USE OF DOUBLE-REPLACEMENT GENE TARGETING TO REPLACE THE MURINE ALPHA-LACTALBUMIN GENE WITH ITS HUMAN COUNTERPART IN EMBRYONIC STEM-CELLS AND MICE

The mouse alpha-lactalbumin gene has been replaced with the human gene by two consecutive rounds of gene. targeting in hypoxanthine phosphor ibosyltransferase (HPRT)-deficient feeder-independent murine embryonic stem (ES) cells. One mouse alpha-lactalbumin allele was first replace d by an HPRT minigene which was in turn replaced by human alpha-lactal bumin. The end result is a clean exchange of defined DNA fragments wit h no other DNA remaining at the target locus. Targeted ES cells at eac h stage remained capable of contributing efficiently to the germ line of chimeric animals. Double replacement using HPRT-deficient ES cells and the HPRT selection system is therefore a powerful and flexible met hod of targeting specific alterations to animal genes. A typical strat egy for future use would be to generate a null mutation which could th en be used to produce multiple second-step alterations at the same loc us.