A BAC-bacterial recombination method to generate physically linked multiple gene reporter DNA constructs
A BAC-bacterial recombination method to generate physically linked multiple gene reporter DNA constructs
复制标题
DOI：
10.1186/1472-6750-9-20
复制
发表时间：
2009-03-13
影响因子：
3.5
通讯作者：
Lichtler, Alexander C.
中科院分区：
文献类型：
--
作者：
Maye, Peter;Stover, Mary Louise;Lichtler, Alexander C.
背景：报告基因小鼠是生物学研究中有价值的动物模型，可以提供基因表达读数，有助于在发育过程中进行细胞表征。随着从BAC基因组克隆中设计报告基因的细菌重组技术的进步，以及光学可区分的荧光蛋白报告基因的产生，相对于传统的可用方法，设计更具信息量的转基因报告鼠模型的能力是前所未有的。结果：我们在这里展示了我们在开发三阶段细菌重组策略方面的首次努力，该策略将多个基因与它们各自的荧光蛋白(FP)报告基因物理地连接在一个DNA片段中。该策略利用细菌重组技术：(1)将感兴趣的基因亚克隆到BAC连接载体中；(2)将所需的报告基因插入到各自的基因中；(3)将不同的报告基因连接在一起。作为概念证明，我们已经生成了一个DNA片段，其中包含分别驱动ECFP、mCherry和Topz FP报告基因表达的基因Trap、Dmp1和Ibsp。结论：将多个基因与其各自的荧光蛋白报告基因相连接的三阶段方法具有较高的效率。此外，基因连锁允许它们共同的染色体整合到一个单一的基因座上。然而，通过转基因对这种多报告DNA结构的测试确实表明，尽管两个不同的基因大小很大，但仍然可以产生位置效应。我们认为基因选择、基因组DNA片段大小和内源性绝缘元件的存在是关键变量。
Background: Reporter gene mice are valuable animal models for biological research providing a gene expression readout that can contribute to cellular characterization within the context of a developmental process. With the advancement of bacterial recombination techniques to engineer reporter gene constructs from BAC genomic clones and the generation of optically distinguishable fluorescent protein reporter genes, there is an unprecedented capability to engineer more informative transgenic reporter mouse models relative to what has been traditionally available.Results: We demonstrate here our first effort on the development of a three stage bacterial recombination strategy to physically link multiple genes together with their respective fluorescent protein (FP) reporters in one DNA fragment. This strategy uses bacterial recombination techniques to: (1) subclone genes of interest into BAC linking vectors, (2) insert desired reporter genes into respective genes and (3) link different gene-reporters together. As proof of concept, we have generated a single DNA fragment containing the genes Trap, Dmp1, and Ibsp driving the expression of ECFP, mCherry, and Topaz FP reporter genes, respectively. Using this DNA construct, we have successfully generated transgenic reporter mice that retain two to three gene readouts.Conclusion: The three stage methodology to link multiple genes with their respective fluorescent protein reporter works with reasonable efficiency. Moreover, gene linkage allows for their common chromosomal integration into a single locus. However, the testing of this multi-reporter DNA construct by transgenesis does suggest that the linkage of two different genes together, despite their large size, can still create a positional effect. We believe that gene choice, genomic DNA fragment size and the presence of endogenous insulator elements are critical variables.