Abstract
Gene manipulation using the Cre/loxP-recombinase system has been successfully employed in zebrafish to study gene functions and lineage relationships. Recently, gene trapping approaches have been applied to produce large collections of transgenic fish expressing conditional alleles in various tissues. However, the limited number of available cell- and tissue-specific Cre/CreERT2-driver lines still constrains widespread application in this model organism. To enlarge the pool of existing CreERT2-driver lines, we performed a genome-wide gene trap screen using a Tol2-based mCherry-T2a-CreERT2 (mCT2aC) gene trap vector. This cassette consists of a splice acceptor and a mCherry-tagged variant of CreERT2 which enables simultaneous labeling of the trapping event, as well as CreERT2 expression from the endogenous promoter. Using this strategy, we generated 27 novel functional CreERT2-driver lines expressing in a cell- and tissue-specific manner during development and adulthood. This study summarizes the analysis of the generated CreERT2-driver lines with respect to functionality, expression, integration, as well as associated phenotypes. Our results significantly enlarge the existing pool of CreERT2-driver lines in zebrafish and combined with Cre–dependent effector lines, the new CreERT2-driver lines will be important tools to manipulate the zebrafish genome.
Highlights
Zebrafish has become an excellent model system to understand gene function in vertebrate development and disease
In order to obtain a wide variety of CreERT2-driver lines a gene trap approach was chosen using a vector containing a splice acceptor (SA) and a mCherry-tagged variant of CreERT2 followed by a polyadenylation (p(A)) signal (Fig 1A)
1479 fish were screened for the different gene trap constructs yielding trapping rates of 8,7%, 14,1% and 11,6% respectively, which is consistent with previous data [5, 35, 57]
Summary
Zebrafish has become an excellent model system to understand gene function in vertebrate development and disease. Several advantages, such as the optical clarity of its embryos, short generation time and large number of offspring enable for large-scale forward mutagenesis [1,2,3,4,5,6] screens as well as real-time in vivo imaging [7,8,9]. Site-specific recombinases (SSRs), which have been an invaluable tool for altering the mouse and fly genome [17,18,19,20], have been successfully applied in zebrafish [21,22,23,24]. PLOS ONE | DOI:10.1371/journal.pone.0129072 June 17, 2015
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