Engineering BAC reporter gene constructs for mouse transgenesis.
Engineering BAC reporter gene constructs for mouse transgenesis.
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用于小鼠转基因的工程 BAC 报告基因构建体。
DOI：
10.1007/978-1-60761-974-1_10
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发表时间：
2011
期刊：
影响因子：
--
通讯作者：
Maye,Peter
中科院分区：
文献类型：
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作者：
Fu,Yu;Maye,Peter
大规模的想法和努力的高潮已经真正允许使用容纳在细菌人工染色体（BAC）载体中的大基因组DNA克隆进行生物研究。实现这一目标的基本进展包括：（1）完成基因组测序项目，建立高度注释的网络访问数据库，允许快速识别和购买含有感兴趣基因的BAC克隆。(2)利用细菌内同源重组的方法对BAC进行遗传修饰，可以快速构建基因靶向构建体或转基因报告基因构建体。我们最近的努力利用这些进展，利用BAC和细菌重组方法制备荧光蛋白报告基因转基因小鼠，用于骨骼生物学研究。使用BAC基因组DNA克隆来工程化报告基因构建体的基本原理是基于它们大得多的尺寸，从而增加了基因的大部分（如果不是全部）各自的异调节元件存在的可能性，从而给出内源基因表达的更真实的表示。在相对较短的时间内，我们已经非常熟练地生成了BAC记者。与人们普遍认为的使用BAC是复杂和困难的观点相反，我们决定撰写本章，以鼓励目前使用传统分子克隆方法设计转基因DNA构建体的实验室强烈考虑学习BAC方法。作为一个例子，我们走过了我们采取的步骤，以产生转基因报告小鼠系，肌腱蛋白C（TNC）-mCherry。
A culmination of large-scale ideas and efforts has truly allowed for the use of large genomic DNA clones housed in Bacterial Artificial Chromosome (BAC) vectors for biological research. Fundamental advances that have allowed this to happen include (1) the completion of genome sequencing projects and the establishment of highly annotated web-accessible databases allowing for the rapid identity and purchase of BAC clones containing genes of interest. (2) The generation of methodologies to modify BACs genetically, allowing for the rapid creation of gene targeting constructs or transgenic reporter gene constructs using homologous recombination in bacteria.Recent efforts on our part have capitalized on these advances by using BACs and bacterial recombination methods to generate fluorescent protein reporter transgenic mice to study skeletal biology. The rationale for using BAC genomic DNA clones to engineer reporter gene constructs is based on their much larger size, thus increasing the likelihood that most, if not all, of a gene’s respectivecisregulator elements are present, giving a truer representation of the endogenous gene’s expression. In a relatively short amount of time, we have become extremely proficient at generating BAC reporters. Contrary to the widely perceived notion that working with BACs is complex and difficult, we decided to write this chapter to encourage laboratories that are currently using traditional molecular cloning methods to engineer transgenic DNA constructs to strongly consider learning BAC methodologies. As an example, we walk through the steps we took to generate the transgenic reporter mouse line,Tenascin C(TNC)-mCherry.