Center for Molecular Neurobiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
FASEB J. 2010 Jun;24(6):1714-24. doi: 10.1096/fj.09-140749. Epub 2010 Jan 28.
Targeting of genes in mice, a key approach to study development and disease, often leaves a neo cassette, loxP, or FRT sites inserted in the mouse genome. Insertion of neo can influence the expression of neighboring genes, but similar effects have not been reported for loxP sites. We therefore performed microarray analyses of mice in which the Ncam or the Tnr gene were targeted either by insertion of neo or loxP/FRT sites. In the case of Ncam, neo, but not loxP/FRT insertion, led to a 2-fold reduction in mRNA levels of 3 genes located at distances between 0.2 and 3.1 Mb from the target. In contrast, after introduction of loxP/FRT sites into introns of Tnr, we observed a 2.5- to 4-fold reduction in the transcript level of the Gas5 gene, 1.1 Mb away from Tnr, most probably due to disruption of a conserved regulatory element in Tnr. Insertion of short DNA sequences such as loxP/FRT can thus influence off-target mRNA levels if these sites are accidentally placed into regulatory elements. Our results imply that conditional knockout mice should be analyzed for genomic positional side effects that may influence the animals' phenotypes.
在小鼠中靶向基因是研究发育和疾病的关键方法，通常会在小鼠基因组中插入新霉素盒、loxP 或 FRT 位点。插入新霉素会影响邻近基因的表达，但尚未报道 loxP 位点有类似的影响。因此，我们对 Ncam 或 Tnr 基因被插入新霉素或 loxP/FRT 位点的小鼠进行了微阵列分析。在 Ncam 的情况下，新霉素而非 loxP/FRT 插入导致距离靶标 0.2 至 3.1 Mb 的 3 个基因的 mRNA 水平降低了 2 倍。相比之下，在 Tnr 的内含子中引入 loxP/FRT 位点后，我们观察到距离 Tnr 1.1 Mb 的 Gas5 基因的转录水平降低了 2.5 至 4 倍，这很可能是由于 Tnr 中的保守调控元件被破坏。如果这些位点意外地放置在调控元件中，那么插入短的 DNA 序列（如 loxP/FRT）可能会影响非靶 mRNA 水平。我们的结果表明，应分析条件性敲除小鼠的基因组位置副作用，这些副作用可能会影响动物的表型。
