Abstract
Transgenes flanked by loxP sites have been widely used to generate transgenic mice where the transgene expression can be controlled spatially and temporally by Cre recombinase. Data from this approach has led to important conclusions in cancer, neurodevelopment and neurodegeneration. Using this approach to conditionally express micro RNAs (miRNAs) in mice, we found that Cre-mediated recombination in neural progenitor cells caused microcephaly in five of our ten independent transgenic lines. This effect was not associated with the types or the quantity of miRNAs being expressed, nor was it associated with specific target knockdown. Rather, it was correlated with the presence of multiple tandem transgene copies and inverted (head-to-head or tail-to-tail) transgene repeats. The presence of these inverted repeats caused a high level of cell death in the ventricular zone of the embryonic brain, where Cre was expressed. Therefore, results from this Cre-loxP approach to generate inducible transgenic alleles must be interpreted with caution and conclusions drawn in previous reports may need reexamination.
Highlights
The Cre-loxP system has been used widely for conditional transgene expression in mice
We used the previously characterized construct pCAG-EGFP/ red fluorescent protein (RFP)-miRNAint (G/R-micro RNAs (miRNAs)) to express miRNAs in transgenic mice (Fig. 1A) [15]. This construct first expresses enhance green fluorescent protein (EGFP), which enables rapid screen of transgenic mouse lines where the transgene is active in the desired tissue
Upon induction by Cre, the EGFP gene is excised, leaving the promoter to drive the expression of red fluorescent protein (RFP) and miRNA
Summary
The Cre-loxP system has been used widely for conditional transgene expression in mice. A common approach is to make transgenic mice by pronuclear injection of transgene constructs where the transgene is flanked with loxP sites. This approach has been adopted for either conditional induction or cessation of transgene expression in a spatially and temporally controllable manner by crossing with various Cre-expressing driver lines. To take advantage of this powerful approach, we applied it for conditional expression of several miRNAs targeting specific genes in mice
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