New techniques for isolation of single prokaryotic cells

Since the 1960s, several new attempts have been made to improve the management of single prokaryotic cells using micromanipulator techniques. In order to facilitate the isolation of pure cultures we have recently developed an improved micromanipulation method for routine work. With the aid of this method single prokaryotic cells can be picked out of a mixed community under direct visual control. The isolated aerobic or anaerobic cells can be grown in pure culture or can be subjected to single cell PCR. Other powerful and completely new approaches are the applications of laser micromanipulation systems, such as optical tweezers or laser microdissection techniques. Of the latter two methods only optical tweezers have been successfully applied to cloning prokaryotic cells.     

Microdissection of banded human chromosomes

Summary Physical dissection of metaphase chromosomes is the most straightforward approach for the isolation of DNA sequences from specific chromosome regions. However, conventional microdissection techniques are too crude and inefficient for analysis of the human genome. Here we describe a technique for the precise dissection of single bands from GTG-banded chromosomes. Cells from normal amniotic fluid cell cultures are harvested by the pipette method. Microdissection is performed on an inverted microscope (magnification 1250 x) with the help of extended siliconized glass needles and an electronically controlled micromanipulator. Enzymatic amplification of the dissected DNA allows the construction of band-specific DNA libraries from as few as 20 dissected chromosome fragments.     

莴苣胚囊细胞分离

用酶解和解剖方法分离了莴苣的卵细胞,助细胞,中央细胞和合子。莴苣子房先在酶液中酶解40～50min,然后在不含酶的分离液中用解剖针解剖子房。在解剖出的胚囊中,可看到卵细胞,两个助细胞和中央细胞的轮廓。将胚囊的合点端切破,轻轻挤压胚囊的珠孔端,四个细胞即可逸出。在最佳条件下,90min可从40个子房中分离出29个胚囊,进一步从中分离出11个卵细胞。分离出的胚囊细胞用显微操作仪收集备用。莴苣卵细胞的成功分离为进行离体受精探索创造了条件。     

TCR‐β repertoire analysis of antigen‐specific single T cells using a high‐density microcavity array

The antigen specificity of cytotoxic T cells, provided by T‐cell receptors (TCRs), plays a central role in human autoimmune diseases, infection, and cancer. As the TCR repertoire is unique in individual cytotoxic T cells, a strategy to analyze its gene rearrangement at the single‐cell level is required. In this study, we applied a high‐density microcavity array enabling target cell screening of several thousands of single cells for identification of functional TCR‐β gene repertoires specific to melanoma (gp100) and cytomegalovirus (CMV) antigens. T cells expressing TCRs with the ability to recognize fluorescent‐labeled antigen peptide tetramers were isolated by using a micromanipulator under microscopy. Regularly arranged cells on the microcavity array eased detection and isolation of target single cells from a polyclonal T‐cell population. The isolated single cells were then directly utilized for RT‐PCR. By sequencing the amplified PCR products, antigen‐specific TCR‐β repertoires for gp100 and human cytomegalovirus antigens were successfully identified at the single‐cell level. This simple, accurate, and cost‐effective technique for single‐cell analysis has further potential as a valuable and widely applicable tool for studies on gene screening and expression analyses of various kinds of cells. Biotechnol. Bioeng. 2010;106: 311–318. © 2010 Wiley Periodicals, Inc.     

胡萝卜精、卵细胞、助细胞和中央细胞的分离

用两个解剖针挤压胡萝卜花粉使其破裂释放出精细胞。用酶解-解剖方法分离胡萝卜胚囊中的卵细胞、助细胞和中央细胞。胡萝卜胚珠先在酶液中酶解40~50min,然后将其转移到不含酶的分离液中用解剖针解剖胚珠。将胚珠的合点端切破,轻轻挤压胚珠的珠孔,卵细胞、助细胞和中央细胞即可逸出。在最佳条件下,20min可从20个胚珠中分离出5个卵细胞。对分离胚囊细胞的渗透压和酶液成分进行了筛选。分离出的卵细胞用显微操作仪收集。胡萝卜精、卵细胞的成功分离为在双子叶植物中进行离体受精探索创造了条件。     

应用激光显微切割技术检测单个结肠细胞的基因表达

目的：探讨检测单个结肠细胞的基因表达的方法。方法：应用激光显微切割技术(1aser micmdissection)从冰冻切片上将单个结肠细胞切下,提取总RNA,将RNA逆转录成cDNA,采用巢式逆转录聚合酶链反应(nested RT—PCR)检测mRNA的表达。结果：在显微镜下用紫外激光显微切割机,将单个结肠细胞成功切下,提取RNA后,逆转录成cDNA,经过巢式RT—PCR扩增后,扩增产物在琼脂糖凝胶上清晰可见。结论：联合应用激光显微切割和巢式RT—PCR可以检测单个结肠细胞的基因表达。     

In vitro separation and development of sheep blastomeres

Four cell embryos collected by laparatomy from Sardinian breed ewes superovulated with FSH-p (16 mg Sigma), were divested of their zonae pellucidae (ZP) by micromanipulation or chemical methods (pronase 0.5%, tyrode pH 2.2). The blastomeres were separated by pipetting using a flame polished pasteur pipette in a Ca free medium (PBS. Sigma) and were inserted into previously evacuated Z.P. using a Leitz micromanipulator. The Z.P. were removed either mechanically or with acid tyrode; pronase was unable to digest them after incubation at 30 degrees C for 120 minutes. The single blastomeres were cocultured on a monolayer of ovine oviductal epithelial cells in TCM 199 + 10 FCS at 38 degrees C in 5% CO2 for 60 hours. No developments were observed in blastomeres obtained by acid digestion of the ZP while 50% of the other blastomeres continued their development until the 16 cell stages. Our results suggest that coculture with oviductal epithelial cell monolayers can support in vitro development of single ovine blastomeres.     

An improved method for single cell isolation of prokaryotes from meso-, thermo- and hyperthermophilic environments using micromanipulation

This study presents an improved system that enables isolation of single viable prokaryotic cells from a mixture of cells. The system is based on an inverted microscope, a microinjector and a micromanipulator. The isolated cell is captured in a microcapillary from a volume of 400 μl and transferred to an appropriate growth medium. Validation of the system was performed using two fluorescent strains: Pseudomonas putida expressing red fluorescent protein (DsRed), and Escherichia coli expressing green fluorescent protein (GFP). A mixture (100:1) of the constructed fluorescent strains was subjected to isolation experiments and nine out of ten individually isolated cells yielded axenic cultures of E. coli. Upon construction and validation, the system was used to isolate and subsequently cultivate axenic cultures of the thermophilic Archaeon Metallosphaera sedula and the hyperthermophilic Archaeon Sulfolobus solfataricus from enriched hot spring samples. The high efficiency of single-cell isolation and cultivation demonstrated over a range of temperatures—90% (30°C), 85% (70°C) and 95% (80°C)—from different environments is probably due to the elimination of osmotic stress and limitation of temperature fluctuations during the isolation process, as a result of the large sample volume from which the cells are isolated.     

Frequent occurrence of diploid a or α sporogenous mater cells and construction of ploidy series in the sake yeast kyokai no. 9

Diploid a or α sporogenous mater cells were frequently obtained in the sake yeast Kyokai no. 9 by heat treatment followed by random spore plating. Polyploid cells were constructed between the opposite mater cells by the mass mating technique, followed by the isolation of the zygotes using a micromanipulator.     

Obtainment of chimeric blastocysts of mice by methods of laser nanosurgery

The procedure of obtainment of chimeric blastocysts of mice by laser nanosurgery methods with-out using any other techniques is described. To perform the experiments, a special laser micromanipulator was invented. The murine embryonic stem cells (ESC), which were transformed with pEF-GFP vector, encoding the green fluorescent protein, were used in the experiments. ESC were introduced into the perivitelline space of murine embryos at the stage of 8 cells using the laser micromanipulator. The operated embryos were cultured in vitro until the stage of emergence from zona pellucida. The fluorescence and its precise localization were registered using a confocal microscope. It was shown for the first time that the inclusions of ESC introduced with the lased micromanipulator were found not only in the inner cell mass (ICM) but also in the trophectoderm of the chimeric blastocyst. The technology of nanosurgical operations at early stage preimplanted mammalian embryos using laser techniques opens great opportunities not only for solution of fundamental tasks of experimental embryology of mammals but also for obtainment of chimeric and transgenic animals with predetermined genotype.     

Selection and microculture of single embryogenic cell clusters in japanese conifers: Picea jezoensis, Larix leptolepis and Cryptomeria japonica

Summary A microculture method for single embryogenic cell clusters was established for several Japanese conifer species, namely Picea jezoensis, Larix leptolepis and Cryptomeria japonica. Individual small, dense cell clusters were identified and picked up by a micromanipulator and cultured in 50 μl liquid medium in a 96-well culture plate. In all three species studied, a majority of the cell clusters actively proliferated within the wells. Maturation of somatic embryos was successful when the newly proliferated cell clusters were transferred to solid abscisic acid-containing medium. Thus, the small, dense cell clusters could be a useful morphological marker for cells capable of proliferation and differentiation.     

改良的PEP方法在无创性产前基因诊断中的应用

应用显微操作技术获取孕妇外周血中的单个有核红细胞,改良的PEP方法扩增单个有核红细胞的全基因组DNA;在此基础上,应用荧光标记聚合酶链反应扩增9个微卫星片段,进行基因型分析判定单个有核红细胞来源。综合性别和DMD基因内的数个STR位点连锁分析进行DMD基因诊断,应用PCR-STR连锁分析进行PKU基因诊断。结果显示,对10例DMD高危胎儿中的6例成功地进行了无创性产前基因诊断。同时对1例PKU也成功地进行了无创性产前基因诊断。改良的PEP方法扩增单个细胞的全基因组可以满足基因诊断的要求,是无创性产前基因诊断中一种极有价值的全基因组扩增的方法。 Abstract：We investigated the feasibility of using improved primer extension preamplificat ion method to diagnose DMD and PKU. The fetal nucleated red blood cells from the peripheral blood of pregnant women were detected and individually retrieved into glass capillary pipettes using a micromanipulator under microscopic observation. The whole genome of a single cell was amplified by improved primer extension preamplification (PEP).Genotypes were analyzed by amplifying the 9 STR fragments using fluorescence?PCR technique and NRBC's(nucleated red blood cell) origin w as determined.We diagnosed DMD prenatally using sex determination and linkage an alysis of several STR sites of dystrophin,and we diagnosed PKU prenatally using PCR?STR linkage analysis.6 of 10 potential DMD patients were diagnosed,includin g 1 male fetal patient,1 potential PKU patient was also diagnosed.The improved P EP method is a very valuable method of amplifying the whole genome of single cel ls,and the products of amplification are enough to the requirements of DNA in no n-invasive prenatal diagnosis.     

Specific single-cell isolation and genomic amplification of uncultured microorganisms

We in this study describe a new method for genomic studies of individual uncultured prokaryotic organisms, which was used for the isolation and partial genome sequencing of a soil archaeon. The diversity of Archaea in a soil sample was mapped by generating a clone library using group-specific primers in combination with a terminal restriction fragment length polymorphism profile. Intact cells were extracted from the environmental sample, and fluorescent in situ hybridization probing with Cy3-labeled probes designed from the clone library was subsequently used to detect the organisms of interest. Single cells with a bright fluorescent signal were isolated using a micromanipulator and the genome of the single isolated cells served as a template for multiple displacement amplification (MDA) using the Phi29 DNA polymerase. The generated MDA product was afterwards used for 16S rRNA gene sequence analysis and shotgun-cloned for additional genomic analysis. Sequence analysis showed >99% 16S rRNA gene homology to soil crenarchaeotal clone SCA1170 and shotgun fragments had the closest match to a crenarchaeotal BAC clone previously retrieved from a soil sample. The system was validated using Methanothermobacter thermoautotrophicus as single-cell test organism, and the validation setup produced 100% sequence homology to the ten tested regions of the genome of this organism.     

Preparation of single cells from aggregated Taxus suspension cultures for population analysis

A method for the isolation of single plant cells from Taxus suspension cultures has been developed for the analysis of single cells via rapid throughput techniques such as flow cytometry. Several cell wall specific enzymes, such as pectinase, pectolyase Y-23, macerozyme, Driselase(R), and cellulase were tested for efficacy in producing single cell suspensions. The method was optimized for single cell yield, viability, time, and representivity of aggregated cell cultures. The best combination for single cell isolation was found to be 0.5% (w/v) pectolyase Y-23 and 0.04% (w/v) cellulase. High viability (>95%) and high yields of single cell aggregates (>90%) were obtained following 4 hours of digestion for four separate Taxus cell lines. In addition, methyl jasmonate elicitation (200 microM) was found to have no effect on three of the four tested Taxus lines. Isolated single cells were statistically similar to untreated cell cultures for peroxidase activity (model cell wall protein) and paclitaxel content (secondary metabolite produced in Taxus cell cultures). In comparison, protoplasts showed marked changes in both peroxidase activity and paclitaxel content as compared to untreated cultures. The use of flow cytometry was demonstrated with isolated cells that were found to have > 99% viability upon staining with fluorescein diacetate. The development of a method for the isolation of single plant cells will allow the study of population dynamics and culture variability on a single cell level for the development of population models of plant cell cultures and secondary metabolism.     

Construction of poplar (Populus tremula) chromosome 1-specific DNA library by using a microdissection technique

A method for single-chromosome microdissection and microcloning was established in forest plants using poplar (Populus tremula) as a model. By use of meristematic cell division in root tip and the wall degradation hypotonic method, well-spread poplar metaphase chromosome spreads showing low contamination were quickly prepared and fitted for chromosome microdissection. An individual chromosome 1 was microdissected from the metaphase spreads of poplar root-tip cells with a fine glass needle controlled by a micromanipulator. The dissected chromosome was amplified in vitro by theSau3A linker adaptor-mediated PCR technique, by which 200- to 3000-bp smear DNA fragments were obtained. Southern hybridization results showed that the PCR products from the single poplar chromosome were homogeneous with poplar genomic DNA, indicating that DNA from the single chromosome has been successfully amplified. Next, the second-round PCR products from the single chromosome 1 were cloned into T-easy vectors to generate a DNA library of the chromosome 1. About 3×10⁵ recombinant clones were obtained. Evaluation based on 160 randomly selected clones showed that the sizes of the cloned inserts varied from 230–2200 bp, with an average of 800 bp. Therefore, this research suggests that microdissection and microcloning of single small chromosomes in forest plants is feasible.     

Preparation of single rice chromosome for construction of a DNA library using a laser microbeam trap

We report the development of a laser micromanipulation system and its application in the isolation of individual rice chromosomes directly from a metaphase cell. Microdissection and flow sorting are two major methods for the isolation of single chromosome. These methods are dependent on the techniques of chromosome spread and chromosome suspension, respectively. In the development of this system, we avoided using chromosome spread and cell suspension was used instead. The cell wall of metaphase rice cell was cut by optical scissors. The released single chromosome was captured by an optical trap and transported to an area without cell debris. The isolated single chromosome was then collected and specific library was constructed by linker adaptor PCR. The average insert size of the library was about 300 bp. Two hundred inserts of chromosome 4 library were sequenced, and 96.5% were aligned to the corresponding sequences of rice chromosome 4. These results suggest the possible application of this method for the preparation of other subcellular structures and for the cloning of single macromolecule through a laser microbeam trap.     

The expression of hex A and hex B isozymes of hexosaminidase in parental and experimental human fibroblast cells and their components

The expression of the two major isozyme forms of hexosaminidase (EC 3.2.1.30), hesoxaminidase A and hexosaminidase B, has been examined. The parental cells and/or cellular components of parental cells are individually fused using inactivated Sendai virus with the aid of a micromanipulator. The progeny cells produced from such hybrids are subjected to a microenzymatic assay which allows measurements at the single cell level. The lysosomal-deficient cells used in this study are Tay-Sachs and Sandhoff fibroblasts, and the normal cells used are WI-38 (fetal lung fibroblasts), amniotic fluid cells (GM 473), and JASD₃ (normal human foreskin). The results show that the ratio of cell components which are fused to form the experimental cell affects the percentage of hexosaminidase A expressed in the progeny cells. Furthermore, our results imply the presence of a “factor” in the Sandhoff cell's cytoplasm which, together with the Tay-Sachs nucleus, is necessary for hexosaminidase A expression in the experimental cell's progeny.     

Microdissection and PCR Amplification of Single Soybean Chromosome

Glass needles were successfully used to dissect the soybean (Glycine max L. ) single chromosome under the micromanipulator in this research. Two dissected soybean chromosomes were digested by Sau3A in two 0.5 mL Eppendorf tubes respectively. The two ends of chromosomal fragments were ligated with Sau3A linker adoptor. After two rounds of PCR amplification, smear DNA fragments ranged from 0.3 to 3 kb were acquired. Southern hybridization result showed the PCR products from the two single soybean chromosomes were homogeneous with the soybean genomic DNA, indicating that DNAs from the two single chromosomes have been successfully amplified. At the same time, the amplified products from the two of the distinguished single chromosome appeared somewhat different. The authors dissected the small chromosomes only by a traditional inverted microscope. Therefore, this research provides a plausible chance for amplification and microcloning of single small chromosomes.