MICE LACKING ALKALINE PHOSPHATASE ISOZYMES.-MOLECULAR GENETICS AND PATHOLOGICAL INVESTIGATION

根据动物种类的不同，碱性磷酸酶(AP)同工酶有3~4种，广泛分布于动物体内。然而，人们对它们的确切生物学功能知之甚少，只留下了一种“神秘的酶”。因此，人们试图通过建立小鼠AP基因((1)D胚胎(EAP)，(2)组织非特异性(TNAP)和(3)肠道(IAP))的“敲除小鼠”，并进一步研究这些基因的基因组和表型表达以及在这些动物中可能出现的各种病理生理现象，来揭示这些生物学功能和意义。由于“IAP基因敲除小鼠”的建立仍在进行中，“EAP基因敲除小鼠”没有表现出任何明显的表型变化，因此观察集中在“TnAP基因敲除小鼠”中出现的变化。(1)大多数“基因敲除小鼠”在表现出典型的癫痫发作后，在断奶期间(平均2周)死亡。由于吡哆醛(维生素B^6=Vb^6)缺陷小鼠出现类似的癫痫发作已是有据可查的事实，我们推测TNAP基因失活会导致Vb^6的耗竭，这一点尚未得到实验证实。TnAP可能在吡哆醛磷酸脱磷中起重要作用。(2)在这些“基因敲除小鼠”中观察到的另一个明显的病理变化是全身骨骼出现异常矿化。成骨细胞中出现明显结构畸形的TNAP的缺失可能是导致这种病理的重要原因。关于IAP基因敲除小鼠的制备，将上述靶向载体导入ES细胞。其中一个克隆经Southern blotts鉴定为同源重组体，并将该克隆注射到杂交瘤细胞中。我们目前正在饲养这四只嵌合雄性小鼠，以获得生殖系杂合子小鼠。

There are 3 to 4, depending on the difference of the animal species, isozymes of alkaline phosphatase (AP), and they are widely distributed in those animal bodies. However, little is known about their exact biological functions and left as a "mysterious enzyme". Attempts were thus made to disclose those biological functions and significances by establishing "knock-out mice" for the genes of mouse APs ((1)D embryonic (EAP), (2) tissue non-specific (TNAP) and (3) intestinal (IAP)) and by further investigations on genomic and phenotypic expression of those genes and diverse patho-physiological phenomena which may appear in those animals. Since the establishment of "IAP knock-out mice" is still in process and "EAP knock-out mice" did not show any significant phenotypic changes, the observations were centered to the changes appeared in the "TNAP knock-out mice" . (1) The majority of these "knock-out mice" died during the weaning period (averagely 2 weeks) after they exhibited characteristic epileptic seizure. Sine it is a well documented fact that pyridoxal (vitaminB^6=VB^6) deficient mice fell in the very similar epileptic seizure, we presume TNAP gene inactivation would cause in VB^6 depletion which is not experimentally proved yet. TNAP could be taking an important role in dephosphorylation of pyridoxal phosphates. (2) Another conspicuous pathological change observed in those "knock-out mice" was abnormal mineralization appeared in bones of almost whole bodies. The deficiency of TNAP in osteoblasts which show marked structural deformity may strongly be responsible for thatpathology. Concerning IAP knockout mouse preparation, ES cells transfected with the targeting vector described before. One of the clones was confirmed as a homologous recombinant by Southern blots, and this clone was injected into the bastocysts. We are currently breeding the four chimeric males in order to obtain germline heterozygote mice.