Analysis of Homologous Recombination in Cultured Mammalian Cells

Direct microinjection of DNA into cultured mammalian cells by glass micropipettes provides a number of advantages as a method for studying recombination in somatic cells. We can choose where in the cell the DNA is introduced, the nucleus, cytoplasm, or even cellular organelles such as a mitochondrion. By injecting the DNA directly into the nucleus, we obviate the need for exogenous DNA to transit the cytoplasm. During the transit from cytoplasm to the nucleus, newly introduced DNA may suffer considerable damage (Calos et al. 1983; Razzaque et al. 1983; Wake et al. 1984). The number of plasmid DNA molecules introduced into a cell by microinjection can be controlled within a range of ± 20%. Even with a very low input of plasmid DNA per cell (i.e., 3–5 copies/cell), a high DNA-mediated transformation efficiency of 20–30% is still achieved (Capecchi 1980; Folger et al. 1982). Finally, the form of DNA introduced...