MOUSE MODEL--TARGETED GENE KNOCK OUT OF CYTOSOLIC PHOSPHOLIPASE A2

同源基因对小鼠编码cPLA2基因的干扰 重组目前正在进行中。功能缺陷的小鼠 CPLA2将通过靶向编码基因的区域而产生 丝氨酸228处的酶活性部位，这是已知必不可少的 用于酶活性。只有编码活性部位的cDNAs序列 区域已经发表，并且没有该区域的基因组序列 吉恩有空。因此，用聚合酶链式反应来确定其近似值。 编码丝氨酸228的外显子及其下一个外显子3‘的位置 活性位点外显子。与这两个外显子对应的探针用于 从E14TG2a小鼠中筛选含DNA的Charon 35基因组文库 胚胎干细胞。从这个文库中，获得了大约15kb的cPLA2 克隆了含有活性部位区域的基因。这个克隆人有 利用Southern杂交和限制性内切酶进行了广泛的定位 片段已经被亚克隆到蓝本中。其他克隆 对丝氨酸228外显子和该外显子的5‘区域进行了编码 分离的和限制性内切酶片段正在被亚克隆到Bluescrip中。 靶向构建体会用新霉素干扰cPLA2基因 抗性基因3‘端剪接接头约10个碱基5’端 编码丝氨酸228的外显子。在这种情况下cPLA2基因的破坏 网站将消除剪接连接，最多允许转录 没有翻译功能的cPLA2的异常消息。

The disruption of the mouse gene encoding cPLA2 by homologous recombination is currently in progress. Mice deficient in functional cPLA2 will be generated by targeting the region of the gene encoding the active site of the enzyme at serine 228 which is known to be essential for enzymatic activity. Only the cDNA sequence encoding the active site region has been published, and no genomic sequence of this region of the gene is available. Therefore, PCR was used to determine the approximate location of the exon encoding serine 228 and the next exon 3' of this active site exon. Probes corresponding to these two exons were used to screen a Charon 35 genomic library containing DNA from E14TG2a mouse embryonic stem cells. From this library, approximately 15 kb of the cPLA2 gene which includes the active site region was cloned. This clone has been extensively mapped by Southern hybridization and restriction fragments have been subcloned into Bluescript. Additional clones which encode for the serine 228 exon and the region 5' of this exon have been isolated and restriction fragments are being subcloned into Bluescript. The targeting construct will disrupt the cPLA2 gene with the neomycin resistance gene approximately 10 bases 5' of the 3' splice junction of the exon encoding serine 228. The disruption of the cPLA2 gene at this site will eliminate the splice junction and at most permit transcription of an aberrant message with no translation of functional cPLA2.