Ainscough J F, John R M, Barton S C
Wellcome/CRC Institute of Cancer and Developmental Biology, University of Cambridge, Cambridge, UK.
Methods Mol Biol. 2001;181:55-65. doi: 10.1385/1-59259-211-2:55.
The production of transgenic mice using small DNA constructs has been widely used for many years to investigate the regulation of gene activity. Small plasmid-based constructs (less than 20 kb) have been favored for a number of reasons, particularly the ease with which they can be manipulated and purified in large quantities. While this approach is powerful, there are some problems associated with the size of these transgenes. In particular, many of these small transgenes do not reproduce accurately the expression seen from the endogenous gene. For some genes the regulatory elements that control activity are located at a distance from the promoter and can be omitted from the transgene. These may be enhancers, repressors, boundary elements, or even locus control regions (LCRs), which are responsible for maintaining the correct spatial and temporal expression patterns of a number of genes, such as the globin clusters in mouse and humans (1). More important, small transgenes are susceptible to position effects from the chromatin environment in which they integrate, which often results in either ectopic expression (from trapping of nearby enhancers for other genes) or suppression of gene activity. Finally, small transgenes usually integrate in a multicopy tandem arrangement that does not accurately reflect the situation seen at the endogenous locus.
多年来，使用小DNA构建体生产转基因小鼠已被广泛用于研究基因活性的调控。基于小质粒的构建体（小于20 kb）因多种原因而受到青睐，特别是它们易于大量操作和纯化。虽然这种方法很强大，但这些转基因的大小存在一些问题。特别是，许多这些小转基因不能准确再现内源基因的表达。对于某些基因，控制活性的调控元件位于离启动子一定距离处，可能会从转基因中省略。这些可能是增强子、阻遏物、边界元件，甚至是位点控制区（LCR），它们负责维持许多基因的正确时空表达模式，如小鼠和人类中的珠蛋白基因簇（1）。更重要的是，小转基因易受其整合的染色质环境的位置效应影响，这通常导致异位表达（由于捕获了其他基因附近的增强子）或基因活性的抑制。最后，小转基因通常以多拷贝串联排列的方式整合，这不能准确反映内源基因位点的情况。
