HIGH-FREQUENCY GERM-LINE TRANSMISSION OF PLASMID DNA-SEQUENCES INJECTED INTO FERTILIZED ZEBRAFISH EGGS
HIGH-FREQUENCY GERM-LINE TRANSMISSION OF PLASMID DNA-SEQUENCES INJECTED INTO FERTILIZED ZEBRAFISH EGGS
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DOI：
10.1073/pnas.88.18.7953
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发表时间：
1991-09-01
影响因子：
11.1
通讯作者：
HOPKINS, N
中科院分区：
文献类型：
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作者：
CULP, P;NUSSLEINVOLHARD, C;HOPKINS, N
为了开发斑马鱼DNA插入突变技术，我们建立了快速获得和注射大量受精卵的程序。使用两种质粒组分中的任何一种，我们将未切割的DNA注入单细胞或两细胞阶段的受精卵。从注射的卵子中孵化出来的鱼被培育到性成熟，通过配对配对，并通过聚合酶链式反应(PCR)和Southern分析检测从16小时大的后代池中提取的DNA，来确定转基因创始人鱼的频率。注射了两种不同质粒之一的鸡蛋没有产生转基因鱼，但注射了另一种质粒的鸡蛋中有7-25%(总共115个)将注射的序列传递给了它们的后代(F1)。在进一步研究的7个品系中，所有品系都能够将外源DNA序列传递给下一代(F2)。在供试的5个品系中，F_2代的遗传均为孟德尔遗传。聚合酶链式反应和Southern分析表明，这些质粒序列以多个拷贝形式存在，很可能是随机排列的。两条方正FISH携带不止一条独立整合的质粒序列。更详细地研究的品系是一个马赛克，在一个生殖细胞中携带两个独立分离的转基因副本，在另一个生殖系前体细胞中携带第三个副本。获得和注射大量斑马鱼卵子的能力与种系整合的高频相结合，可能是朝着能够对这种生物进行插入突变的目标迈出的一步。
With the goal of developing techniques for DNA insertional mutagenesis in zebrafish, we established procedures for rapidly obtaining and injecting large numbers of fertilized eggs. Using either of two plasmid constructs, we injected uncut DNA into fertilized eggs at the one- or two-cell stage. Fish hatched from injected eggs were raised to sexual maturity, and the frequency of transgenic founder fish was determined by pair-mating the fish and testing DNA extracted from pools of their 16-hr-old offspring by the polymerase chain reaction (PCR) and then Southern analysis. Eggs injected with one of two different plasmids yielded no transgenic fish, but 7-25% (19 of 115 overall) of the eggs injected with the other plasmid transmitted the injected sequences to their offspring (F1). Of seven lines studied further, all were able to pass the foreign DNA sequences to the next (F2) generation. Inheritance in the F2 generation was Mendelian in the five lines tested. PCR and Southern analysis indicated that the plasmid sequences were present in multiple copies, probably tandemly arranged. Two founder fish carried more than one independent integration of the plasmid sequences. The line studied in more detail was a mosaic carrying two independently segregating copies of the transgene in one germ cell and a third copy in another germ-line precursor cell. The ability to obtain and inject large numbers of zebrafish eggs combined with a high frequency of germ-line integration may be steps toward the goal of being able to perform insertional mutagenesis with this organism.