One key bottleneck in understanding the human genome is the relative under-characterization of 90% of protein coding regions. We report a collection of 1,200 transgenic zebrafish strains made with the gene-break transposon (GBT) protein trap to simultaneously report and reversibly knockdown the tagged genes. Protein trap-associated mRFP expression shows previously undocumented expression of 35% and 90% of cloned genes at 2 and 4 days post-fertilization, respectively. Further, investigated alleles regularly show 99% gene-specific mRNA knockdown. Homozygous GBT animals in ryr1b, fras1, tnnt2a, edar and hmcn1 phenocopied established mutants. 204 cloned lines trapped diverse proteins, including 64 orthologs of human disease-associated genes with 40 as potential new disease models. Severely reduced skeletal muscle Ca2+ transients in GBT ryr1b homozygous animals validated the ability to explore molecular mechanisms of genetic diseases. This GBT system facilitates novel functional genome annotation towards understanding cellular and molecular underpinnings of vertebrate biology and human disease.
2The zebrafish is a powerful model to explore the molecular genetics and expression of the vertebrate 3 genome. The gene break transposon (GBT) is a unique insertional mutagen that reports the expression of 4 the tagged member of the proteome while generating Cre-revertible genetic alleles. This 1000+ locus 5 collection represents novel codex expression data from the illuminated mRFP protein trap, with 36% 6 and 87% of the cloned lines showcasing to our knowledge the first described expression of these genes 7 at day 2 and day 4 of development, respectively. Analyses of 183 molecularly characterized loci indicate 8 a rich mix of genes involved in diverse cellular processes from cell signaling to DNA repair. The 9 mutagenicity of the GBT cassette is very high as assessed using both forward and reverse genetic 10 approaches. Sampling over 150 lines for visible phenotypes after 5dpf shows a similar rate of discovery 11 of embryonic phenotypes as ENU and retroviral mutagenesis. Furthermore, five cloned insertions were 12 in loci with previously described phenotypes; embryos homozygous for each of the corresponding GBT 13 alleles displayed strong loss of function phenotypes comparable to published mutants using other 14 mutagenesis strategies (ryr1b, fras1, tnnt2a, edar and hmcn1). Using molecular assessment after 15 positional cloning, to date nearly all alleles cause at least a 99+% knockdown of the tagged gene. 16 Interestingly, over 35% of the cloned loci represent 68 mutants in zebrafish orthologs of human disease 17 loci, including nervous, cardiovascular, endocrine, digestive, musculoskeletal, immune and integument 18 systems. The GBT protein trapping system enabled the construction of a comprehensive protein codex 19 including novel expression annotation, identifying new functional roles of the vertebrate genome and 20 generating a diverse collection of potential models of human disease.21 22 23
The prognosis of acute myeloid leukemia (AML) remains poor in part due to the leukemic bone marrow microenvironment. Our lab has found that CXCL12, a chemokine abundant within the leukemic bone marrow microenvironment, induces apoptosis of AML cells expressing CXCR4, the receptor for CXCL12. However, this CXCL12/CXCR4-induced apoptosis is inhibited by differentiating osteoblasts, which protect AML cells from apoptosis in the bone marrow. Tipifarnib is a farnesyltransferase inhibitor shown to increase progression-free survival in AML patients that express high levels of CXCL12. Here, we report that tipifarnib inhibits the CXCL12/CXCR4-directed migration of AML cells via an ERK independent pathway. Furthermore, tipifarnib enhances CXCL12/CXCR4-mediated AML cell apoptosis via a mechanism that alters expression of apoptosis-regulating proteins. In addition, tipifarnib disrupts AML protection by osteoblasts, increasing AML cell apoptosis. Tipifarnib inhibits the osteoblast-mediated protection of AML cells via disrupting COL1A1 and TNAP, proteins essential for extracellular matrix production. In conclusion, tipifarnib alters the bone marrow microenvironment which is predicted to enhance eradication of AML via inhibiting CXCL12/CXCR4 directed cellular migration of AML cells, reducing the protective effects of differentiating osteoblasts by disrupting matrix protection proteins, and increasing CXCL12/CXCR4-mediated apoptosis.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.