Home About us Contact
|
Home About us Contact | ||
|
|
||
Enhancer Trap (enhancer + trap)
Selected AbstractsTargeted gene expression by the Gal4-UAS system in zebrafishDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 6 2008Kazuhide Asakawa Targeted gene expression by the Gal4-UAS system is a powerful methodology for analyzing function of genes and cells in vivo and has been extensively used in genetic studies in Drosophila. On the other hand, the Gal4-UAS system had not been applied effectively to vertebrate systems for a long time mainly due to the lack of an efficient transgenesis method. Recently, a highly efficient transgenesis method using the medaka fish Tol2 transposable element was developed in zebrafish. Taking advantage of the Tol2 transposon system, we and other groups developed the Gal4 gene trap and enhancer trap methods and established various transgenic fish expressing Gal4 in specific cells. By crossing such Gal4 lines with transgenic fish lines harboring various reporter genes and effector genes downstream of UAS (upstream activating sequence), specific cells can be visualized and manipulated in vivo by targeted gene expression. Thus, the Gal4 gene trap and enhancer trap approaches together with various UAS lines should be important tools for investigating roles of genes and cells in vertebrates. [source] Adaptation of GAL4 activators for GAL4 enhancer trapping in zebrafishDEVELOPMENTAL DYNAMICS, Issue 3 2009Eri Ogura Abstract An enhancer trap-based GAL4-UAS system in zebrafish requires strong GAL4 activators with minimal adverse effects. However, the activity of yeast GAL4 is too low in zebrafish, while a fusion protein of the GAL4 DNA-binding domain and the VP16 activation domain is toxic to embryonic development, even when expressed at low levels. To alleviate this toxicity, we developed variant GAL4 activators by fusing either multimeric forms of the VP16 minimal activation domain or the NF-,B activation domain to the GAL4 DNA-binding domain. These variant GAL4 activators are sufficiently innocuous and yet highly effective transactivators in developing zebrafish. Enhancer-trap vectors containing these GAL4 activators downstream of an appropriate weak promoter were randomly inserted into the zebrafish genome using the Sleeping Beauty transposon system. By the combination of these genetic elements, we have successfully developed enhancer trap lines that activate UAS-dependent reporter genes in a tissue-specific fashion that reflects trapped enhancer activities. Developmental Dynamics 238:641,655, 2009. © 2009 Wiley-Liss, Inc. [source] High-throughput enhancer trap by remobilization of transposon Minos in Ciona intestinalisGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 5 2007Satoko Awazu Abstract The enhancer trap approach utilizing transposons yields us information about gene functions and gene expression patterns. In the ascidian Ciona intestinalis, transposon-based transgenesis and insertional mutagenesis were achieved with a Tc1/mariner transposon Minos. We report development of a novel technique for enhancer trap in C. intestinalis. This technique uses remobilization of Minos in the Ciona genome. A Minos vector for enhancer trap was constructed and a tandem array insertion of the vector was introduced into the Ciona genome to create a mutator line. Minos was remobilized in Ciona chromosomes to create new insertions by providing transposases. These transposase-introduced animals were crossed with wild-type animals. Nearly 80% of F1 families showed novel GFP expression patterns. This high-throughput enhancer trap screen will be useful to create new marker transgenic lines showing reporter gene expression in specific tissues and to identify novel patterns of gene expression. genesis 45:307,317, 2007. © 2007 Wiley-Liss, Inc. [source] Multiple GUS expression patterns of a single Arabidopsis geneANNALS OF APPLIED BIOLOGY, Issue 1 2009Ekrem Dündar Abstract Ten independent transposant lines with gene or enhancer traps (ET) inserted into the same gene (At2g01170) were identified in Arabidopsis thaliana. Transposon insertions were confirmed for each line. Only three of five ET lines and only one of the five gene trap (GT) lines displayed uidA (GUS) staining. The GUS (,-glucuronidase) expression patterns of the ET lines were different in all three lines. In the GT line, the GUS expression was restricted to the vascular tissue under all conditions examined. The variation in ET GUS expression suggests that each ET was controlled by different enhancer elements or the different elements of the trapped locus may give rise to different GUS expression patterns. Of five GT lines, three have the GUS gene in the same orientation as the At2g01170 open reading frame, yet only one yielded GUS staining. Regardless of the insertion construct, only those transposants with an insertion at the 3, end of the gene yielded GUS staining. Some transposants displayed a longer root phenotype in the presence of kanamycin that was also observed in 3, insertion sites in At2g01170. Taken together, these data show that insertions in the 5, end of the gene disrupted expression and emphasise the complexity encountered with ET and GT constructs to characterise the expression patterns of genes of interest based solely on GUS expression patterns. [source] | ||||||||||