A Non-Specific Effect Associated with Conditional Transgene Expression Based on Cre-loxP Strategy in Mice

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.

Partial loss of TDP-43 function causes phenotypes of amyotrophic lateral sclerosis

“…S1B), which led to aberrant chromosome segregation (SI Appendix, Fig. S1C) and death of proliferating cells during brain development (45). Because of this artifact, we were precluded from inducing the transgene expression selectively in the CNS.…”

“…As a control, we also generated a line of Tg mice that constitutively expressed a scrambled amiRNA (amiR-SCR) using the same construct design and the same strategy to convert from the GFP-expressing parent line to the constitutive RFP-expressing and amiR-SCR-expressing line (45). Western blot analysis revealed that these Tg mice expressed a higher level of transgene than the amiR-TDP43 mice in the CNS (SI Appendix, Fig.…”

“…S1A). The Tg lines were created and screened as described (45). An artifact associated with multiple transgene integration into the mouse genome precluded a selective induction of transgene expression in the CNS (45) (SI Appendix, Fig.…”

Nitric Oxide-Mediated Oxidative Damage and the Progressive Demise of Motor Neurons in ALS

“…These animal models demonstrate that mutant SOD1 expression within non-neuronal cells can be a determinant of disease progression, while the mutant enzyme’s presence in motor neurons contributes to disease onset. However, a recent study showed that such models utilizing conditional transgene expression based on a Cre-loxP strategy was associated with microencephaly, which may cause the loss of SOD1-expressing cells during development leading to observed phenotype (Qiu et al , 2011)…”

Creating conditional dual fluorescence labeled transgenic animals for studying function of small noncoding RNAs

“…The approach was applied for CNS knockdown (37). But it did not work due to unintended effects from inverted tandem integrations.…”