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Establishment of transgenic rat expressing human A or B Transferase gene and cloning of rat paralogous genes equivalent of human histo-blood group ABO gene.

表达人A或B转移酶基因的转基因大鼠的建立以及相当于人组织血型ABO基因的大鼠旁系同源基因的克隆。

基本信息

批准号：
13670435
负责人：
IWAMOTO Sadahiko
金额：
$ 2.24万
依托单位：
Jichi Medical School
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670435/
关键词：
rat ABO homologue paralogous gene ABH antigen expression transgenic rat ラットABOオーソログ

项目摘要

Histo-blood group ABH antigens are important not only for blood transfusion but also for organ transplantation. To improve the clinical management of transplantation, development of ABO-mismatched animal models is desirable. Rats are more suitable for ordinary examination of organ transplantation because of the larger body size compared with mice. To evaluate the availability of rats as an animal model, we studied rat ABO homologue and established human A- and B-transferase transgenic rats. A DNA fragment corresponding to exon 7 of the human ABO gene was amplified from Wistar rat genomic DNA and sequenced. Using the amplified fragments as a probe for Southern blotting, multiple hybridized bands appeared on both EcoRI and BamHI digested genomes of seven rat strains, which showed variations in the band numbers among the strains. Four cDNAs were cloned from a Wistar rat, three of which showed A-transferase activity and one of which showed B-transferase activity. These activities were dependent on the equivalent residues at 266 and 268 of human ABO transferase. Wild Wistar rats expressed A-antigen in salivary gland, intestine, and urinary bladder tissue, but B-antigen was not stained in any organs studied, while a transcript from the ABO homologue with B-transferase activity was ubiquitous. Human A-transferase and B-transferase were transferred into Wistar rats. A-transgenic rats expressed A-antigen in ectopic tissue of the brain plexus, type II lung epithelium, pancreas, and epidermis. B-antigen in the B-transgenic rat was expressed in the same organs as A-transgenic rats. If the hominoid ABO ancestral genes consisted of multicopy genes with A and B transferase activity like rats, they must donate or accept gene fragments from each other and may produce a locus composed of A-alleles and B-alleles. This estimation must be resolved by further genome projects in rats and other mammals.

组织血型ABH抗原不仅对输血而且对器官移植都很重要。为了改善移植的临床管理，需要开发ABO不匹配的动物模型。由于大鼠的体型比小鼠大，因此更适合于器官移植的普通检查。为了评估大鼠作为动物模型的可用性，我们研究了大鼠ABO同源物并建立了人A-和B-转移酶转基因大鼠。从Wistar大鼠基因组DNA中扩增出与人ABO基因外显子7对应的DNA片段并进行测序。以扩增片段为探针进行Southern杂交，在7个品系大鼠基因组的EcoRI和BamHI酶切位点上均出现了多条杂交带，表明不同品系大鼠基因组间的杂交带数存在差异。从Wistar大鼠中克隆了4个cDNA，其中3个显示A-转移酶活性，1个显示B-转移酶活性。这些活性依赖于人ABO转移酶266和268位的等效残基。野生Wistar大鼠的唾液腺，肠，膀胱组织中表达A-抗原，但B-抗原没有染色的任何器官研究，而转录的ABO同源物与B-转移酶活性是无处不在的。将人A-转移酶和B-转移酶转移到Wistar大鼠中。A转基因大鼠在脑丛、II型肺上皮、胰腺和表皮的异位组织中表达A抗原。B-转基因大鼠中的B-抗原在与A-转基因大鼠相同的器官中表达。如果类人猿ABO祖先基因像大鼠一样由具有A和B转移酶活性的多拷贝基因组成，它们必须相互捐赠或接受基因片段，并可能产生由A等位基因和B等位基因组成的基因座。这一估计必须通过进一步的大鼠和其他哺乳动物基因组计划来解决。