The biophysical aspects of reconstructing a single cell by the methods of cell engineering

The problem of the low efficiency of mammalian cloning is discussed with emphasis on the need of expert assessment of every step in single cell reconstruction, beginning with microsurgical manipulations. Experimental proof is provided for the impairment of cell integrity upon its fixation for microsurgery by the negative pressure in a conventional holding pipette. The ensuing leakage of the cell contents is shown to depend on the value of negative pressure, the duration of holding, and the size of the holder orifice. An alternative method of cell fixation is proposed, taking advantage of the capillary forces in the holding micropipette. This reduces the holding effort by two orders of magnitude and raises the cell survival upon microsurgery at least to 92%. To alleviate cell damage by instrumental invasion, a new technique is proposed for making micropipettes. Another novel method is offered for pipette filling with viscous liquid such as DNA solution, which allows continuous injection of more than 1000 cells.