Abstract
Genetic redundancy poses a major problem to the analysis of gene function. RNA interference allows the down-regulation of several genes simultaneously, offering the possibility to overcome genetic redundancy, something not easily achieved with traditional genetic approaches. Previously we have used a polycistronic miR155-based framework to knockdown expression of three genes of the early B cell factor family in cultured cells. Here we develop the system further by generating transgenic mice expressing the RNAi construct in vivo in an inducible manner. Expression of the transgene from the strong CAG promoter is compatible with a normal function of the basal miRNA/RNAi machinery, and the miR155 framework readily allows inducible expression from the Rosa26 locus as shown by Gfp. However, expression of the transgene in hematopoietic cells does not lead to changes in B cell development and neuronal expression does not affect cerebellar architecture as predicted from genetic deletion studies. Protein as well as mRNA levels generated from Ebf genes in hetero- and homozygous animals are comparable to wild-type levels. A likely explanation for the discrepancy in the effectiveness of the RNAi construct between cultured cells and transgenic animals lies in the efficiency of the sequences used, possibly together with the complexity of the transgene. Since new approaches allow to overcome efficiency problems of RNAi sequences, the data lay the foundation for future work on the simultaneous knockdown of several genes in vivo.
Highlights
One of the surprising findings of gene targeting in mice are knock-out animals with no obvious phenotype
Despite a strong expression in tissues like the olfactory epithelium and in many neuronal cells, deletion of Ebf1 results in a distinct phenotype, i.e. the complete block of early B cell development [13]. This finding was unexpected, but Ebf1 is the only family member expressed in hematopoietic cells, whereas the tissues mentioned above express other early B cell factor (Ebf) genes in a largely overlapping manner [14,15,16,17]
The single shRNAs bind to regions of the Ebf mRNAs corresponding to the DNA binding or the adjacent IPT/TIG-domain of the encoded proteins (Figure S1A) and are concatemerised as indicated (Figure S1B)
Summary
One of the surprising findings of gene targeting in mice are knock-out animals with no obvious phenotype. In an effort to overcome functional redundancy and examine the contribution of Ebf factors in general to the support of hematopoietic stem cells, we employed the SIBR (synthetic inhibitory BIC-derived RNA) vectors, a new approach to downregulate several genes simultaneously based on RNA interference [24]. In this system shRNA sequences are embedded into the framework of miR155/BIC including the flanking sequences, which are cleaved by the RNAseIII enzyme Drosha. We report the generation of transgenic mice with a targeted insertion of the SIBR-based EbfRNAi construct into the murine Rosa locus, and describe the effects of its induced expression
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