KOSEN 한인과학기술자네트워크

Tools for Insertional Mutagenesis in the Mouse: SBIR/STTR Initiative Primary Sponsor: National Institute on Drug Abuse Deadline: 3/11/2001; 4/11/2001 KEYWORDS RFA: DA-01-012 National Institute on Drug Abuse National Institute on Aging National Institute on Dental and Craniofacial Research National Institute on General Medical Sciences National Institute of Mental Health National Eye Institute National Institute on Deafness and Other Communication Disorders National Human Genome Research Institute National Center for Research Resources National Institute of Diabetes and Digestive and Kidney Diseases National Institute on Alcohol Abuse and Alcoholism National Institute of Child Health and Human Development National Institute of Environmental Health Sciences National Institute of Allergy and Infectious Diseases National Institute of Neurological Disorders and Stroke Letter of Intent Receipt Date: March 11, 2001 Application Receipt Date: April 11, 2001 PURPOSE This Request for Applications (RFA) solicits proposals for development of tools and techniques for the establishment of random and targeted sequence-tagged insertion libraries of embryonic stem (ES) cells that can be used to generate mutant mice in which the expression of the tagged gene could be controlled temporally and spatially. The development of such a resource for wide distribution to the scientific community would make it possible to scan the sequence database for any gene of interest and order the corresponding targeted ES cell line. Ideally, the insertional mutagenesis system developed would permit a wide range of genetic analyses and manipulations, including enhancer-trapping, conditional knockouts, conditional expression or overexpression, etc. It also would permit the larger community of investigators to utilize genomic resources efficiently. This effort complements ongoing National Institutes of Health (NIH) efforts to create and characterize induced point mutations in mice using ethylnitrosourea (ENU) and provides a functional genomics tool to translate the information from the Mouse Genome Sequencing Project. Further information about NIH initiatives on mouse genomics and genetics resources is available at www.nih.gov/science/models/mouse/. Because this initiative deals with the development of technology-driven commercial products, this initiative will use the Small Business Innovation Research/Small Business Technology Transfer Research (SBIR/STTR) programs. This RFA provides a flexible system within the SBIR/STTR programs to cover the research steps needed to develop and validate technology to generate insertional mutations in mouse ES cells. It will be run in parallel with a program of identical scientific scope utilizing the research project grant (R01) and for exploratory/development grant (R21) (see grants.nih.gov/grants/guide/rfa-files/RFA-DA-01-011.html) mechanisms. HEALTHY PEOPLE 2010 The Public Health Service (PHS) is committed to achieving the health promotion and disease prevention objectives of "Healthy People 2010," a PHS-led national activity for setting priority areas. This RFA, "Tools for Insertional Mutagenesis in the Mouse: SBIR/STTR Initiative," is related to several of the priority areas. Potential applicants may obtain a copy of "Healthy People 2010" at www.health.gov/healthypeople/. ELIGIBILITY REQUIREMENTS Eligibility requirements are described in the Omnibus Solicitation of the Public Health Service, Centers for Disease Control and Prevention, and Food and Drug Administration for Phase I SBIR/STTR Grant Applications (PHS 2000-2) (Omnibus Solicitation) (grants.nih.gov/grants/funding/sbirsttr1/index.pdf). Each organization submitting an SBIR/STTR grant application must qualify as a small business concern in accordance with the definition given in Section III of the Omnibus Solicitation. MECHANISM OF SUPPORT This RFA invites grant applications for Small Business Innovation Research/Small Business Technology Transfer (SBIR/STTR) projects with award duration and amounts greater than those routinely allowed under the SBIR program. This RFA must be read in conjunction with the Omnibus Solicitation (grants.nih.gov/grants/funding/sbirsttr1/index.pdf) and with the instructions for Phase II SBIR/STTR Grant Applications grants.nih.gov/grants/funding/sbir2/index.htm and grants.nih.gov/grants/funding/sttr2/index.html). All instructions and information in these documents also apply to applications in response to this RFA. The NIH has announced that applicants may request a larger budget and period of support if necessary for completion of the project. See NIH Guide for Grants and Contracts, February 13, 1998 at: grants.nih.gov/grants/guide/notice-files/not98-014.html. This RFA is a one-time solicitation. Responsibility for the planning, direction, and execution of the proposed project will be solely that of the applicant. Awards will be administered under NIH grants policy stated in the NIH Grants Policy Statement, NIH publication 99-8, October 1998. A. Fast-Track Applications. Applications may be submitted for the Fast- Track review option. Information on the Fast-Track process may be found at: grants.nih.gov/grants/funding/sbirsttr1/index.pdf. To be eligible for the Fast-Track option, the Phase I SBIR/STTR application must include well-defined, quantifiable milestones that will be used to judge the success of the proposed research, as well as a credible plan to apply the selected technology in a pilot study of interest to exposure assessment research for the Phase II R44 or R42 application. The Fast-Track application must have a section labeled Milestones at the end of the Research Plan Phase I. This section must include well-defined, quantifiable milestones for completion of Phase I, a discussion of the suitability of the proposed milestones for assessing the success in Phase I, and a discussion of the implications of successful completion of these milestones on the proposed Phase II. Applications submitted through the Fast-Track option are subject to the same total cost limits per year as when submitted outside of the Fast-Track option, as described below in the PROJECT PERIOD AND AMOUNT OF AWARD section. B. Individual Phase I Applications. Phase I applications in response to this RFA will be funded as Phase I SBIR Grants (R43) or STTR Grants (R41) with modifications as described below. Applications for Phase I grants should be prepared following the directions for Phase I SBIR/STTR applications as described in the Omnibus Solicitation. The Omnibus Solicitation is available on the Internet at grants.nih.gov/grants/funding/sbirsttr1/index.pdf. C. Individual Phase II Applications. Phase II applications will not be accepted in response to this RFA unless they are submitted for Fast-Track review. They will only be accepted as competing continuations of Phase I SBIR awards funded under this initiative. PROJECT PERIOD AND AMOUNT OF AWARD Applicants planning to propose research in excess of the limits (duration and amount) stated below must obtain agreement from Institute staff (named under INQUIRIES below) that the application will be accepted for consideration. The applicant must identify, in the cover letter that is sent with the application, the staff member and Institute who agreed to accept the application. Phase I: Because of the challenges that may be involved in the development and evaluation of insertional mutagenesis technology, the participating Institutes will entertain well-justified Phase I applications for an SBIR/STTR award with a project period up to two years and a budget not to exceed a total cost of $100,000 per year. Consultant and contractual costs associated with Phase I: The total amount of all consultant costs and contractual costs normally may not exceed 33 percent of the total costs requested for Phase I SBIR/STTR applications. However, well-justified Phase I applications for an SBIR/STTR award with greater than 33 percent contractual costs will be considered when those costs are necessary to support the development and evaluation of tools for insertional mutagenesis. Page limitations: The 25-page limitation for Phase I applications applies (see Omnibus Solicitation). Phase II: This solicitation is only for Phase I or Fast-Track SBIR applications. Phase II applications that result from funded Phase I studies under this RFA will be awarded as Phase II SBIR (R44) or STTR (R42) grants with modifications as described below. In addition, Fast-Track Phase I and Phase II applications will be accepted as described above. Project period and amount of award: Because of the challenges that may be involved in the development and evaluation of insertional mutagenesis technology, the participating Institutes will entertain well-justified Phase II applications for an SBIR/STTR award with a project period up to two years and a budget of no more than $500,000 per year for STTR and $750,000 for SBIR awards. Consultant and contractual costs: For SBIR projects, the total amount of all contractual costs and consultant fees normally may not exceed 33 percent of the total costs requested for Phase I projects and 50 percent of total costs requested for Phase II projects. However, well-justified Phase II applications for an SBIR award with greater contractual costs will be considered when those costs are necessary to support the development, evaluation, and validation of tools for insertional mutagenesis. FUNDS AVAILABLE It is expected that 10-20 awards will be made during FY 2001 and FY 2002, and approximately $3.15 million from the SBIR set-aside of the participating NIH Institutes and Centers will be designated for this purpose. The number of awards will be dependent upon receipt of a sufficient number and diversity of applications with high scientific merit. RESEARCH OBJECTIVES Background The use of transposon tagging and retroviral insertional mutagenesis in model organisms such as Drosophila, C. elegans, and zebrafish has greatly facilitated the characterization of gene function and permitted rapid cloning of the mutated gene. This approach has complemented analysis of gene function using chemically and X-ray-induced mutations where great effort is expended in positional cloning of the mutant gene. Insertional mutagenesis in mice is made practical by the availability of efficient methods of transfecting ES cells, the production of a 2.5 to 3.5X draft of the mouse genome using C57BL/6 by February 2001, polymerase chain reaction (PCR), and automated sequencing methods. The development of both random and targeted sequence-tagged insertion libraries in mouse ES cells would greatly facilitate analysis of gene function in mice and permit the rapid development of mouse models for human genetic disease. Not only would such an approach create induced mutation methodologies, but it would also permit analysis of patterns of gene expression. The value of this resource would be greatly enhanced by the use of site-specific gene recombination systems or trans-acting factor binding sites that would allow the expression of the tagged gene to be controlled temporally and spatially. Two recombination systems currently used to create conditional mutations or knockouts in mice are the cre-lox and FLP-FRT site-specific recombination systems. The usefulness of these recombination systems in vertebrate systems is dependent on the activity of the recombinase and the ability to drive the expression of the recombinase with non-mammalian promoters, such as ecdysone or tetracycline-sensitive promoters. The ability to control the spatial expressions of the recombinase is limited by the lack of well-characterized enhancers that control gene expression. To overcome these obstacles, modifications of these recombinase systems, as well as the development of new ones, are needed. In addition, the flexibility of existing systems can be enhanced through the use of inducible promoters or fusion-protein recombinases that are activated by ligands such as steroids. The creation of appropriate insertional mutagenesis vectors containing site-specific recombination targets will also aid in the generation of chromosomal deletions, duplications, and inversions when another genetic locus is tagged with a vector containing the same target sequence. Chromosomal aberrations are an important tool for selecting and mapping mutations in a specific chromosomal region and for positional cloning, as well as for the study of position effects and contiguous gene syndromes. In addition, inversions can be combined to produce balancer chromosomes. Balancer chromosomes carrying dominant phenotypic markers simplify the maintenance of recessive mutations and combinations of alleles from generation to generation because the balancer prevents recombination. Balancer chromosomes also facilitate isolation and high-throughput screening for new recessive mutations. Current estimates are that a total of 500,000 ES cell lines may be needed to tag every single mouse gene. Thus, high-throughput methods are needed to automate the processing of large numbers of clones and to identify the site of insertion. The use of C57BL/6 ES cells will speed the distribution of mice in a defined isogenic background by eliminating the need to cross sv129 mice over successive generations into C57BL/6. Many of the current sv129 cell lines in use are derived from different strains of sv129 mice, making comparisons among the various targeted mutations difficult until transferred to a defined genetic background. Moreover, much of the public chemical mutagenesis and sequencing effort is being done in C57BL/6. Thus, crosses between C57BL/6 mice carrying mutations created by ENU and C57BL/6 mice carrying a targeted deletion can be performed in a single generation without concern about the effects of genetic background. It is anticipated that the results of funded research projects will eventually lead to production of new libraries of ES cells with random or targeted insertional mutations for wide distribution to the research community. Examples of research that may be considered responsive to this RFA include, but are not limited to, those listed below. o Feasibility studies for the establishment of sequence-tagged insertional libraries of C57BL/6 ES cells in which the expression of the tagged gene can be controlled temporally and spatially. o The development of new or modified site-specific recombination systems for efficient random and targeted insertional mutagenesis and enhanced control of conditional expression. o The development of novel vectors that allow imaging of specific cell types or tissues, metabolic activity, or other cellular or physiological functions. o The invention of efficient systems for transposon tagging in mammalian systems for the wide use of the scientific community. o The development of vectors for identification ("trapping") of promoters and enhancers that could be used for tissue-specific and temporal expression of recombinases and for the study of gene expression patterns. o Methods to automate the processing of large numbers of clones and to identify the sites of insertion. INQUIRIES Written and phone inquiries are encouraged. The opportunity to clarify any issues or questions from potential applicants is welcome. Inquiries regarding programmatic issues may be directed to: Dr. Rebekah S. Rasooly Division of Neuroscience and Behavioral Research National Institute on Drug Abuse/NIH 6001 Executive Boulevard, Room 4260, MSC 9555 Bethesda, MD 20892-9555 Phone: 301-443-6300 Fax: 301-594-6043 Email: rr185i@nih.gov