Characterization and Targeting of the Murine α2-Antiplasmin Gene
Characterization and Targeting of the Murine α2-Antiplasmin Gene
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鼠 α2-抗纤溶酶基因的表征和靶向
DOI：
10.1055/s-0038-1657694
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发表时间：
1997
影响因子：
6.7
通讯作者：
R. Lijnen
中科院分区：
文献类型：
--
作者：
R. Lijnen
α-2-抗纤溶酶(α-2-AP)是哺乳动物血浆中主要的生理性纤溶酶抑制物。作为建立α2-AP基因缺陷小鼠的第一步，我们鉴定了小鼠α2-AP 1基因，并构建了胚胎干细胞同源重组的靶向载体。从基因组亚克隆获得的核苷酸序列比对允许定位α2-AP 1基因的外显子2到10，但未能确定外显子1的5‘边界。与人类基因相比，小鼠基因的外显子2到9具有相同的大小和符合GT/AG规则的内含子-外显子边界。外显子10的5‘边界在两个基因中是相同的，而3’非编码区在人类基因中比长。内含子2、3、6和8在小鼠和人类基因中具有相似的大小；内含子1小6倍，内含子5、7和9小2到3倍，而内含子4在小鼠基因中大约大2倍。与人类5‘侧翼序列相比，发生了具有序列(TGG)n的简单重复区域的插入。小鼠α2-AP基因的开放阅读框编码一个491个氨基酸的蛋白质，包括实验确定的成熟蛋白质Val-Asp-Leu-Pro-Gly-的NH2端。在亲本载体pPNT中引入8.3kb的同源序列，构建了靶向载体pPNT.α2-AP。在pPNT.α2-AP中，新霉素抗性表达盒取代了一个7kb的基因组片段，该片段包括外显子2到外显子10的一部分(包括终止密码子)，代表了编码成熟蛋白的整个序列，包括纤维蛋白结合域、反应部位肽键和纤溶酶(原)结合区。用线性化载体pPNT-α2-AP对129R1胚胎干细胞进行电穿孔，获得了3个目的克隆，在5‘端和3’端进行了正确的同源重组，用合适的限制性内切酶和探针对纯化的基因组进行了Southern杂交分析。这些靶向克隆将用于产生α2-AP缺陷小鼠。
Summary α2-Antiplasmin (α2-AP) is the main physiological plasmin inhibitor in mammalian plasma. As a first step toward the generation of α2-AP deficient mice, the murine α2-AP 1 gene was characterized and a targeting vector for homologous recombination in embryonic stem (ES) cells constructed. Alignment of nucleotide sequences obtained from genomic subclones allowed location of exons 2 through 10 of the α2-AP 1gene, but failed to identify the 5’ boundary of exon 1. Compared to the human gene, exons 2 through 9 in the murine gene have identical size and intron-exon boundaries obeying the GT/AG rule. The 5’ boundary of exon 10 is identical in both genes while the 3’ non-coding region is 64 bp longer in the human gene. Introns 2,3,6 and 8 have similar sizes in the mouse and human genes; intron 1 is 6-fold smaller, introns 5, 7 and 9 are 2- to 3-fold smaller, whereas intron 4 is about 2-fold larger in the mouse gene. Compared to the human 5’ flanking sequence, an insertion of a simple repeat region with sequence (TGG)n has occurred. The open reading frame of the mouse α2-AP gene encodes a 491-amino acid protein comprising the experimentally determined NH2-terminus of the mature protein Val-Asp-Leu-Pro-Gly-. A targeting vector, ppPNT.α2-AP, was constructed by introducing a homologous sequence of 8.3 kb in total in the parental pPNT vector. In pPNT.α2-AP, the neomycin resistance expression cassette replaces a 7 kb genomic fragment comprising exon 2 through part of exon 10 (including the stop codon), which represents the entire sequence encoding the mature protein, including the fibrin-binding domain, the reactive site peptide bond and the plasmin(ogen)-binding region. Electroporation of 129R1 embryonic stem (ES) cells with the linearized vector pPNT.α2-AP yielded three targeted clones with correct homologous recombination at the 5’- and 3’-ends, as confirmed by Southern blot analysis of purified genomic DNA with appropriate restriction enzymes and probes. These targeted clones will be used to generate α2-AP deficient mice.
影响因子：
5.6
作者：
GUIGO, R;KNUDSEN, S;SMITH, T
通讯作者：
SMITH, T
影响因子：
15.9
作者：
Yamamoto, K;Loskutoff, DJ
通讯作者：
Loskutoff, DJ
大鼠穹窿RNA基因含有独特的RNA聚合酶III启动子，该启动子由协同作用的外部和内部元件组成。
DOI：
--
发表时间：
1994
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Vilalta,A;Kickhoefer,VA;Rome,LH;Johnson,DL
通讯作者：
Johnson,DL