Gene introduction into mouse blastocysts via “pricking”
Gene introduction into mouse blastocysts via “pricking”
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通过“刺破”将基因导入小鼠囊胚
DOI：
10.1002/mrd.1080340402
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发表时间：
1993
影响因子：
2.5
通讯作者：
E. Amann
中科院分区：
文献类型：
--
作者：
M. Sato;N. Tada;R. Iwase;E. Amann
在外来DNA存在的情况下，培养的哺乳动物细胞被刺破，这是一个众所周知的现象。这项微操作技术被应用于小鼠的囊胚，以确定胚胎是否会摄取外源DNA。在含有线性化的质粒DNA的培养液中，在每个囊胚中刺三次内细胞团的中间区域(ICM)。将处理后的60个囊胚移植到假孕妇女的子宫角，获得了30个发育中的胚胎(50%)。聚合酶链式反应-Southern分析显示，30个胎儿中有22个(73%)每个二倍体细胞的外源DNA少于1个拷贝，这是一种结合PCR产物的Southern印迹处理的敏感技术。其余8例胎儿的外源DNA均为阴性。将大肠杆菌β-半乳糖苷酶(β-GAL)基因与小鼠金属硫蛋白-I(MT-I)启动子偶联，在含有1β-Gal基因的1μ氯化镉溶液中培养1天和5天后，对囊胚进行β-GAL活性组织化学检测，发现至少65%的胚胎表现出β-GAL活性，且主要分布在ICM区。这些结果表明，小鼠胚泡在穿孔后可以相对较高的效率进行转基因，并发生了导入基因的表达。这种方法提供了一种方法来定位小鼠胚泡中活跃的ICM基因的调控元件，并通过穿刺法对ICM细胞进行标记，从而有助于研究ICM细胞在完整胚泡中的命运。©1993 Wiley-Liss，Inc.
It is a well‐known phenomenon that cultured mammalian cells that have been pricked in the presence of foreign DNA can be transformed. This micromanipulation ‘pricking’ technique was applied to mouse blastocysts to determine whether uptake of exogenous DNA would occur in the embryos. The middle region of the inner cell mass (ICM) was pricked three times in each blastocyst in a medium containing a linearized plasmid DNA. When the 60 treated blastocysts were transferred to the uterine horns of pseudopregnant females, 30 developing fetuses (50%) at the mid‐gestation stage were obtained. Twenty‐two of the 30 fetuses (73%) had less than 1 copy of the foreign DNA per diploid cell, as revealed by polymerase chain reaction (PCR)‐Southern analysis, a sensitive technique combined with Southern blot processing of the PCR products. The 8 other fetuses were negative for the foreign DNA. When blastocysts were pricked in the presence of vector DNA coupling E. coli β‐galactosidase (β‐gal) gene to a mouse metallothionein‐I (MT‐I) promoter and assessed for β‐gal activity histochemically after 1 and 5 days of culture in the presence of 1 μM CdCI2, at least 65% of the embryos exhibited β‐gal activity mainly in the ICM region. These results indicate that mouse blastocysts can be transfected with a relatively high efficiency after pricking, and that the introduced gene expression occurs. This approach provides a means of mapping the regulatory elements of genes that are active in the mouse blastocyst ICM, and may be useful in investigating the fate of the ICM cells in an intact blastocyst by labeling them via pricking technique. © 1993 Wiley‐Liss, Inc.
新型多功能质粒载体，用于表达由与编码 β-半乳糖苷酶酶活性羧基末端部分的 lacZ 基因序列融合的克隆基因编码的杂合蛋白。
影响因子：
3.5
作者：
Shapira,SK;Chou,J;Richaud,FV;Casadaban,MJ
通讯作者：
Casadaban,MJ