INTRODUCTION AND EXPRESSION OF RECOMBINANT GENES IN ASCIDIAN EMBRYOS
INTRODUCTION AND EXPRESSION OF RECOMBINANT GENES IN ASCIDIAN EMBRYOS
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DOI：
10.1111/j.1440-169x.1992.tb00031.x
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发表时间：
1992-12-01
影响因子：
2.5
通讯作者：
MAKABE, KW
中科院分区：
文献类型：
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作者：
HIKOSAKA, A;KUSAKABE, T;MAKABE, KW
为了检测外源基因导入海鞘卵后的表达情况，将两个重组质粒pmiwZ和pHrMA4aCAT分别显微注射入海鞘和罗氏海鞘受精卵的细胞质中。质粒pmiwZ含有与动物基因启动子融合的细菌β-半乳糖苷酶基因（lac-Z）的编码序列，而pHrMA4aCAT是由约1.4kb长的H.鱼际肌肌动蛋白基因HrMA 4a与细菌氯霉素乙酰转移酶基因（CAT）。将10 μ g/ml的pmiwZ DNA注射到海鞘卵中，约160 μ l不影响胚胎的发生，但注射30 μ g/ml的pmiwZ DNA则导致卵发育异常。当通过酶活性的组织化学检测来检查lac-Z的表达时，无论质粒的线性或环状形式，在早期尾芽胚胎和后期胚胎以及幼虫中的表达都是明显的。酶活性出现在各种细胞类型中，包括表皮、神经系统、内胚层、间充质、脊索和肌肉。相反，当将pHrMA4aCAT引入盐爪蟾卵中，随后通过抗CAT抗体检查CAT蛋白的出现时，CAT表达仅限于肌肉细胞。这些结果表明，导入海鞘卵的重组基因可以在胚胎发生期间表达，并且HrMA4a的1.4 kb长的5 ′侧翼区包含足够的调控序列，以使该基因在适当的空间和时间表达。
In order to examine the expression of exogenous genes introduced into ascidian eggs, two recombinant plasmids pmiwZ and pHrMA4aCAT were microinjected into the cytoplasm of fertilized eggs ofCiona savignyiandHalocynthia roretzi, respectively. The plasmid pmiwZ contains the coding sequence of bacterial β‐galactosidase gene (lac‐Z) fused with animal gene promoters, while pHrMA4aCAT was constructed by fusing about 1.4‐kb long 5′ flanking region ofH. roretzimuscle actin geneHrMA4awith bacterial chloramphenicol acetyltransferase gene (CAT). Injection of approximately 160 pl of 10 μg/ml pmiwZ DNA intoCionaeggs did not affect the embryogenesis, although introduction of the same volume of 30 μg/ml pmiwZ DNA resulted in abnormal development of injected eggs. When the expression oflac‐Zwas examined by histochemical detection of the enzyme activity, the expression was evident in the early tailbud embryos and later stage embryos, and larvae, irrespective of linear or circular form of the plasmid. The enzyme activity appeared in various cell‐types including epidermis, nervous system, endoderm, mesenchyme, notochord, and muscle. In contrast, when pHrMA4aCAT was introduced intoHalocynthiaeggs and the appearance of CAT protein was examined later by the anti‐CAT antibody, theCATexpression was restricted to muscle cells. These results indicate that the recombinant genes introduced into ascidian eggs could express during embryogenesis and that the 1.4‐kb long 5′ flanking region of HrMA4a contains regulatory sequences enough for the appropriate spatial and temporal expression of the gene.