ABSTRACT
The Bacterial Artificial Chromosome (BAC) cloning system has been developed to stably maintain large fragments of genomic DNA in E. coli. The quantity of BAC DNA recovered from 1–2 ml of culture is sufficient for restriction digestion and analytical or preparative agarose gel electrophoresis using ethidium bromide staining. The largest sized clones can be used when the location of the gene is not certain, or in other cases, the smallest clone known to contain the intact gene and its regulatory segments can be used. When complementation can be readily scored, the preparation of transgenic animals can be used to assist gene identification during positional cloning. Linearized DNA is mixed with synthetic cationic lipid forming vesicles, which are then fused to the plasma membrane of the recipient ES cells. In comparison with other ES cell transfection procedures (electroporation, calcium phosphate precipitation), lipofection gives higher efficiency and also minimizes mechanical breakage.
