Efficient transgenic rat production by a lentiviral vector
Efficient transgenic rat production by a lentiviral vector
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DOI：
10.1152/ajpheart.00060.2007
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发表时间：
2007-07-01
影响因子：
4.8
通讯作者：
Cowley, Allen W., Jr.
中科院分区：
文献类型：
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作者：
Michalkiewicz, Mieczyslaw;Michalkiewicz, Teresa;Cowley, Allen W., Jr.
用鸡β-肌动蛋白启动子、巨细胞病毒增强子和兔β-珠蛋白（CAG）内含子序列驱动增强型绿色荧光蛋白（eGFP）的慢病毒构建体产生大鼠转基因系，以评价该方法在基因功能研究中的有用性。从近交系Dahl S和远交系Sprague-Dawley大鼠中收集受精卵，并将约100 μ l浓缩病毒显微注射到单细胞胚胎的玻璃体周隙中。在注射的121个胚胎中，出生了60只幼崽（49.6%）。Dahl S大鼠的转基因率平均为22%，Sprague-Dawley大鼠的转基因率平均为14%。拷贝数范围从1到4的创始人，和遗传的转基因在随后的F-1人口为48.2%。少量的插入位点使我们能够在一代内获得具有单拷贝转基因的近交转基因系。对每个转基因插入位点的测序显示，它们以单拷贝形式插入，优先选择基因的内含子。CAG启动子驱动了脑、肾、心脏和血管系统中高水平的eGFP表达，使其非常适合探索新发现基因的心血管功能。eGFP的表达模式在5个不同的F-1转基因株系中是相似的，表明转基因的表达与其染色体位置无关。因此，慢病毒转基因提供了一个强大的工具，生产转基因近交系大鼠，并将提高这种物种的基因发现和目标验证研究的有用性。
A lentiviral construct for an enhanced green fluorescent protein (eGFP) driven by a chicken beta-actin promoter, cytomegalovirus enhancer, and intronic sequences from rabbit beta-globin (CAG) was used to produce transgenic lines of rats for evaluation of the usefulness of this approach in gene function studies. Fertilized eggs were collected from inbred Dahl S and outbred Sprague-Dawley rats, and similar to 100 pl of concentrated virus were microinjected into the perivitrelline space of one-cell embryos. Of 121 embryos injected, 60 pups (49.6%) were born. Transgenic rates averaged 22% in Dahl S and 14% in Sprague-Dawley rats. Copy number ranged from one to four in the founders, and the inheritance of the transgene in a subsequent F-1 population was 48.2%. The small number of insertion sites enabled us to derive inbred transgenic lines with a single copy of the transgene within one generation. Sequencing of each transgene insertion site revealed that they inserted as single copies with a preference for the introns of genes. The CAG promoter drove high levels of eGFP expression in brain, kidney, heart, and vasculature, making it very suitable for exploring the cardiovascular function of newly discovered genes. The pattern of eGFP expression was similar across five different F-1 transgenic lines, indicating that the expression of the transgene was independent of its chromosomal position. Thus lentiviral transgenesis provides a powerful tool for the production of transgenic inbred rats and will enhance the usefulness of this species in gene discovery and target validation studies.