1999 — 2021 |
Seydoux, Geraldine Catherine Joelle |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R37Activity Code Description: To provide long-term grant support to investigators whose research competence and productivity are distinctly superior and who are highly likely to continue to perform in an outstanding manner. Investigators may not apply for a MERIT award. Program staff and/or members of the cognizant National Advisory Council/Board will identify candidates for the MERIT award during the course of review of competing research grant applications prepared and submitted in accordance with regular PHS requirements. |
Regulation of Germ Cell Fate During Embryogenesis @ Johns Hopkins University
? DESCRIPTION (provided by applicant): The long-term goal of this project is to characterize the mechanisms that specify the fate of the germline, a fundamental problem in developmental biology. In all animals, the founder cells of the germline (primordial germ cells) display two conserved characteristics: they possess germ granules, RNA granules specific to the germline, and they express members of the Nanos family of RNA-binding proteins. The goal of this proposal is to understand how germ granules and Nanos function together to specify the fate of primordial germ cells. In preliminary work, we have found that PGCs that lack Nanos activity prematurely activates the expression of ~1000 genes normally expressed in oocytes and somatic cells, a phenotype also seen in mutants that lack the Polycomb Repressive Complex. Specific Aim I will identify the mechanisms used by Nanos to prevent this massive gene mis-regulation. We have already identified one promising candidate Nanos target: LIN-15B, a likely component of the DRM transcription factor complex that activates gene expression in oocytes. Specific Aim II will examine the role of a new group of germ granule proteins we recently characterized. The MEG proteins form stabilizing scaffolds around the central cores of each germ granule. Our genetic analyses indicate that the MEGs, but not the proteins in the core, are essential for PGC fate. We will investigate how the MEGs, predicted to be intrinsically-disordered with no recognizable domain, function with Nanos to specify PGCs. Two technical breakthroughs support this application. First we have worked out methods to isolate PGCs in large enough numbers to determine their transcriptome by RNAseq. We will use this method to define how Nanos and germ granule proteins affect the PGC transcriptome at a genome-wide level. Second we have developed a highly efficient genome editing method, using CRISPR/Cas9, that allows us to mutate, tag, delete and replace any gene of interest in just 4 days. This method gives us unprecedented genetic and biochemical access to the genes and proteins that are the focus of this application.
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1 |
2002 — 2005 |
Seydoux, Geraldine |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Control of Cell Polarity in the C. Elegans Zygote @ Johns Hopkins University
DESCRIPTION: (provided by applicant): The long-term goal of this project is to elucidate the mechanisms that control cell polarity using the C. elegans 1-cell embryo (zygote) as model system. C. elegans offers two key advantages for this work: 1) the ability to visualize specific protein components of the polarity machinery in live cells, and 2) the ability to rapidly identify genes required for polarity using both forward and reverse genetic approaches. Establishment of the anterior-posterior (AlP) body axis of C. elegans depends on polarization of the zygote shortly after fertilization. The relevance of this system to other polarized cells was firmly established last year, when homologues of the core components of the C. elegans A/P polarity machinery were discovered in Drosophila and mammals, and shown to have similar polarity functions. Recently, we demonstrated that A/P polarity in the C. elegans zygote is triggered by microtubules emanating from the sperm asters; earlier studies also indicated a role for the actin cytoskeleton. The goal of this proposal is to identify the molecular mechanisms that link the core A/P polarity machinery to the microtubule and actin cytoskeletons. To this end, we have developed a method to monitor the establishment of A/P polarity directly in live zygotes. We will use this method to characterize the dynamics of AfP polarity, and to identify new genes involved in this process. Our screening strategy will use a functional genomics approach as well as standard genetic methods. In a pilot screen, we identified a new gene, porn-I, required for maintenance of the polarity axis during mitosis and alignment of the spindle along that axis. POM-1 is related to Pomip, a protein kinase required for polarized growth and cell division in S. pombe. Our initial findings with porn-i reveal the existence of a previously unrecognized link between spindle alignment and the A/P polarity machinery in the zygote. Cell polarity is at the core of many essential processes in animals including asymmetric cell divisions, the functioning of polarized cell types (e.g. neurons and epithelial cells), and inhibition of unregulated cell proliferation. We anticipate that our studies will provide significant insights into these processes by taking advantage of the experimental attributes of a simple model system.
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1 |
2003 — 2004 |
Seydoux, Geraldine |
R13Activity Code Description: To support recipient sponsored and directed international, national or regional meetings, conferences and workshops. |
2003/2005 International C. Elegans Meetings @ Johns Hopkins University
DESCRIPTION (provided by applicant): Research on the nematode worm C. elegans ranges from investigating such global problems as the circuitry and function of an entire nervous system and the organization of a genome, to investigating elements involved in control of gene expression and specification of the fates of individual cells. C. elegans has become an important experimental organism for the study of many aspects of animal biology, particularly the genetic and molecular bases of development and behavior. The international meeting has run biennially since 1979 and is the only International meeting dedicated to C. elegans. We are requesting funds to help cover costs and travel of six percent of participants to attend the international C. elegans Meeting to be held at the University of California in Los Angeles, CA, in 2003. We are also requesting similar funds for the 2005 meeting to be held at a location to be determined. The Organizing Committee will rank applications for support, basing their funding decisions on 1) the potential contributions of the applicants to the meeting, 2) the benefits the applicants will receive from attending the meeting and 3) minority status. As this is the only international meeting dedicated to C. eIegans research, the committee is particularly anxious to encourage participation of women and minorities. Previous C. elegans meetings have led to the exchange of knowledge, ideas, methods, mutants, and clones and have been vital in fostering the sense of excitement and the collegiality and cooperativity that characterize this field.
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1 |
2017 — 2018 |
Rougvie, Ann E. [⬀] Seydoux, Geraldine Catherine Joelle Sternberg, Paul Warren (co-PI) [⬀] |
R24Activity Code Description: Undocumented code - click on the grant title for more information. |
Enhancing the C. Elegans Animal Resource Through Genome Editing @ University of Minnesota
Project Summary/Abstract C. elegans is a premier model organism that has proven highly useful for discovery of gene function and embedding genes into functional pathways, many of which were discovered in this transparent animal and are conserved in humans. In addition, other nematodes are crucial parasites of humans, infecting roughly a third of the world's population and plant-parasitic nematodes are recognized as one of the greatest threats to crops throughout the world. Despite these extensive ties to human health and disease, nematode specific genes are vastly understudied. We will use the latest CRISPR technology to knockout genes and provide a set of high value genetic tools to the communities of C. elegans researchers, human geneticists, and parasitic nematologists. Targets chosen will be 1000 C. elegans orthologs of genes implicated in human disease as well as 500 conserved genes about which essentially nothing is known, and 500 nematode-specific genes present in human parasites. We will develop an efficient pipeline of gene disruption that includes target choice, oligonucleotide design and ordering, molecular biology, microinjection into worms, selection or screening of conversion events, homozygosing or balancing alleles and verification. Edited strains will be grossly phenotyped and deposited in the CGC for distribution and advertised though the CGC and WormBase websites. We will begin by evaluating two approaches. One approach selects for gene conversion and disruption using a selectable insertion cassette; the other uses insertion of GFP as a zero-length translation fusion and will be screened by PCR. Over the first year we will identify and focus on the approach that is of higher throughput. We will continuously refine targeting methods and improve the pipeline to increase the range and efficiency as well as decrease the cost of production. We will develop Cas9 variants to increase the availability of editing sites within genes. We will develop a PCR bridge method to allow cloning free addition of homology arms. To efficiently screen for in frame GFP edits we will develop a low cost ELISA assay for GFP. Finally, we will also explore using the transcriptional activator Gal4 as a marker. This is a multi-PI project which includes the lead-PIs of the CGC, of WormBase and of the Knockout Consortium; two of the PIs have made important contributions to CRISPR technology development in C. elegans.
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0.91 |