oocytes, however, was much lower (P less than 0.05) than that of controls (14% vs. 42% and 40%, respectively). Sham operation by puncturing the zona pellucida of the sucrose-treated oocytes with the microinjection pipette did not increase parthenogenesis (P greater than 0.05). In Experiment 2 a smaller-size injection pipette and shorter sucrose exposure time after sperm microinjection resulted in 41% (n = 42) of the oocytes developing into blastocysts for the microinjection group, whereas only 21% (n = 24) developed to blastocysts in the control IVF group (P less than 0.05). When relatively older oocytes (17 hr post ovulation injection) were used to test if microinjection could reduce the time to fertilization and cleavage (Expt. 3), an average of 27% (n = 63) blastocysts resulted from microinjection vs. 0% (n = 28) for the control IVF group.     

Human sperm chromosome complements after microinjection of hamster eggs

A technique was developed for microinjection of human spermatozoa into golden hamster (Mesocricetus auratus) eggs to obtain human pronuclear chromosome complements. Before microinjection the spermatozoa were treated by brief sonication or incubation in TEST-yolk buffer to reduce motility. Very few sperm chromosome complements developed after sperm treatment with sonication and the frequency of spermatozoa with structural chromosomal abnormalities was exceedingly high (91%). The majority of sperm chromosome complements analysed had multiple breaks and rearrangements. Sperm incubation in TEST-yolk buffer before microinjection provided more analysable sperm karyotypes with a significantly lower frequency of structural chromosomal abnormalities (39%, P less than 0.001). Our results therefore suggest that sonication induces structural chromosomal abnormalities in spermatozoa. Since the frequency of chromosomal abnormalities after microinjection was higher than after sperm fertilization of hamster eggs, it appears that microinjection per se may also increase the frequency of chromosomal abnormalities in spermatozoa. These results are based on small numbers and must be confirmed on larger sample sizes, but our study suggests that microinjection of spermatozoa into eggs should not be recommended for clinical use until fully evaluated.     

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.     

High throughput easy microinjection with a single-cell manipulation supporting robot

A single-cell manipulation supporting robot (SMSR) has been developed for the high throughput and easy microinjection. Its concept is to let an experimenter concentrate his/her attention only on the microinjection by facilitating other associated works. SMSR was applied to the microinjection into rice protoplasts and mouse embryonic stem (ES) cells. The microinjection into these cells is exceptionally difficult than usual animal cells such as fibroblasts. In the case of rice protoplast, for example, non-stop microinjection into 100 cells could be done within 1h that was 17-times faster than that of the robot-less work. The success rate was 7-8% that was same level obtained by the robot-less work. The present results indicate that SMSR is a useful machine for the microinjection of specific genes and proteins in living cells to analyze their respective functions, which is an urgent and important subject in the post-genome era.     

Antibodies to a nucleolar protein are localized in the nucleolus after red blood cell-mediated microinjection

To determine whether red blood cell-mediated microinjection of antibodies can be used to study nuclear protein localization and function, we microinjected antibodies that have been shown to react specifically with nucleolar acidic phosphoprotein C23 into Walker 256 cells. The intracellular distribution of microinjected anti-C23 antibodies and preimmune immunoglobulins were determined by immunofluorescence. At 3 h after microinjection, affinity-purified anti- C23 antibodies were localized in the cytoplasm and nucleolus. At 17 h after microinjection, the affinity-purified antibody was localized to those nucleolar structures previously shown to contain protein C23. Furthermore, the antibody remained localized in the nucleolus for at least 36 h after microinjection. In contrast to the results obtained with specific antibodies, preimmune immunoglobulins remained in the cytoplasm 36 h after microinjection. These results indicate that red blood cell-mediated microinjection of antibodies can be used to study nucleolar and nuclear antigens.     

Injection of An. stephensi embryos to generate malaria-resistant mosquitoes

The introduction of exogenous genes into the genomes of mosquitoes requires microinjection techniques tailored to the specific species of interest. This video protocol demonstrates a method used by the James laboratory to microinject DNA constructs into Anopheles stephensi embryos for the generation of transformed mosquitoes. Techniques for preparing microinjection needles, collecting and preparing embryos and performing the microinjection are illustrated.     

Red blood cell-mediated microinjection: methodological considerations

Red blood cell-mediated microinjection is a powerful approach to introducing proteins into the cytoplasm of cultured cells. In the course of our microinjection studies of intracellular protein degradation, we have encountered several potential problems with certain proteins. The microinjection procedure may be accompanied by denaturation of protein by radiolabeling procedures, binding of protein to red cell ghosts during loading, degradation of protein by the red cell ghost prior to microinjection, and adsorption of protein that leaks from red cell ghosts in the presence of fusogen to the fibroblast monolayer. We conclude with a list of points that must be considered prior to use of red cell-mediated microinjection to study a particular protein.     

Requirement of phospholipase C-catalyzed hydrolysis of phosphatidylcholine for maturation of Xenopus laevis oocytes in response to insulin and ras p21

Recent studies have demonstrated the activation of phospholipase C-mediated hydrolysis of phosphatidylcholine both by growth factors and by the product of ras oncogene, ras p21. Also, evidence has been presented indicating that the stimulation of this phospholipid-degradative pathway is sufficient to activate mitogenesis in fibroblasts. In Xenopus laevis oocytes, microinjection of transforming ras p21 is a potent inducer of maturation, whereas microinjection of a neutralizing anti-ras p21 antibody specifically inhibits maturation induced by insulin but not by progesterone. The results presented here demonstrated that microinjection of phosphatidylcholine-hydrolyzing phospholipase C is sufficient to induce maturation of Xenopus laevis oocytes. Furthermore, microinjection of a neutralizing anti-phosphatidylcholine-hydrolyzing phospholipase C specifically blocks the maturation program induced by ras p21/insulin but not by progesterone.     

Factors related to successful sperm microinjection of hamster eggs: The effect of sperm species, technical experience, needle dimensions, and incubation medium on egg viability and sperm decondensation following microinjection

A series of experiments was designed to identify factors in a sperm microinjection system that could influence egg viability and decondensation of sperm nuclei after microinjection. Egg viability and sperm decondensation rates were not different among eggs microinjected with rodent sperm. The microinjection of ram sperm required a larger diameter needle for injection, which resulted in low egg viability and sperm decondensation in the first 3 mo of the study but improved greatly after 9 mo of technical experience. The degree of technical experience (3 vs 9 mo) also improved (P<0.05) egg viability after microinjection with rodent sperm; however, the rate of sperm decondensation remained unaffected. Altering the dimensions of the injection needle from a tapered needle barrel to a more uniform needle barrel increased egg viability from 61 to 96% and sperm decondensation from 3 to 27%. The use of medium 199 for incubating microinjected eggs further increased (P<0.05) the percentage of eggs containing decondensed sperm nuclei (52%) compared to eggs incubated in Holmes defined medium (28%). By altering the dimensions of the injection needle, by selecting an appropriate incubation medium, and by gaining technical experience in microinjection, the efficiency of a sperm microinjection system was improved for both rodents and domestic animals.     

中缝大核在臂旁内侧核区注射吗啡所致呼吸抑制效应中的作用

实验在33只浅麻醉、肌肉麻痹、人工呼吸及切断双侧颈迷走神经的家兔上进行。观察中缝大核区电解损毁或微量注射利多卡因对呼吸活动及臂旁内侧核区微量注射吗啡所致呼吸抑制效应的影响。结果是:电解损毀中缝大核区,使呼吸频率增加,膈神经放电的幅度和频率均无明显变化,而臂旁内侧核区微量注射吗啡抑制呼吸的程度减轻;中缝大核区微量注射利多卡因,则部分消除臂旁内侧核区微量注射吗啡的呼吸抑制效应。中缝大核旁网状结构电解损毁或微量注射利多卡因,不影响吗啡的呼吸抑制效应。上述结果提示,中缝大核区可能在脑桥臂旁内侧核区微量注射吗啡抑制呼吸的机制中起一定作用。     

ATP-gamma-S (adenosine 5'-0(3-thiotriphosphate)) blocks progesterone-induced maturation of the Xenopus oocyte

ATP-gamma-S microinjection into Xenopus oocyte prevents progesterone induced maturation. Inhibition is time and dose dependent; 50% inhibition occurs when 50 nl of 0.5 mM ATP-gamma-S solution are microinjected/oocyte 1 hr prior to the hormonal trigger. ATP-gamma-S inhibited oocytes can be induced to mature (100%) following microinjection of extracts containing maturation promoting factor (MPF). Our results suggest that the maturation protein(s) has been stabilized in ovo by ATP-gamma-S microinjection, in its phosphorylated inhibitory form.     

The diagonal band of Broca is involved in the pressor pathway activated by noradrenaline microinjected into the periaqueductal gray area of rats

AimsThe dorsal periaqueductal gray area (dPAG) is involved in cardiovascular modulation. Previously, we reported that noradrenaline (NA) microinjection into the dPAG caused a pressor response that was mediated by vasopressin release into the circulation. However, the neuronal pathway that mediates this response is as yet unknown. There is evidence that chemical stimulation of the diagonal band of Broca (dbB) also causes a pressor response mediated by systemic vasopressin release. In the present study, we evaluated the participation of the dbB in the pressor response caused by NA microinjection into the dPAG as well as the existence of neural connections between these areas.Main methodsWith the above goal, we verified the effect of the pharmacological ablation of the dbB on the cardiovascular response to NA microinjection into the dPAG of unanesthetized rats. In addition, we microinjected the neuronal tracer biotinylated-dextran-amine (BDA) into the dPAG and looked for efferent projections from the dPAG to the dbB.Key findingsThe pharmacologically reversible ablation of the dbB with local microinjection of CoCl₂ significantly reduced the pressor response caused by NA microinjection (15 nmol/50 nL) into the dPAG. In addition, BDA microinjection into the dPAG labeled axons in the dbB, pointing to the existence of direct connections between these areas.SignificanceThe present results indicate that synapses within the dbB are involved in the pressor pathway activated by NA microinjection into the dPAG and direct neural projection from the dPAG to the dbB may constitute the neuroanatomic substrate for this pressor pathway.     

头端延髓腹外侧区注射5—羟色胺对应激性高血粘度...

Experiments were carried out on 62 wistar rats. The hyperviscosity and elevation of blood pressure were induced by hanging and restraining the rats with their four limbs tied on a frame. It was found that microinjection of 5-HT (25 micrograms/10 microliters) into the 4th ventricle of the brain or bilateral microinjection of 5-HT (4 micrograms/0.5 microliters/site) into rostral ventrolateral medulla (rVLM) reduced stress-induced hyperviscosity (p < 0.01) and elevation of blood pressure (p < 0.01). The effect of 5-HT injected into the 4th ventricle or rVLM was blocked by bilateral microinjection of cinanserine (4 micrograms/0.5 microliter/site) into rVLM. These results suggest that microinjection of 5-HT into 4th ventricle and rVLM could reduce stress-induced hyperviscosity and elevation of blood pressure and these effects were probably mediated via 5-HT receptors in the rVLM.     

Effects of forebrain microinjection of cholecystokinin on dopamine cell firing rate.

In anesthetized rats, midbrain dopamine (DA) neuronal firing rate was differentially sensitive to focal brain microinjection of cholecystokinin peptides (CCK-4 and CCK-8) and N-methyl-D-aspartate (NMDA) into nucleus accumbens, amygdala and prefrontal cortex. Whereas changes in DA neuronal firing rate were frequently observed in response to intra-amygdalar microinjection of CCK peptides, NMDA was most effective in eliciting changes in DA neuronal activity following intra-accumbal microinjection. Thus, stimulation of amygdalar CCK receptors and accumbal excitatory amino acid receptors may participate in the afferent regulation of midbrain DA neuronal function.     

Preparation of intact yeast artificial chromosome DNA for transgenesis of mice

Transgenesis with large DNA molecules such as yeast artificial chromosomes (YACs) has an advantage over smaller constructs in that an entire locus and all its flanking cis-regulatory elements are included. The key to obtaining animals bearing full-length transgenes is to avoid physical shearing of the DNA during purification and microinjection. This protocol details how to prepare intact YAC DNA for transgenesis of mice and involves separation of YAC DNA from yeast chromosomal DNA by pulsed field gel electrophoresis, concentration to a range suitable for microinjection by second dimension electrophoresis and enzymatic digestion of matrix-embedded YAC DNA to produce a solution that can be injected. The YAC is maintained in an agarose gel matrix to avoid damage until the final steps before microinjection. Special precautions are also taken during the microinjection protocol. Transgenesis efficiency is approximately 15%; most animals carry 1-5 copies of the desired locus. This method takes 6 d for completion.     

Intranuclear microinjection for transformation of tomato callus cells

An efficient method, called the culture plate method, was devised for microinjection of foreign materials into nuclei of tomato callus cells. The culture plate method, used in this study, is advantageous because cells suitable for microinjection can be selected microscopically and the injected cells subsequently cultured in the same plate. With this microinjection system, some foreign materials were injected into nuclei of callus cells without causing detrimental effects. Kanamycin-resistant callus clones were obtained 1 month after injection from single cells whose nuclei were microinjected with a NPT II DNA fragment of the pE2KX plasmid.     

Identification of embryogenic microspores of barley (Hordeum vulgare L.) by individual selection and culture and their potential for transformation by microinjection

Summary In order to identify microspores, suitable for transformation via microinjection of DNA, single microspores of barley (Hordeum vulgare L.) were selected after initial preculture of anthers floating on liquid media and analysed for their development in individual culture in microdroplets of culture medium. Conditions for microculture and plant regeneration from single selected embryogenie microspores were established. The technical feasability of intranuclear microinjection was demonstrated by injecting the fluorescent dye Lucifer Yellow. All essential procedures for a transformation system of barley based on microinjection into microspores have thus been performed successfully. Further efforts to increase efficiencies of culture and microinjection procedures are necessary, however, in order to improve the suitability of this approach towards transformation of barley.Abbreviations MES 2 (N-morpholino) ethanesulfonic acid - PEG polyethylene glycol     

Microinjection of A. aegypti embryos to obtain transgenic mosquitoes

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever. The techniques for correctly preparing microinjection needles, desiccating embryos, and performing microinjection are demonstrated.