Relevance of BAC transgene copy number in mice: transgene copy number variation across multiple transgenic lines and correlations with transgene integrity and expression
Relevance of BAC transgene copy number in mice: transgene copy number variation across multiple transgenic lines and correlations with transgene integrity and expression
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DOI：
10.1007/s00335-007-9056-y
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发表时间：
2007-10-01
期刊：
影响因子：
2.5
通讯作者：
Mortlock, Douglas P.
中科院分区：
文献类型：
--
作者：
Chandler, Kelly J.;Chandler, Ronald L.;Mortlock, Douglas P.
细菌人工染色体（BAC）是操纵大DNA片段的优秀工具，因此，越来越多地被用于通过原核注射来工程化转基因小鼠。对BAC转基因小鼠的需求强调了在转基因后仔细检查BAC完整性和保真度的必要性，这对于解释转基因功能可能是至关重要的。因此，必须有评估这些参数的可靠方法。然而，关于BAC转基因是否常规地作为完整分子整合在小鼠基因组中，BAC转基因在连续世代中通过种系时如何表现，以及BAC转基因拷贝数的变化如何与转基因表达相关的数据有限。为了解决这些问题，我们使用TaqMan实时PCR来估计BAC转基因胚胎和品系中的BAC转基因拷贝数。在这里，我们证明了重复性的拷贝数定量与这种方法，并描述了在独立的转基因株系的拷贝数的变化。此外，多态性标记分析表明，大多数BAC转基因株系含有完整的分子。值得注意的是，含有多个BAC拷贝的所有品系也含有所有BAC特异性标记。分析的23个创始人中有3个含有整合到一个以上基因组位置的BAC转基因。最后，我们发现增加的BAC转基因拷贝数与增加的BAC转基因表达相关。总之，我们的努力提供了一个可靠的方法来测定BAC转基因的完整性和保真度，和数据，应该是有用的研究人员使用BAC作为转基因载体。
Bacterial artificial chromosomes (BACs) are excellent tools for manipulating large DNA fragments and, as a result, are increasingly utilized to engineer transgenic mice by pronuclear injection. The demand for BAC transgenic mice underscores the need for careful inspection of BAC integrity and fidelity following transgenesis, which may be crucial for interpreting transgene function. Thus, it is imperative that reliable methods for assessing these parameters are available. However, there are limited data regarding whether BAC transgenes routinely integrate in the mouse genome as intact molecules, how BAC transgenes behave as they are passed through the germline across successive generations, and how variation in BAC transgene copy number relates to transgene expression. To address these questions, we used TaqMan real-time PCR to estimate BAC transgene copy number in BAC transgenic embryos and lines. Here we demonstrate the reproducibility of copy number quantification with this method and describe the variation in copy number across independent transgenic lines. In addition, polymorphic marker analysis suggests that the majority of BAC transgenic lines contain intact molecules. Notably, all lines containing multiple BAC copies also contain all BAC-specific markers. Three of 23 founders analyzed contained BAC transgenes integrated into more than one genomic location. Finally, we show increased BAC transgene copy number correlates with increased BAC transgene expression. In sum, our efforts have provided a reliable method for assaying BAC transgene integrity and fidelity, and data that should be useful for researchers using BACs as transgenic vectors.