Mosaic analysis and tumor induction in zebrafish by microsatellite instability-mediated stochastic gene expression.

ABSTRACT: Mosaic analysis, in which two or more populations of cells with differing genotypes are studied in a single animal, is a powerful approach to study developmental mechanisms and gene function in vivo. Over recent years, several genetic methods have been developed to achieve mosaicism in zebrafish, but despite their advances, limitations remain and different approaches and further refinements are warranted. Here, we describe an alternative approach for creating somatic mosaicism in zebrafish that relies on the instability of microsatellite sequences during replication. We placed the coding sequences of various marker proteins downstream of a microsatellite and out-of-frame; in vivo frameshifting into the proper reading frame results in expression of the protein in random individual cells that are surrounded by wild-type cells. We optimized this approach for the binary Gal4-UAS expression system by generating a driver line and effector lines that stochastically express Gal4-VP16 or UAS:H2A-EGFP and self-maintaining UAS:H2A-EGFP-Kaloop, respectively. To demonstrate the utility of this system, we stochastically expressed a constitutively active form of the human oncogene H-RAS and show the occurrence of hyperpigmentation and sporadic tumors within 5 days. Our data demonstrate that inducing somatic mosaicism through microsatellite instability can be a valuable approach for mosaic analysis and tumor induction in Danio rerio.

SUBMITTER: Koole W 

PROVIDER: S-EPMC4073281 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature