Transferring isolated mitochondria into tissue culture cells

We have developed a new method for introducing large numbers of isolated mitochondria into tissue culture cells. Direct microinjection of mitochondria into typical mammalian cells has been found to be impractical due to the large size of mitochondria relative to microinjection needles. To circumvent this problem, we inject isolated mitochondria through appropriately sized microinjection needles into rodent oocytes or single-cell embryos, which are much larger than tissue culture cells, and then withdraw a ‘mitocytoplast’ cell fragment containing the injected mitochondria using a modified holding needle. These mitocytoplasts are then fused to recipient cells through viral-mediated membrane fusion and the injected mitochondria are transferred into the cytoplasm of the tissue culture cell. Since mouse oocytes contain large numbers of mouse mitochondria that repopulate recipient mouse cells along with the injected mitochondria, we used either gerbil single-cell embryos or rat oocytes to package injected mouse mitochondria. We found that the gerbil mitochondrial DNA (mtDNA) is not maintained in recipient rho0 mouse cells and that rat mtDNA initially replicated but was soon completely replaced by the injected mouse mtDNA, and so with both procedures mouse cells homoplasmic for the mouse mtDNA in the injected mitochondria were obtained.     

Microinjection of deoxynucleotides into mouse cells. No evidence that precursors for DNA synthesis are channeled

The technique of microinjection was applied for the introduction of radioactive, phosphorylated precursors of DNA synthesis into living mouse cells in culture. Autoradiographs proved that DNA was well labeled by injected dTTP, dCTP, dATP, dGTP, and (to a minor extent) dTMP, but the efficiency was much lower when CDP, ADP, or GDP was used. For practical reasons, injections into nuclei were preferred, but injections into cytoplasm showed no principal difference with the autoradiographed nuclei. The kinetics of the uptake of the injected material agreed neatly with the calculation (from pool sizes and polymerization rate) that the intracellular deoxytriphosphates are sufficient for about 10 min of DNA synthesis. All this evidence strongly argues against the concept that precursors of DNA synthesis are channeled in vivo within a multienzyme complex and suggests a free diffusion of deoxynucleotides within the cell. Injected thymidine was less able to enter the deoxy nucleotide metabolism compared with thymidine from the culture medium. A mutant cell line deficient in thymidine kinase did not accumulate intracellular thymidine. These data indicate that thymidine kinase is a membrane associated enzyme and that uptake and phosphorylation of thymidine are coupled reactions.     

Microinjection of living adherent cells by using a semi-automatic microinjection system

Testing in vitro is an alternative to animal experimentation. The capillary pressure microinjection technique is a supporting technology for efficient in vitro testing. The main benefit of the technique is the possibility of injecting large molecules into a single living cell. The ultimate goal of the research discussed in this paper is to increase the cell survival rate in capillary pressure microinjection. A method to reliably evaluate cell survival rate is therefore needed. A three-phase evaluation process is presented in this paper. The first phase determines the success rate of the injection capillary to penetrate the cell membrane. The second phase studies the success rate of delivering the injection substance inside the cell, while the third phase studies cell survival after the microinjection. In addition to the three-phase evaluation process, this paper describes the initial results of penetration and injection tests performed by using a semi-automatic capillary pressure microinjection system developed by the research group. Three adherent cell lines, namely, retinal pigment epithelial cells, MCF-7 human breast cancer cells and SH-SY5Y neuroblastoma cells, were used in the experiments. The results of the penetration tests show that the average success rate of penetrating the cell membrane using the micromanipulator was 87%. The goal of the injection tests was to demonstrate the successful microinjection of living cells and to study the injection success rate. Fluorescein dextran was injected into MCF-7 cells, and preliminary results showed an injection success rate of 49%. In the survival tests, the neuronal cells were microinjected with KCl. During long-term observation after the microinjection, the microinjected cells first decreased their adhesion to the plate, but later adhered to the bottom of the plate and even grew some dendrites. In the next phase of the study, more tests will be performed in order to obtain a statistically reliable value for the survival rate.     

The Correlation between the Chromosome Variation in Callus and Genotype of Explants of Arabidopsis thaliana

Twelve callus lines of Arabidopsis thaliana were derived from four types of explants excised from diploid plants of two ecotypes (Columbia and Wilna) and autotetraploid plants of the Wilna ecotype. Cytogenetic analysis of the chromosome variation in particular callus lines was carried out for primary culture and callus during 5 months of culture. Ploidy levels of interphase nuclei were estimated by counting the number and size of chromocentres and nuclei of interphase cells. The first polyploid cells in all callus lines were observed during callogenesis. In primary culture the ploidy level ranged between 2 and 15x (10-75 chromosomes). The frequency of polyploid cells was higher in the 5-month old callus culture, but the ploidy level was the same. In the callus lines derived from autotetraploid plants, cells with reduced chromosome number appeared quite frequently along with diploid and polyploid cells.     

Transfer of macromolecules into living adult cardiomyocytes by microinjection

Among techniques commonly used to deliver bioactive molecules into living cells, microinjection is a very efficient method. Microinjection has been used extensively for gene transfer into different cell types. We applied the microinjection technique to the adult rat ventricular cardiac muscle cells (AVC) in primary culture and optimized microinjection parameters and the appropriate cell culture conditions. We also optimized the use of particular agents (i.e. 2,3-butanedione monoxime, verapamil) for the prevention of the cell damage caused by the micropuncture. We obtained the expression of a CMV--galactosidase reporter gene in up to 20% of the injected cells with efficient maintenance of long term cell viability. Under our experimental conditions direct microinjection is a very advantageous technique to transfer macromolecules into living adult cardiac muscle cells and a powerful system to study and manipulate the biochemistry and molecular biology of the cardiac myocyte.     

A simple and highly efficient transgenesis method in mice with the Tol2 transposon system and cytoplasmic microinjection

Creating transgenic mice is an important technology for genetic studies and is currently performed by pronuclear microinjection of plasmid DNA into fertilized eggs. Since survival of injected embryos and integration of plasmid DNA are not efficient, total efficiency is only around 3% with a standard protocol. To circumvent this problem, here we describe a novel transgenesis method, the Tol2-mediated cytoplasmic injection method (Tol2:CI). We injected a foreign DNA cloned in a Tol2-transposon vector together with the transposase mRNA into the cytoplasm of fertilized eggs. As expected, the survival rate of the injected embryos was increased drastically. Also, the foreign DNA was transposed from the plasmid to the genome and transmitted to the next generation very efficiently. Together, the overall transgenic efficiency became more than 20%. Considering its simplicity and perfect compatibility with existing pronuclear microinjection facilities, we propose that the Tol2:CI method is applicable to high throughput functional genomics studies.     

Transformation of L cells with virus thymidine kinase genes introduced by red cell-mediated microinjection

Fusion of red cell ghosts containing foreign materials with cells results in the introduction of the materials into the cells (red cell-mediated microinjection). Until now, 'two-step dialysis' has mainly been used for trapping proteins in the ghosts. Large-sized materials such as DNA, however, are rarely trapped in the ghosts, since the holes in the red cell membrane caused by osmotic shock are too small for such materials to pass through. In this study, we improved the trapping technique. Some of the Hind III fragments of lambda phage DNA as well as proteins could be trapped in the ghosts when the mixture of these materials and red cells were frozen at -80 degrees C for a short period followed by quick thawing. Red cell-mediated microinjection using ghosts containing plasmid pBR322 linked with a Herpes simplex viral thymidine kinase (tk) gene brought about transformation of tk-defective L cells, the efficiency of transformation was 1 out of 20 000-60 000 cells fused with the ghosts.     

Development of a positive method for male stem cell-mediated gene transfer in mouse and pig

Classical approaches for producing transgenic livestock require labor-intensive, time-consuming, and expensive methods with low efficiency of transgenic production. A promising approach for producing transgenic animals by using male stem cells was recently reported by Brinster and Zimmermann (1994: Proc Natl Acad Sci 91:11298-11302) and by Brinster and Avarbock (1994: Proc Natl Acad Sci USA 91:11303-11307). However, in order to apply this technique to producing transgenic animals, some difficulties have to be overcome. These include a satisfactory method for short-term in vitro culture for drug selection after transfection with exogenous DNA, and methods for the use of livestock such as pigs. We developed a new method for transferring foreign DNA into male germ cells. Mice and pigs were treated with busulfan, an alkylating agent, to destroy the developing male germ cells, and liposome/bacterial LacZ gene complexes were introduced into each seminiferous tubule by using a microinjection needle. As a control, lipofectin was dissolved in phosphate-buffered saline at a ratio of 1:1, and then injected into seminiferous tubules. In mice, 8.0–14.8% of seminiferous tubule expressed the introduced LacZ gene, and 7–13% of epididymal spermatozoa were confirmed as having foreign DNA by polymerase chain reaction. The liposome-injected testes were all negative for X-gal staining. These results indicate that some spermatozoa were successfully transformed in their early stages by liposome/DNA complexes. In pigs, foreign DNA was also incorporated efficiently into male germ cells, and 15.3–25.1% of the seminiferous tubules containing germ cells expressed the LacZ gene. The data suggest that these techniques can be used as a powerful tool for producing transgenic livestock. Mol. Reprod. Dev. 46:515–526, 1997. © 1997 Wiley-Liss, Inc.     

Integration of foreign DNA following microinjection of tobacco mesophyll protoplasts

Summary DNA from a bacterial plasmid containing the T-DNA border sequences of Agrobacterium tumefaciens was transferred into the nucleus or the cytoplasm of tobacco mesophyll protoplasts by microinjection. Following culture in hanging drops, some of these protoplasts produced calli containing the foreign DNA sequences. Evidence for the presence of the injected plasmid DNA in these calli was provided by Southern hybridization analysis. The results demonstrated that random portions of the bacterial plasmid were linked to plant DNA and that integration did not occur at the T-DNA borders present on the injected plasmid. The average number of integrated copies ranged from less than one to 1–2 per tobacco genome. The frequency of integration averaged 14% with intranuclear injections compared to 6% with cytoplasmic injections. With further refinement, the use of microinjection may allow the introduction of many different types of genetic elements into plants.     

Comparative studies on microinjected high-mobility-group chromosomal proteins, HMG1 and HMG2

The nonhistone chromosomal proteins, HMG1 and HMG2, were iodinated and introduced into HeLa cells, bovine fibroblasts, or mouse 3T3 cells by erythrocyte-mediated microinjection. Autoradiographic analysis of injected cells fixed with glutaraldehyde consistently showed both molecules concentrated within nuclei. Fixation with methanol, on the other hand, resulted in some leakage of the microinjected proteins from the nuclei so that more autoradiographic grains appeared over the cytoplasm or outside the cells. Both injected and endogenous HMG1 and HMG2 partitioned unexpectedly upon fractionation of bovine fibroblasts, HeLa, or 3T3 cells, appearing in the cytoplasmic fractions. However, in calf thymus, HMG1 and HMG2 molecules appeared in the 0.35 M NaCl extract of isolated nuclei, as expected. These observations show that the binding of HMG1 and HMG2 to chromatin differs among cell types or that other tissue-specific components can influence their binding. Coinjection of [125I]HMG1 and [131I]HMG2 into HeLa cells revealed that the two molecules display virtually equivalent distributions upon cell fractionation, identical stability, identical intracellular distributions, and equal rates of equilibration between nuclei. In addition, HMG1 and HMG2 did not differ in their partitioning upon fractionation nor in their stability in growing vs. nongrowing 3T3 cells. Thus, we have not detected any significant differences in the intracellular behavior of HMG1 and HMG2 after microinjection into human, bovine, or murine cells.     

SV40 chromatin structure is not essential for viral gene expression

The biological activity and the fate of SV40 DNA (minichromosomes, DNA I, DNA II, DNA III) were tested in culture cells by immunofluorescence staining and blot analysis. Following microinjection of 2-4 circular SV40 molecules (minichromosomes, DNA I, DNA II) into the cytoplasm or the nuclei of monkey and rat cells, T- and V-antigen synthesis was demonstrable in nearly every recipient cell. Only linear DNA induced T-antigen synthesis with a very low efficiency after cytoplasmic injection. This low activity correlates with a rapid degradation of DNA III in the recipient cells. Further modifications observed immediately after injection are relaxation of superhelical molecules and formation of high-Mr DNA. Assembly of the injected DNA into SV40 chromatin-like structure, however, occurred only late after early viral gene expression.     

Transient correction of genetic defects in cultured animal cells by introduction of functional proteins.

Material was introduced into cultures of cells by using the method of scrape loading, in which cells are simply rubbed from the surface of a plastic tissue culture dish by a rubber-tipped rod in the presence of a macromolecule of interest. The volume of solution introduced into cells was comparable to that generally injected in the direct microinjection method with glass capillaries, that is, about 50 to 100 fl per cell. Genetic defects (lack of hypoxanthine-guanine phosphoribosyltransferase and thymidine kinase) in several cell lines were transiently corrected by scraping the cells in the presence of crude cell extracts prepared from wild-type cells.     

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     

Somatic histone H1 microinjected into fertilized mouse eggs is transported into the pronuclei but does not disrupt subsequent preimplantation development

We injected somatic subtypes of histone H1 into newly fertilized mouse eggs, which do not naturally contain this chromosomal protein, and examined the fate of the injected protein and its effect on preimplantation development of recipient eggs. Rhodamine-labelled H1 injected into the cytoplasm of 53 eggs was transported into the pronuclei in 51 cases, and this nuclear accumulation could be detected within 15 min of injection. Unlabelled histone H1, which was detected using immunofluorescence, was also transported following microinjection to the pronuclei, where it colocalized with the chromatin and remained associated with the nuclei following cleavage to the two-cell stage. Nuclear accumulation of injected H1 was inhibited when injected eggs were incubated in the presence of drugs that prevent mitochondrial electron transport or glycolysis, which indicates that nuclear transport occurs through an energy-dependent process, as previously observed in tissue culture cells. To determine whether the presence of somatic H1 in early embryonic nuclei would influence subsequent development, fertilized eggs were injected with an approximately physiological quantity (1–5 pg) of somatic H1 or, as controls, with another small basic protein, cytochrome c. Fifty-three eggs were injected with cytochrome c, of which 51 divided to the two-cell stage, and 32 (60%) reached the blastocyst stage, after 5 days in culture. One hundred and eleven eggs were injected with somatic H1, of which 95 divided to the two-cell stage, and 53 (48%) reached the blastocyst stage, after 5 days in culture. The two groups did not differ statistically (X², P > 0.1) with respect to the fraction of injected embryos that developed to the blastocyst stage. These results show that, although mouse embryos lack the somatic subtypes of histone H1 until the four-cell stage of development, they are able to progress through preimplantation development when these subtypes are present beginning at the one-cell stage. This may imply that the distinctive chromatin composition that characterizes early embryos of a variety of species is not essential for early development in mammals. © 1996 Wiley-Liss, Inc.     

Determination of the mitotic index by microinjection of fluorescently labelled tubulin

The microneedle injection technique is one of the most established procedures for the introduction of proteins into living cells. To analyse injected proteins which are important in cell cycle progression it is often necessary to determine the mitotic index. Measuring the mitotic index after microinjection is complicated because only a limited number of cells of the whole cell population is microinjected. Therefore, we attempted to establish a new method to determine the mitotic index using microinjection of fluorescently labelled alpha/beta-tubulin into mammalian cells which allows to monitor the injected cells simultaneously with the determination of the mitotic index. We demonstrated that fluorescently labelled tubulin incorporates efficiently into the mitotic spindle apparatus. Fluorescence remains stable for several hours which is sufficient to observe the progression of cells through the M-phase of the cell cycle. The determination of the mitotic index with the method presented here gave similar results to those determined using other methods. With this method also different stages of mitosis can be visualized by analysing various steps of spindle formation. Thus, this rapid method allows the monitoring of the injected cells after microneedle injection and simultaneously the determination of the mitotic index.     

Extrachromosomal DNA transformation of Caenorhabditis elegans.

DNA was introduced into the germ line of the nematode Caenorhabditis elegans by microinjection. Approximately 10% of the injected worms gave rise to transformed progeny. Upon injection, supercoiled molecules formed a high-molecular-weight array predominantly composed of tandem repeats of the injected sequence. Injected linear molecules formed both tandem and inverted repeats as if they had ligated to each other. No worm DNA sequences were required in the injected plasmid for the formation of these high-molecular-weight arrays. Surprisingly, these high-molecular-weight arrays were extrachromosomal and heritable. On average 50% of the progeny of a transformed hermaphrodite still carried the exogenous sequences. In situ hybridization experiments demonstrated that approximately half of the transformed animals carried foreign DNA in all of their cells; the remainder were mosaic animals in which some cells contained the exogenous sequences while others carried no detectable foreign DNA. The presence of mosaic and nonmosaic nematodes in transformed populations may permit detailed analysis of the expression and function of C. elegans genes.     

In vitro culture ofBellevalia romana (L.) Rchb.

Summary Bellevalia romana (L.) Rchb., a monocotyledonous plant characterized by few (2 n=2 x=8) and very large chromosomes, is a useful subject for studying developmental problemsin vitro. Cytological analysis of callus revealed that the majority of cells were diploid, but the remaining cells had aneuploid nuclei with a wide range of chromosome numbers, tetraploid and haploid nuclei. The frequency of aneuploid and polyploid cells was higher in callus grown in the presence of 2,4-D than in callus grown in NAA plus BAP. These nuclei seemed to increase with the duration of culture. The chromosome number distribution as determined by chromosome counts in calli at different culture times was confirmed by DNA cytophotometry. Chromosome number mosaicism (mixoploidy and aneusomaty) also occurred in all root apices of 9 out of 46 plantlets regenerated from callusvia adventitious shoots.     

Somatic hybridization in higher plants.

Somatic hybridization in higher plants has come into focus since methods have been established for protoplast fusion and uptake of foreign DNA and organelles by protoplasts. Polyethylene glycol (PEG) was an effective agent for inducing fusion. Treatment of protoplasts with PEG resulted in 5 to 30% heterospecific fusion products. Protoplasts of different species, genera and even families were compatible when fused. A number of protoplast combinations (soybean + corn, soybean + pea, soybean + tobacco, carrot + barley, etc.) provided fusion products which underwent cell division and callus formation. Fusion products initially were heterokaryocytes. In dividing heterokaryocytes, random distribution of mitotic nuclei was observed to be accompanied by multiple wall formation and to result in chimeral callus. Juxtaposition of mitotic nuclei suggested nuclear fusion and hybrid formation. Fusion of heterospecific interphase nuclei was demonstrated in soybean + pea and carrot + barley heterokaryons. Provided parental protoplasts carry suitable markers, the fusion products can be recognized. For the isolation and cloning of hybrid cells, fusion experiments must be supplemented with a selective system. Complementation of two non-allelic genes that prevent or inhibit growth under special culture conditions appears as the principle on which to base the selection of somatic hybrids. As protoplasts of some species have been induced to regenerate entire plants, the development of hybrid plants from protoplast fusion products is feasible and has already been demonstrated for tobacco.     

Embryonic and genetic manipulation in fish

Fishes,the biggest and most diverse community in vertebrates are good experimental models for studies of cell and developmental biology by many favorable characteristics.Nuclear transplantation in fish has been thoroughly studied in China since 1960s.Fish nuclei of embryonic cells from different genera were transplanted into enucleated eggs generating nucleo-cytoplasmic hybrids of adults.Most importantly,nuclei of cultured goldfish kidney cells had been reprogrammed in enucleated eggs to support embryogenesis and ontogenesis of a fertile fish.This was the first case of cloned fish with somatic cells.Based on the technique of microinjection,recombinant MThGH gene has been transferred into fish eggs and the firsh batch of transgenic fish were produced in 1984.The behavior of foreign gene was characterized and the onsed of the foreign gene replication occurred between the blastula to gastrula stages and random integration mainly occurred at later stages of embryogenesis.This eventually led to the transgenic mosaicism.The MThGH-transferred common carp enhanced growth rate by 2-4 times in the founder juveniles and doubled the body weight in the adults.The transgenic common carp were more efficient in utilizing dietary protein than the controls.An “all-fish” gene construct CAgcGH has been made by splicing the common carp β-actin gene (CA) promoter onto the grass carp growth hormone gene (grGH) coding sequence.The CAgcGH-transferred Yellow River Carp have also shown significantly fast-growth trait.Combination of techniques of fish cell culture,gene transformation with cultured cells and nuclear transplantation should be able to generate homogeneous strain of valuable transgenic fish to fulfil human requirement in 21^st century.     

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.