| Abstract: | The problems of a low efficiency of mammalian cloning are discussed with emphasis on the necessity of the expertise of each step of single cell reconstruction, beginning with microsurgical manipulations. The fact of cell content leakage when the cell is held during microsurgery or microinjections with the help of the conventional method using negative pressure in the holding micropipette was demonstrated in experiments on murine embryos. It was shown that the rate of cell content efflux depends on the value of negative pressure generated in the holding micropipette, and is directly proportional to the dimensions of its orifice and the duration of micromanipulations. An alternative method of cell fixation using the capillary forces of the holding micropipette was proposed. The method optimizes the process of cell fixation, reducing the holding effort by two orders of magnitude. As a result, 92% of embryos remain viable after fixation of embryo, as compared with 39% in the conventional technique. In order to diminish the cell damage produced by the tip of a microinstrument, a new technique of fabricating micropipettes was proposed. The improved method of filling the micropipette with viscous liquids, including DNA, which is described in details in the paper, enabled constant (non-stop) microinjection of more than 1000 cells by hand, without any special automatic device. |
