Daphne Georlette

Background: External development and optical transparency of embryos make zebrafish exceptionally suitable for in vivo insertional mutagenesis using fluorescent proteins to visualize expression patterns of mutated genes. Recently developed Gene Breaking Transposon (GBT) vectors greatly improve the fidelity and mutagenicity of transposon-based gene trap vectors. Results: We constructed and tested a bipartite GBT vector with Gal4-VP16 as the primary gene trap reporter. Our vector also contains a UAS:eGFP cassette for direct detection of gene trap events by fluorescence. To confirm gene trap events, we generated a UAS:mRFP tester line. We screened 270 potential founders and established 41 gene trap lines. Three of our gene trap alleles display homozygous lethal phenotypes ranging from embryonic to late larval: nsf ^tpl6 , atp1a3a ^tpl10 and flr ^tpl19 . Our gene trap cassette is flanked by direct loxP sites, which enabled us to successfully revert nsf ^tpl6 , atp1a3a ^tpl10 and flr ^tpl19 gene trap alleles by injection of Cre mRNA. The UAS:eGFP cassette is flanked by direct FRT sites. It can be readily removed by injection of Flp mRNA for use of our gene trap alleles with other tissue-specific GFP-marked lines. The Gal4-VP16 component of our vector provides two important advantages over other GBT vectors. The first is increased sensitivity, which enabled us to detect previously unnoticed expression of nsf in the pancreas. The second advantage is that all our gene trap lines, including integrations into non-essential genes, can be used as highly specific Gal4 drivers for expression of other transgenes under the control of Gal4 UAS. Conclusions: The Gal4-containing bipartite Gene Breaking Transposon vector presented here retains high specificity for integrations into genes, high mutagenicity and revertibility by Cre. These features, together with utility as highly specific Gal4 drivers, make gene trap mutants presented here especially useful to the research community.

Background: The Positive Transcription Elongation Factor b (P-TEFb) is a complex of Cyclin Dependent Kinase 9 (CDK9) with either cyclins T1, T2 or K. The complex phosphorylates the C-Terminal Domain of RNA polymerase II (RNAPII) and negative elongation factors, stimulating productive elongation by RNAPII, which is paused after initiation. P-TEFb is recruited downstream of the promoters of many genes, including primary response genes, upon certain stimuli. Flavopiridol (FVP) is a potent pharmacological inhibitor of CDK9 and has been used extensively in cells as a means to inhibit CDK9 activity. Inhibition of P-TEFb complexes has potential therapeutic applications.Results: It has been shown that Lipopolysaccharide (LPS) stimulates the recruitment of P-TEFb to Primary Response Genes (PRGs) and proposed that P-TEFb activity is required for their expression, as the CDK9 inhibitor DRB prevents localization of RNAPII in the body of these genes. We have previously determined the effects of FVP in global gene expression in a variety of cells and surprisingly observed that FVP results in potent upregulation of a number of PRGs in treatments lasting 4-24 h. Because inhibition of CDK9 activity is being evaluated in pre-clinical and clinical studies for the treatment of several pathologies, it is important to fully understand the short and long term effects of its inhibition. To this end, we determined the immediate and long-term effect of FVP in the expression of several PRGs. In exponentially growing normal human fibroblasts, the expression of several PRGs including FOS, JUNB, EGR1 and GADD45B, was rapidly and potently downregulated before they were upregulated following FVP treatment. In serum starved cells re-stimulated with serum, FVP also inhibited the expression of these genes, but subsequently, JUNB, GADD45B and EGR1 were upregulated in the presence of FVP. Chromatin Immunoprecipitation of RNAPII revealed that EGR1 and GADD45B are transcribed at the FVP-treatment time points where their corresponding mRNAs accumulate. These results suggest a possible stress response triggered by CDK9 inhibition than ensues transcription of certain PRGs.Conclusions: We have shown that certain PRGs are transcribed in the presence of FVP in a manner that might be independent of CDK9, suggesting a possible alternative mechanism for their transcription when P-TEFb kinase activity is pharmacologically inhibited. These results also show that the sensitivity to FVP is quite variable, even among PRGs.