62) Methods of producing chimeras

Chimera is the animal consisting of different genotype cells which originated more than 1 zygotes.

Methods of creating chimeras:

• Embryonic aggregation chimeras

• Injection of embryonic cells to blastocyst

• Therato-carcinomas (for example, the introducton of embryonic cells to the adult)

• Xenotransplantation (transplantations of cells, tissue, organs from one adult to another)

57) Methods of genetic engineering: agrobacterial genetic transformation

The ability of Agrobacterium to transfer genes to plants and fungi is used in biotechnology, in particular, genetic engineering for plant improvement. A modified Ti or Ri plasmid can be used. The plasmid is 'disarmed' by deletion of the tumor inducing genes; the only essential parts of the T-DNA are its two small (25 base pair) border repeats, at least one of which is needed for plant transformation. Marc Van Montagu and Jozef Schell at the University of Ghent (Belgium) discovered the gene transfer mechanism between Agrobacterium and plants, which resulted in the development of methods to alter Agrobacterium into an efficient delivery system for gene engineering in plants.^[7][8] A team of researchers led by Dr Mary-Dell Chilton were the first to demonstrate that the virulence genes could be removed without adversely affecting the ability of Agrobacterium to insert its own DNA into the plant genome (1983).

The genes to be introduced into the plant are cloned into a plant transformation vector that contains the T-DNA region of the disarmed plasmid, together with a selectable marker (such as antibiotic resistance) to enable selection for plants that have been successfully transformed. Plants are grown on media containing antibiotic following transformation, and those that do not have the T-DNA integrated into their genome will die. An alternative method is agroinfiltration.

Transformation with Agrobacterium can be achieved in two ways. Protoplasts, or leaf-discs can be incubated with the Agrobacterium and whole plants regenerated using plant tissue culture. A common transformation protocol for Arabidopsis is the floral-dip method: the flowers are dipped in anAgrobacterium culture, and the bacterium transforms the germline cells that make the female gametes. The seeds can then be screened for antibiotic resistance (or another marker of interest), and plants that have not integrated the plasmid DNA will die.

Agrobacterium does not infect all plant species, but there are several other effective techniques for plant transformation including the gene gun.

59)Cryopreservation of plant cell and tissue Cryopreservation - freezing at very low temperatures. Usually it is carried out in liquid nitrogen at-196oC.

Freezing Plant cells from animals differs mainly presence preculture stage.

Cryoprotectants - substances that reduce the damaging effects of physical and chemical factors in cryopreservation. These include sucrose, dextran, ethylene glycol, polyvinyl pyrrolidone, dimethylsulfoxide (DMSO), glycerol. To determine the toxicity of the cryoprotectant cell was kept at room temperature in its various concentrations for 30 - 50 minutes, then determine their viability.

Typically, cooling is carried out in two phases (Fig. 26):

Stage 1: from-28oC to +20 at a rate of 1 degree per minute (plant cell freezing rate of 0.5 degrees per minute to -35 ° C), maintained at this temperature for 15 minutes.

2nd stage: immersion in liquid nitrogen (instantaneous cooling to - 196oC).

Freezing produced in special vehicles. In their absence, - an alcohol bath (0.5 - 1 liter alcohol is poured into the metal thermos flask was immersed in it for 15 minutes vial was added with stirring and liquid nitrogen or dry ice, the temperature is brought to-32oC (temperature should not exceed -28 or below-32C.) Next transferred into liquid nitrogen ampoule.

When thawing ampules forceps transferred into a water bath at 37 - 40 ° C, an ampoule of 1 mL were thawed for 0.5 - 1 minutes.

After thawing, the cells were washed in either growth medium (animal) or supportive environment. Plant cells can also be laundered 3 - 10% sucrose solution.

The cells were checked for viability using the vital dye staining dead cells. The final criterion is a clear return to growth on standard culture media used for the culture.

Immortalized mammalian cells after thawing have an increased susceptibility to viruses, which manifests itself in the first two passages. Next, the sensitivity of returns to the original.

The slowdown

Slowdown can be achieved by the following methods:

1. Storage under a layer of mineral oil (for fungal and bacterial cultures).

2. Change in gas composition and atmospheric pressure inside the culture vessel.

3. Changing light conditions.

4. Cooling stop temperature to active growth.

5. Hormonal and osmotic inhibitors. From hormonal inhibitors most often used hlorholinhlorida (for plant cells) of osmotic - mannitol in a concentration of 3-6%.

6. Replacement of CaCl2 to Ca (NO3) 2 in the culture media.

For the potato as a method allowing to keep the gene pool, it is recommended nodulation in test tubes.

61 Rules on keeping and breeding facilities in the laboratory. Proper housing and management of animal facilities are essential to animal well-being, to the quality of research data and teaching or testing programs in which animals are used.The primary enclosure (usually a cage, pen, or stall) provides the limits of an animal's immediate environment.* Allow for the normal physiologic and behavioral needs of the animals, including urination and defecation, maintenance of body temperature, normal movement and postural adjustments, and, where indicated, reproduction. * Allow conspecific social interaction and development of hierarchies within or between enclosures. * Make it possible for the animals to remain clean and dry (as consistent with the requirements of the species). * Allow adequate ventilation. * Allow the animals access to food and water and permit easy filling, refilling, changing, servicing, and cleaning of food and water utensils. * Provide a secure environment that does not allow escape of or accidental entrapment of animals or their appendages between opposing surfaces or by structural openings. * Are free of sharp edges or projections that could cause injury to the animals. * Allow observation of the animals with minimal disturbance of them. * Room air is mixed with at least 50% fresh air (that is, the supply air does not exceed 50% recycled air). * Husbandry practices, such as bedding-change and cage-washing frequency, and the preparation of recycled air used are sufficient to minimize toxic gases and odors. * Recycled air is returned only to the room or area from which it was generated, except if it comes from other than animal-housing areas. * Recycled air is appropriately conditioned and mixed with sufficient fresh air to address the thermal and humidity requirements of animals in that space. In general, lighting should be diffused throughout an animal holding area and provide sufficient illumi nation for the well-being of the animals and to allow good housekeeping practices, adequate inspection of animals including the bottom-most cages in racks and safe working conditions for personnel. Some conditions might require increased environmental temperatures, such as postoperative recovery, maintenance of chicks for the first few days after hatching, housing of some hairless rodents, and housing of neonates that have been separated from their mothers. Noise produced by animals and animal-care activities is inherent in the operation of an animal facility. Therefore, noise control should be considered in facility design and operation. Noisy animals such as dogs, swine, goats, and nonhuman primates should be housed away from quieter animals, such as rodents, rabbits, and cats. Radios, alarms, and other sound generators should not be used in animal rooms unless they are parts of an approved protocol or an enrichment program.