Gerd Weber

Manipulation of Cells, Organelles, and Genomes by Laser Microbeam and Optical Trap

Publisher Summary This chapter discusses manipulation of cells, organelles, and genomes by laser microbeam and optical trap. It examines how lasers of intermediate and high-power densities are influencing the micromanipulation techniques and finding use in cellular and molecular biology. Lasers are light sources providing light from ultraviolet, green, red, infrared, and up into the microwave region. A specific laser works at a well-defined wavelength. Lasing media may be gases, liquids, crystals, glass, or semiconductors. The performance of a laser is described by several typical parameters: working wavelength and average power are important for continuous wave (cw) lasers and for pulsed lasers. Additional parameters for the pulsed lasers are the energy delivered by a single pulse, the number of pulses per second, and the peak power in a single pulse. A laser pulse can deliver DNA into specific cells through cell walls and membranes. The high spatial accuracy can deliver energy to fuse membranes or perform surgery or micromanipulations in cells, tissues, or chromosomes. Particles of the size of 1 μm, such as polystyrene beads, can also be manipulated by laser light. A focused laser is required and the particles are balanced on such a focused laser beam. Dielectric particles are pulled into the focus of a laser. The experimental arrangement for such an experiment is called an “optical trap” or “optical tweezers.”

The light microscope on its way from an analytical to a preparative tool

Light intensities of up to 1013 W/cm2 can be generated by focusing light, particularly laser pulses, into a microscope. Such power densities can be used to cut, perforate, or fuse microscopic objects with submicrometre accuracy. Suitable light sources for such a ‘microbeam’ are nitrogen lasers with a working wavelength of 337 nm, frequency‐multiplied Neodym YAG lasers (266 or 355 nm) or excimer lasers (308 nm). In combination with dye lasers, tunable microbeams covering the wavelength range from the ultraviolet to the infra‐red can be constructed. Such laser microbeams can be used to modify microchip substrates. Micro‐injection of materials into biological cells or fusion of selected cell pairs under total microscopic control is also possible. Using the same equipment, elongated biological objects can be microdissected with submicrometre precision, for example in attempts to isolate DNA from a specific region of the human genome.

Microperforation of plant tissue with a UV laser microbeam and injection of DNA into cells

Direct injection of DNA into mammalian cells has become a standard procedure in studying gene expression [1]. In plant cells the rigid cell wall prevents DNA introduction. This drawback can only be bypassed by digesting the cell wall to yield relatively fragile protoplasts. DNA can be introduced into protoplasts by microinjection [2, 3] or electroporation [4, 5]. Some major problems are associated with this approach. In particular, protoplasts of crops of which many belong to monocotyledons do not grow in tissue culture. The regeneration of plants from individual protoplasts is only possible in some species [6]. Furthermore, prolonged tissue culture associated with protoplast regeneration often is associated with somaclonal alterations [7]. Therefore, it would be desirable to introduce DNA directly into selected cells of plant tissue. DNA was injected into plant tissue by using high-velocity petlets [8]. However, this technique permitted DNA to be entered into cells in a rather random way. In contrast, UV laser microbeams can be aimed very accurately. They have been used successfully to induce selective cell fusion [9, 10], to microdisseet chromosomes [11], and introduce DNA into chloroplasts [12]. Laser beams based on Nd-YAG (more powerful in the visible part o f the spectrum) have been used to manipulate subcellular structures [13] and to introduce DNA into mammalian cells

Storage of Cell Suspensions and Protoplasts of Glycine max (L.) Mem, Brassica napus (L.), Datura innoxia (Mill.), and Daucus carota (L.) by Freezing

Summary The effect of freezing on survival was studied in fast growing cell suspensions of Glycine max (L.) Merr., Datura innoxia (Mill.), Brassica napus (L.) and Daucus carota (L.). The cells were pretreated by partial plasmolysis in culture medium containing 1.0 M sorbitol. The pretreated cells were subjected to a decrease in temperature at a rate of 1 dgC/min from 0° to -35°C. The frozen cells were stored at either -80°C or -196°C. Thawing of the cells was performed in the presence of 1.0 M sorbitol. After storage in liquid nitrogen for up to 6 months at least 19 % of the colony forming capacity was retained, as compared to non-frozen controls. After cryopreservation the four cell lines retained their growth characteristics and chromosome number. An auxotrophic mutant of soybean preserved in liquid nitrogen displayed its phenotype after thawing. Cryopreserved cells of Daucus carota were regenerated to normal plants at a high frequency. Protoplasts of G. max were frozen in medium containing 0.7 M sorbitol under otherwise identical conditions. After storage in liquid nitrogen clones developed from individual protoplasts with an efficiency of 10-3 as compared to non-frozen controls.

Individual Culture of Selected Single Cells and Protoplasts of Higher Plants in Microdroplets of Defined Media

Summary Single cells from suspension cultures of Datura innoxia (Mill.) and mesophyll protoplasts of Nicotiana tabacum (L.) cv. xanthi were cultured individually in 10–25 nl microdroplets of synthetic and unconditioned culture medium under a thin layer of mineral oil. More than 50 % of individually cultivated single cells of Datura innoxia (Mill.) survived for more than two weeks, divisions were found in more than 30 % of the cells, callus formation occured at a frequency of up to 30 %. Buffering of media is strictly required for individual culture of single cells under our conditions. The pH of the culture medium has a more pronounced influence on the frequency of callus formation than on the proportions of surviving cells and cells undergoing one division only. More than 40 % of individually cultivated mesophyll protoplats of Nicotiana tabacum (L.) cv. xanthi survive for more than two weeks. Divisions and callus formation occur at frequencies of more than 30 %, and 30 %, respectively.

Genetic manipulation of plant cells and organelles with a laser microbeam

Direct introduction of cloned genes into eukaryotic cells has proven to be a powerful tool to study the molecular mechanisms of gene regulation. Direct transformation was mainly accomplished by delivering cloned genes into protoplasts or tissue (1, 2). Protoplasts, however, are comparatively fragile. Furthermore, in many species they cannot be regenerated into plants (3). Introduction of DNA into organelles like chloroplasts is even more difficult. DNA destined for the chloroplast has to be delivered through plasma membrane and chloroplast envelope into the stroma of the organelle.

Synchronization of protoplasts from Glycine max (L.) Merr. and Brassica napus (L.)

Cells of Glycine max originating in a suspension culture and cells of Brassica napus prepared from hypocotyls were synchronized. Synchronization was achieved by preparing protoplasts in the usual way and subsequently letting the protoplasts regenerate into cells by removing the cell-wall-digesting enzymes. More than 70% of the cells had divided synchronously at the end of the first cycle as determined by the mitotic index. The high frequency of mitosis critically depended on the osmolality of the medium. The duration of the S-phase was estimated by measuring the activity of thymidylate kinase as well as incorporation of [3H]deoxythymidine into acid-insoluble material. The data indicate that synchronization is induced by resetting the cell cycle.

GENETIC CHANGES INDUCED IN HIGHER PLANTS BY A UV LASER MICROBEAM

Plant cell walls can be perforated by a UV laser microbeam. Cells and immature pollen grains (microspores) of Brassica napus L. continued to grow in culture after being punctured by a laser pulse. During laser treatment, uptake of buffer into the cytoplasm of cells could be achieved by plasmolyzing cells in a hypertonic solution. Using this approach, plasmid DNA carrying a gene for bacterial glucuronidase was introduced into B. napus cells. The marker gene was expressed by 70% of the treated cells. Likewise, the gene for resistance to the antibiotic hygromycin B was introduced into B. napus cells and was stably integrated into growing cells. Furthermore, DNA was also incorporated into chloroplasts. First, DNA was injected into the cytoplasm and then the organelles were punctured by laser pulses. When a marker gene coding for triazine resistance was introduced into the organelles, transient expression at the level of callus cultures was observed.

Synchronization of cells from higher plants

Controle de la regeneration de cellules a partir de protoplastes par remplacement du milieu de culture en eliminant les enzymes de degradation des parois

Methotrexate-resistant somatic cells of Glycine max (L.) Merr.: selection and characterization

A total of 70 methotrexate-resistant cell lines were isolated from UV-irradiated fast growing suspensions of Glycine max(L.) Merr. The resistant cells grew in 4 X 10(-4) M MTX without significant reduction in growth rate, whereas 100 % of wild type cells died in 2.2 x 10 (-7) M within 24 h. The frequency of resistant cell clones was 2 x 10(-8) per cell generation. Two different selection strategies, a single-step and a multiple-step protocol yielded phenotypically identical resistant lines. All resistant lines have retained their resistance over more than 500 cell generations in the absence of selection. Resistant cells plated on MTX could be selected from an excess of wild type cells. Ten percent of the resistant lines internalized MTX at a reduced rate. DHFR activity in wild type and resistant cells was equally sensitive to inhibition by MTX. Resistant cells did not display an increased activity of DHFR. Likewise MTX binding to cell extracts was the same for all cell lines. In the wild type MTX-blockage of cell division could be partially released by 2 x 10(-4) M thymidine. In the presence of MTX thymidine had no influence on the growth of resistant cells.