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

碱性磷酸酶（AP）同工酶根据动物种类的不同，有3 ~ 4种，它们广泛分布于这些动物体内。然而，人们对其确切的生物学功能知之甚少，并将其作为一种“神秘的酶”。因此，通过建立小鼠ap ((1)D胚胎（EAP），(2)组织非特异性（TNAP）和(3)肠道（IAP）基因的“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这些动物可能出现的各种病理生理现象，试图揭示这些生物学功能和意义。由于“IAP敲除小鼠”的建立还在进行中，“EAP敲除小鼠”没有出现明显的表型变化，所以我们的观察主要集中在“TNAP敲除小鼠”出现的变化上。(1)这些“敲除小鼠”在表现出特征性癫痫发作后，大多数在断奶期间（平均2周）死亡。由于有充分的证据表明吡哆醛（维生素b ^6=VB^6）缺乏的小鼠在非常相似的癫痫发作中下降，我们假设TNAP基因失活会导致VB^6耗竭，这尚未得到实验证明。TNAP可能在吡哆醛磷酸的去磷酸化过程中起重要作用。(2)“基因敲除小鼠”另一个显著的病理变化是几乎全身骨骼出现异常矿化。在表现出明显结构畸形的成骨细胞中缺乏TNAP可能是造成这种病理的重要原因。关于IAP敲除小鼠的制备，用之前描述的靶向载体转染ES细胞。其中一个克隆经Southern blots证实为同源重组，并将该克隆注入bastocysts。我们目前正在培育这四只嵌合雄性，以获得种系杂合子小鼠。

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.