Proceedings of the 10th International

ABSTRACT Intensive Programs (IP) have been organized by four European partner universities. The main idea is to gather approximately 40 students and 15 teachers together for three weeks to conceive, design, implement, and operate embedded system prototypes. Self-evaluation is an integrated part of the IP. The results of the evaluations are used to improve the concept, content, and practical arrangements for the next IP. The same partner network has organized similar intensive projects with different topics, but using the same internal evaluation method. We can recognize issues which make the IP successful and are common to the intensive project concept, independent of the topic. Based on the evaluation material, we will make some recommendations that can help organize similar intensive projects in the future

0812-9869-9940 (WA), Jual Keranda Mayat Jungke

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Generation of a conditional lima1a allele in zebrafish using the FLEx switch technology

Gene trapping has emerged as a valuable tool to create conditional alleles in various model organisms. Here we report the FLEx‐based gene trap vector SAGFLEx that allows the generation of conditional mutations in zebrafish by gene‐trap mutagenesis. The SAGFLEx gene‐trap cassette comprises the rabbit β‐globin splice acceptor and the coding sequence of GFP, flanked by pairs of inversely oriented heterotypic target sites for the site‐specific recombinases Cre and Flp. Insertion of the gene‐trap cassette into endogenous genes can result in conditional mutations that are stably inverted by Cre and Flp, respectively. To test the functionality of this system we performed a pilot screen and analyzed the insertion of the gene‐trap cassette into the lima1a gene locus. In this lima1a allele, GFP expression faithfully recapitulated the endogenous lima1a expression and resulted in a complete knockout of the gene in homozygosity. Application of either Cre or Flp was able to mediate the stable inversion of the gene trap cassette and showed the ability to conditionally rescue or reintroduce the gene inactivation. Combined with pharmacologically inducible site specific recombinases the SAGFLEx vector insertions will enable precise conditional knockout studies in a spatial‐ and temporal‐controlled manner

CreER^T2-driver lines expressing in various embryonic tissues.

<p>(A) Ubiquitous expression of CreER^T2 in tud20Gt at 24 hpf. (B-E) CreER^T2 is expressed in the developing heart in tud35Gt and tud36Gt at 24 and 48 hpf. (F-P) Expression of CreER^T2 can be detected in the anlagen of (F-I) the inner ear in tud37Gt and tud38Gt (J) blood island in tud16Gt, (K,L) kidney in tud28Gt (M-O) fin buds in tud28Gt, tud29Gt and tud35Gt and (P) the reproductive system in tud29Gt at 24 and 48 hpf. Bold letters indicate CreER^T2-driver lines with known gene trap integrations. <i>(See text for description of expression patterns</i>.<i>)</i></p

Summary of currently available Cre/CreER^T2-driver lines in zebrafish.

<p>Cre- and CreER^T2-driver lines available in zebrafish are listed in chronological order including citation.</p><p>Summary of currently available Cre/CreER^T2-driver lines in zebrafish.</p

CreER^T2-driver lines expressing in the embryonic neural tube.

<p>(A, B) Pan-neural expression of CreER^T2 revealed by <i>in situ</i> hybridization in tud20Gt and tud30Gt at 24 hpf. (C,D) Expression of CreER^T2 in the spinal cord in tud17Gt at 24 and 48 hpf. (E,F) Hindbrain expression of CreER^T2 in tud18Gt and tud35Gt at 24 hpf. (G-J) Fore-/Midbrain expression and (K-Q) other restricted patterns of CreER^T2 in tud15Gt, tud19Gt, tud27Gt, tud28Gt, tud34Gt, tud35Gt and tud37Gt at 24 and 48 hpf, respectively. Bold letters indicate CreER^T2-driver lines with known gene trap integrations. (<i>See text for detailed description of expression patterns</i>.<i>);</i> ep: epiphysis; fb: forebrain; hb: hindbrain; mb: midbrain; mhb: mid-hindbrain-boundary; t: tectum; tc: telencephalon.</p

Molecular identification of functional CreER^T2-driver lines.

<p>Full names of functional CreER^T2-driver lines are designated as transgenic lines according to ZFIN nomenclature. Information of the gene trap integrations are shown with gene names, NCBI gene ID, chromosomal insertion (linkage group) and the insertion site relative to the gene architecture.</p><p>Molecular identification of functional CreER^T2-driver lines.</p

Gene trap insertion into the <i>otx1b</i> locus of tud37Gt.

<p>(A) Schematic drawing of the <i>otx1b</i> locus comprising of four exons (E1-E4) encoding a homeobox domain transcription factor. White boxes represent the 5’ and 3’ untranslated regions separated by the open reading frame in pink. The mCT2aC-cassette integrated into E4. (B) Bright field images of homozygous mutant tud37Gt embryos and heterozygous siblings. In comparison to heterozygous siblings, homozygous mutant tud37Gt embryos show defects in the developing eye, fore-/midbrain, ear and heart (white arrowheads) as well as a bend body shape.</p

Isolation of Novel CreER^T2-Driver Lines in Zebrafish Using an Unbiased Gene Trap Approach

<div><p>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/CreER^T2-driver lines still constrains widespread application in this model organism. To enlarge the pool of existing CreER^T2-driver lines, we performed a genome-wide gene trap screen using a <i>Tol2</i>-based mCherry-T2a-CreER^T2 (mCT2aC) gene trap vector. This cassette consists of a splice acceptor and a mCherry-tagged variant of CreER^T2 which enables simultaneous labeling of the trapping event, as well as CreER^T2 expression from the endogenous promoter. Using this strategy, we generated 27 novel functional CreER^T2-driver lines expressing in a cell- and tissue-specific manner during development and adulthood. This study summarizes the analysis of the generated CreER^T2-driver lines with respect to functionality, expression, integration, as well as associated phenotypes. Our results significantly enlarge the existing pool of CreER^T2-driver lines in zebrafish and combined with Cre–dependent effector lines, the new CreER^T2-driver lines will be important tools to manipulate the zebrafish genome.</p></div