Stable lines of transgenic zebrafish exhibit reproducible patterns of transgene expression.
Stable lines of transgenic zebrafish exhibit reproducible patterns of transgene expression.
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发表时间：
1990-07
期刊：
影响因子：
4.6
通讯作者：
Gary W. Stuart;Juergen R. V 1 Elkind;James V. McMURRAY;Monte;Westerfield
中科院分区：
文献类型：
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作者：
Gary W. Stuart;Juergen R. V 1 Elkind;James V. McMURRAY;Monte;Westerfield
为了研究生殖系转化的频率，并检查组织特异性转基因表达的再现性，我们生产了几条表达重组氯霉素乙酰转移酶（CAT）基因的转基因斑马鱼品系。在第一次卵裂之前，将含有劳斯肉瘤病毒和CAT编码区上游的SV-40启动子序列的超螺旋质粒注射到斑马鱼胚胎中。CAT活性可以检测到在批次注射胚胎早在8小时，最多至少12天后受精。大约18%饲养至成熟的注射鱼的鳍中表现出CAT活性，大约5%的注射鱼成为稳定的种系转化体。育种研究表明，虽然转基因创始人鱼经常生殖系镶嵌，转基因个体的后代完全半合子的转基因标记。四个独立品系的F1代中存在的转基因在鳍和皮肤中相对较好地表达，而在心脏、鳃和肌肉中观察到较低水平的表达。在脑、肝和性腺中观察到很少或没有CAT表达。针对CAT基因产物的单克隆抗体一致地揭示了这些品系中的三个中的外胚层衍生的鳍表皮细胞中CAT表达的杂色图案。这些结果表明，有可能有效地产生稳定的斑马鱼生殖系转化体，并在这种生物体中观察到可重复的组织特异性转基因表达模式。组织内观察到的杂色表达的可能机制也被考虑。
To study the frequency of germ-line transformation and to examine the reproducibility of tissue-specific transgene expression, we produced several lines of transgenic zebrafish expressing a recombinant chloramphenicol acetyltransferase (CAT) gene. Supercoiled plasmids containing both Rous sarcoma virus and SV-40 promoter sequences upstream of the CAT coding region were injected into zebrafish embryos prior to first cleavage. CAT activity could be detected in batches of injected embryos as early as 8 h and up to at least 12 days post-fertilization. Approximately 18% of injected fish raised to maturity exhibited CAT activity in their fins, and approximately 5% of injected fish became stable germ-line transformants. Breeding studies indicated that although transgenic founder fish were frequently germ-line mosaics, transgenic individuals of subsequent generations were fully hemizygous for the transgene marker. The transgenes present in the F1 progeny of four independent lines were relatively well expressed in fin and skin, while lower levels of expression were observed in heart, gill and muscle. Little or no CAT expression was observed in the brain, liver and gonad. A monoclonal antibody directed against the CAT gene product consistently revealed variegated patterns of CAT expression in ectodermally derived fin epidermal cells in three of these lines. These results show that it is possible to efficiently produce stable germ-line transformants of the zebrafish and to observe reproducible tissue-specific patterns of transgene expression in this organism. Possible mechanisms for the variegated expression observed within tissues are also considered.