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DVDY_21863_sm_SuppFigS1.eps672KSupp. 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.tif7374KSupp. 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.eps12904KSupp. 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.xls53KSupplementary Table 1. Screens for enhancer-trap lines
DVDY_21863_sm_SuppTableS2.xls19KSupplementary Table 2. Transposon insertion sites of selected enhancer-trap lines

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