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A novel strategy for a splice-variant selective gene ablation: the example of the versican v0/v2 knockout

By M T Dours-Zimmermann and D R Zimmermann


The complete knockout of genes that give rise to alternative splice products can often provide only an integral view of the dominant function(s) of all the isoforms they encode. If one of these isoforms is indispensable for life, a constitutive and complete inactivation may even preclude any in vivo studies of later expressed splice-variants in mice. To explore function of the tissue-restricted versican V2 isoform during central nervous system maturation, for instance, we had to circumvent the early embryonic lethality of the complete knockout by employing a novel splice-variant-specific gene ablation approach. For this purpose, we introduced a preterm translational stop codon preceded by an ER-retention signal (KDEL) into the alternatively spliced exon 7 of the VCAN gene. This way the synthesis of the V2- and the V0-forms of the proteoglycan was entirely abolished in the mutant mice, most likely mediated by a KDEL-promoted intracellular degradation of the mutant fragment and by a nonsense-mediated decay mechanism. The expression of the vitally important V1-isoform and the smallest V3-variant remained, however, unaffected. Here we provide the details of our targeting strategy, the screening procedure, the generation of isoform-specific antibodies, and the transcript analysis and we supply the experimental protocols for the biochemical and immunohistological examinations of the mutant mouse strain Vcan(tm1.1Dzim)

Topics: Institute of Pathology and Molecular Pathology, 610 Medicine & health
Publisher: Springer
Year: 2012
DOI identifier: 10.1007/978-1-61779-498-8_5
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Provided by: ZORA
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