Adaptation of GAL4 activators for GAL4 enhancer trapping in zebrafish
Version of Record online: 2 FEB 2009
Copyright © 2009 Wiley-Liss, Inc.
Volume 238, Issue 3, pages 641–655, March 2009
How to Cite
Ogura, E., Okuda, Y., Kondoh, H. and Kamachi, Y. (2009), Adaptation of GAL4 activators for GAL4 enhancer trapping in zebrafish. Dev. Dyn., 238: 641–655. doi: 10.1002/dvdy.21863
- Issue online: 22 FEB 2009
- Version of Record online: 2 FEB 2009
- Manuscript Accepted: 19 DEC 2008
- The Ministry of Education, Culture, Sports, Science and Technology of Japan. Grant Numbers: 19-5118, 16027229, 20017019, 18570197, 17107005
- Japan Society for the Promotion of Science for Japanese Junior Scientists
Additional supporting information may be found in the online version of this article.
|DVDY_21863_sm_SuppFigS1.eps||672K||Supp. Fig. 1. Effects of the EF1a enhancer on the TK, TKBA, and GATA2 promoters in zebrafish embryos. A: Schematic representation of the constructs used in this assay. The Xenopus EF1a enhancer fragment (175 bp) from pXeX Johnson and Krieg, 1994, Gene, 147:223–226) was placed upstream of the promoter of each Venusluc construct. B: Approximately 1 nl of a solution containing each of the Venusluc vectors (20 pg/nl) was injected with the reference Renilla luciferase vector (phRG-TK, 5 pg/nl) into one-cell-stage embryos of wild-type zebrafish. Injected embryos were lysed at 10 hpf and luciferase activities were measured. The luciferase activity generated by the TK-Venusluc vector was arbitrarily assigned a value of 1. Data are the mean values with standard errors from two independent injection experiments.|
|DVDY_21863_sm_SuppFigS2.tif||7374K||Supp. Fig. 2. GAL4VP16 expressed by trapped endogenous enhancers strongly activates UAS:DsRedExDR reporter expression, but disrupts normal development. A–S: F1 embryos derived from crossing of the GAL4VP16 enhancer-trap founders with the UAS:DsRedExDR reporter line demonstrated strong DsRedExDR reporter expression at 30 to 36 hpf. In these F1 embryos, two classes of DsRedExDR-expression were recognized: strong, region-specific expression, and weak, widespread expression. The first class of DsRedExDR expression probably resulted from trapped enhancer-dependent GAL4VP16 expression because crossing with different driver fish led to unique expression patterns. The second class of DsRedExDR expression was likely caused by low-level GAL4VP16 expression mediated by the basal promoter activity in the transgene. GAL4VP16-positive F1 embryos except TKBAsi-GAL4VP16#14m did not survive beyond 5 dpf. Note that the embryos showing the most intense reporter expression levels were often already malformed (B,E,N,P,Q).|
|DVDY_21863_sm_SuppFigS3.eps||12904K||Supp. Fig. 3. Comparison of the toxicity and transactivation potentials of the variant GAL4 activators including GAL4VP16413-470 in zebrafish embryos. A: Comparison of the toxicity levels of the various GAL4 activators. The indicated GAL4 activator mRNAs (10, 20, or 30 pg) were injected into one- to two-cell-stage embryos (TL line) and morphological abnormalities at 28–30 hpf were classified according to their severity into five groups: normal, weakly malformed, moderately malformed, severely malformed, and lethal phenotypes. The affected tissues varied depending on the activators as shown in B. EGFP mRNA (30 pg) was also injected as a control. B: Typical morphologies for each phenotypic class. GAL4NFkB (a, 10 pg; b–d, 30 pg), GAL4VPmad2 (e–h, 20 pg), and GAL4VP16413-470 (i, 20 pg; j,k, 30 pg). Because GAL4VP16413-470 caused defects in the eye and the anterior CNS (insets of j, k) without significantly affecting posterior development, the anterior phenotype was mainly used as a criterion of phenotypic classes. C: Comparison of the transactivation potentials of GAL4VP16, GAL4VPmad3, GAL4VPmad2, and GAL4VP16413-470. Co-injection of GAL4 activator mRNAs (2 pg) with the UAS:Venusluc reporter vector was carried out as described in Figure 3. A low amount of activator mRNAs was used, as the adverse effects of GAL4VP16 are negligible at this expression level.|
|DVDY_21863_sm_SuppTableS1.xls||53K||Supplementary Table 1. Screens for enhancer-trap lines|
|DVDY_21863_sm_SuppTableS2.xls||19K||Supplementary Table 2. Transposon insertion sites of selected enhancer-trap lines|
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