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Elucidation of Biological Function of Mouse Peptidylarginine Deiminase by Gene Targeting

通过基因打靶阐明小鼠肽基精氨酸脱亚胺酶的生物学功能

基本信息

批准号：
10660071
负责人：
TAKAHARA Hidenari
金额：
$ 2.3万
依托单位：
IBARAKI UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10660071/
关键词：
Gene Targeting mouse peptidylarginine deiminase knockout mouse 粒子間反発力

项目摘要

Peptidylarginine deiminases (PAD), a group of post-translational enzymes, catalyze the conversion of protein-bound arginine residues to citrulline residues in a calcium ion dependent manner and are widely distributed in various organs of vertebrates. We found the existence of four isoforms of PAD (types I, II, III, and IV) in mouse. However, the relative functional consequences of the isoforms with respect to their co-location in tissues have yet to be explored. Recently we succeeded to clone the full-length cDNAs of every mouse PAD isoforms and deduced their primary structures. In order to elucidate the functions of each PAD isoform in murine cells, we make strategies by using knockout mouse whose PAD gene(s) is (are) destroyed by replacing the PAD gene to a vector harboring a partial of the PAD gene and a resistant gene against an antibiotic. From 1998 to 1999 we succeeded to clone the genes of every PAD isoform and determined their nucleotide sequences of 5'-flanking region, and we also extended the cloning of long region of each PAD isoform from bacterial artificial chromosome (BAC) libraries of embryonic stem cell lines (ES-129/SvJ). In this year, the found clone was derived from the type II gene and was useful to make targeting vectors for homologous recombination of PAD genes. Therefore, we focused on the PAD types II gene for the knockout experiment and succeeded to isolate the recombinant ES cells. Now, we are establishing the knock out mouse whose PAD type II gene is destroyed.

肽精氨酸脱亚胺酶（Peptidylarginine deiminases， PAD）是一类翻译后酶，以钙离子依赖的方式催化蛋白质结合的精氨酸残基向瓜氨酸残基的转化，广泛分布于脊椎动物的各个器官中。我们在小鼠中发现了四种PAD亚型（I型、II型、III型和IV型）。然而，相对于它们在组织中的共定位的功能后果的同工异构体尚未被探索。最近，我们成功克隆了所有小鼠PAD亚型的全长cdna，并推导了它们的初级结构。为了阐明每个PAD异构体在小鼠细胞中的功能，我们通过将PAD基因被破坏的敲除小鼠的PAD基因替换为含有部分PAD基因和抗抗生素抗性基因的载体来制定策略。1998 - 1999年，我们成功克隆了每个PAD异构体的基因，并测定了其5'-侧翼区的核苷酸序列，并从胚胎干细胞（ES-129/SvJ）细菌人工染色体（BAC）文库中扩展了每个PAD异构体长片段的克隆。今年发现的克隆来源于II型基因，可用于制作PAD基因同源重组的靶向载体。因此，我们针对PAD II型基因进行敲除实验，成功分离出重组ES细胞。现在，我们正在建立被敲除的小鼠，其II型PAD基因被破坏。