Blood 94/9

WITH THE INCREASING integration of molecular genetics into the study of human diseases we have come to almost expect that, whenever a new gene is cloned, the next paper will report the consequences of ‘knocking out’ the gene. In a way, we are thus witnessing the logical updating of a century-old approach to the study of physiology. To define the function of an organ, a simple way was to remove it surgically; today, targeted homologous recombination has proven a powerful micro-surgical technique to remove the function of one gene at a time.1 However, lest we lose sight of our purpose, it is useful to draw a distinction. When a gene is isolated by positional cloning, we often have no idea of its normal function: therefore, knocking it out can suddenly provide an entirely new insight. When a gene is isolated instead on the basis of its known biochemical function, and the abnormality of that function is already known to cause a certain disease, no immediate surprise is to be expected. However, the knock-out approach can be still used to identify subtle pathophysiological features of the disease concerned, and especially to obtain an animal model lending itself to experimental manipulations and ultimately to investigating new forms of therapy.2 The PIG-A gene was cloned by the group of Miyata et al3 in 1993 by an elegant approach: the functional complementation of a cell line with a known defect in the biosynthesis of glycosyl phosphatidyl inositol (GPI) anchors. At the time it was also already known that the biochemical lesion in paroxysmal nocturnal hemoglobinuria (PNH) cells was in this metabolic pathway,4 and it became immediately clear that the ‘PNH gene’ had been really identified.5,6 We did not need to wait for any knock-out experiment to understand that a frameshift mutation of PIG-A would completely abrogate GPI biosynthesis,7 which would prevent CD59 from appearing on the surface of red blood cells (RBCs), which would cause these RBCs to be highly vulnerable to the final stage of the complement activation cascade. Complement activation can be triggered by an antigenantibody reaction or, through the alternative pathway, by the flimsiest of excuses: if CD59 is lacking this ends up in a large number of RBCs being destroyed. However, there are of course two important differences between PNH and other hemolytic anemias due to intracorpuscular abnormalities of the RBCs: (1) Because PNH is caused by a somatic mutation in a hematopoietic stem cell,8 rather than by an inherited mutation, the other cells in the body are not affected. (2) Even within the hematopoietic system, because only one or very few9 stem cells can be expected to have PIG-A mutations, there is always a residual population of cells with normal PIG-A and, therefore, normal GPI-linked molecules. Indeed, as originally suggested a decade ago,10 and as more recently highlighted in several recent reviews,11-15 a central issue to understanding PNH is to pinpoint the factors that determine the balance between the normal and the PNH hematopoietic populations. Thus, knock-out animals were needed not so much to prove that PIG-A mutations cause PNH, or to define more precisely the function of PIG-A, but very much to provide an animal model in which other components of the disease process could be investigated. Three groups promptly targeted pig-a in male mouse embryonic stem (ES) cells and, thanks to the fact that this gene is X-linked, they promptly obtained ES cells that were genetically and phenotypically GPI-negative and that proved viable.16-18 However, when these cells were used to produce pig-a null mice, the problem arising was similar to that confronting an over-enthusiastic physiologist who, a century ago, having successfully defined the function of the pancreas and of the pituitary gland by surgically removing them, decided to do the same with the liver or the heart. We all found that highcontribution chimeric embryos died in utero and, therefore, germ-line transmission could not be obtained. Low-contribution pig-a null mice were born, but their low numbers of GPInegative blood cells failed to provide a valid model of the human disease PNH. In this issue of Blood two papers report success in producing such a model, based on the technique of conditional knock-out, using the system known in jargon as cre-lox. Murakami et al19 have crossed male mice, in which two lox sequences had been engineered to flank exon 6 of pig-a, with female mice transgenic for cre driven by the universally active cytomegalovirus (CMV) promoter. The resulting females, heterozygous for a pig-a gene vulnerable to inactivation, died late in fetal life with brain abnormalities: but before they died, hematopoietic cells from their liver were used to reconstitute hematopoiesis in