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STUDY OF BIOLOGICAL SIGNIFICANCE OF ANTIZYMES IN KNOCKOUT MICE

抗酶在基因敲除小鼠中的生物学意义研究

基本信息

批准号：
12470031
负责人：
MATSUFUJI Senya
金额：
$ 4.29万
依托单位：
Jikei University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2003
项目状态：
已结题

项目摘要

1) Antizymes are polyamine-induced proteins that negatively regulate cellular polyamines. We previously reported that homozygous AZ1 knockout mice with a mixed genetic background are partially embryonic lethal. Homozygous embryos with the C57BL/6J background showed a high mortality rate at embryonic day (E) 13.5-16.5. Hepatic hypoplasia was the earliest abnormality ever detected. Subsequent analysis revealed a reduction of hepatic hemopoietic cells with apoptotic morphology at E12.5-13.5 and anemia with increased erythroblasts in the peripheral circulation, indicating that the secondary embryonic hematopojesis in the liver was a most affected process in the AZ1 knockout mice. Presence of modifier gene(s) for the lethal phenotype of AZ1 knockout mice was indicated in our genetic analysis. To identify the modifier gene(s) using positional assignment, we have prepared a congenic line in MSKR mice, a strain originated from wild molossinus mice in Japan.2) Studies using primary and immortalized cultured cells derived from AZ1 knockout mice demonstrated that ODC repression by polyamines was retarded in these cells and that AZ2 partially compensated the polyamine regulation. Thus AZ2 has activities to repress ODC and inhibit polyamine uptake both in a polyamine-dependent manner.3) We also prepared AZ2 knockout mice. Homozygous AZ2 knockout mice showed no apparent phenotype. AZ1-AZ2 double homozygous knockout mice were complete embryonic lethal by E14.5. The systemic developmental retardation started by E10.5 before the formation of liver, suggesting that the cause of death in the double knockout embryos differs from AZ1 knockouts.4) Long term observation of survivors failed to show the increase in the tumorigenesis in AZ1 homozygotes. However, crossing of AZ1 knockout mice and APC-deficient mm mice demonstrated increases in the number and size of intestinal adenomas in the presence of the heterozygous AZ1 mutation in min mice.

1)抗酶是多胺诱导的蛋白质，其负调节细胞多胺。我们以前报道过，具有混合遗传背景的纯合子AZ 1基因敲除小鼠是部分胚胎致死的。具有C57 BL/6 J背景的纯合胚胎在胚胎日（E）13.5-16.5显示高死亡率。肝发育不全是最早发现的异常。随后的分析显示，在E12.5-13.5时，肝脏造血细胞减少，细胞凋亡形态，外周循环中贫血伴成红细胞增加，表明肝脏中的继发性胚胎造血是AZ 1敲除小鼠中最受影响的过程。在我们的遗传分析中，AZ 1基因敲除小鼠的致死表型的修饰基因的存在被指示。为了使用位置分配来鉴定修饰基因，我们在MSKR小鼠中制备了同源系，MSKR小鼠是源自日本野生molossinus小鼠的品系。2）使用来自AZ 1敲除小鼠的原代和永生化培养细胞的研究表明，在这些细胞中，多胺对ODC的抑制被延迟，并且AZ 2部分补偿了多胺的调节。因此，AZ 2具有抑制ODC和抑制多胺摄取的活性，两者均以多胺依赖的方式进行。3）我们还制备了AZ 2敲除小鼠。纯合子AZ 2基因敲除小鼠没有表现出明显的表型。AZ 1-AZ 2双纯合子基因敲除小鼠在E14.5时胚胎完全致死。在E10.5时开始出现全身发育迟缓，肝脏形成之前，提示双基因敲除胚胎的死亡原因与AZ 1基因敲除胚胎不同。4）对存活胚胎的长期观察未能显示AZ 1纯合子的肿瘤发生增加。然而，AZ 1基因敲除小鼠和APC缺陷型mm小鼠的杂交表明，在min小鼠中存在杂合AZ 1突变的情况下，肠腺瘤的数量和大小增加。