SUPPLEMENTS FOR EMBRYONIC CRYOPRESERVATION OF HYPERTENSIVE RATSTRAINS NIH GUIDE, Volume 21, Number 38, October 23, 1992 PA NUMBER: PA-93-010 P.T. 34 Keywords: Preservation of Organs/Tissue Hypertension Biological Resources National Heart Lung and Blood Institute PURPOSE The objective of this supplement program is to preserve embryos from phenotypically and genotypically unique rat strains used in hypertension research. Funds will be provided to investigators with active grants from the National Heart, Lung, and Blood Institute (NHLBI) so that embryos of hypertensive and normotensive control rat strains with well-defined phenotypic and genotypic characteristics that are of value to the study of high blood pressure can be cryopreserved for future revitalization and use. Limited research may also be supported through this mechanism, but only if it clearly relates to the optimization of protocols for cryopreservation. HEALTHY PEOPLE 2000 The Public Health Service (PHS) is committed to achieving the health promotion and disease prevention objectives of "Healthy People 2000," a PHS-led national activity for setting priority areas. This program announcement, Supplements for Embryonic Cryopreservation of Hypertensive Rat Strains, is related to the priority area of heart disease and stroke. Potential applicants may obtain a copy of "Healthy People 2000" (Full Report: Stock No. 017-001- 00474-0) or "Healthy People 2000" (Summary Report: Stock No. 017-001-00473-1) through the Superintendent of Documents, Government Printing Office, Washington, DC 20402-9325 (telephone 202-783-3238). ELIGIBILITY REQUIREMENTS Supplemental applications may be submitted by foreign and domestic, for-profit and non-profit organizations, public and private, such as universities, colleges, hospitals, laboratories, units of State and local governments, and eligible agencies of the Federal government. A respondent to this announcement must have an active grant from the NHLBI with at least two full years remaining at the time a supplemental award is made. Supplemental awards will be considered only for regular research project (R01) and program project (P01) grants. MECHANISM OF SUPPORT Administrative supplements will be made for active R01 and P01 grants as described under ELIGIBILITY REQUIREMENTS. RESEARCH OBJECTIVES Summary The objective of this program is to preserve phenotypically and genotypically unique rat strains used in hypertension research. This effort is necessary due to extensive genetic heterogeneity associated with many hypertensive rat strains and to the emergence of new molecular genetic technologies that will result in the availability of many new strains in the near future. The administrative supplement program will allow unique and valuable strains to be preserved so that both existing and newly developed models of hypertension, including those derived from traditional breeding techniques as well as molecular genetic technologies, will be available to investigators for future use. The major emphasis of the initiative will be on embryonic cryopreservation of phenotypically and genotypically well-characterized hypertensive and normotensive control rat strains. Much of the methodology and technology for cryopreservation of rat embryos has been adopted from similar work done with mouse embryos. Therefore, while a certain amount of research and technology development for rat embryo cryopreservation may be included as part of the goals of this program, the research component of the initiative will be limited to optimizing protocols for cryopreservation of embryos. Examples of the types of research that would be supported include: maximization of the production of embryos in immature female rats and synchronization of embryo production in mature females rats via superovulation protocols; determination of the exact number of female rats needed to produce a bank of approximately 1,000 embryos per strain; optimization of procedures for the most efficient transfer of embryos to the revitalization state in situ; and establishment of methods of cryopreservation that ensure the maintenance of genetic integrity in stored embryos. Background The impetus for this program comes from deliberations by the NHLBI Arteriosclerosis, Hypertension, and Lipid Metabolism Advisory Committee (AHLMAC) and two separate expert panels of scientists that were convened to evaluate the current status and need for genetically defined animal models for the study of hypertension. The expert panels noted the lack of standardized genetic rat models of hypertension due to independent breeding and the absence of appropriate inbred controls. The panels assessed the extent to which phenotypic and genotypic variability among genetic rat models of hypertension is impeding research in the field. The panels commented that genetic heterogeneity can hamper the use of advanced molecular biological approaches, such as gene targeting, transgenic studies, and gene mapping; can compromise the scientific value of many studies; and can lead to wasteful and inefficient experiments that render inter-laboratory comparisons difficult. It was also pointed out that preservation efforts will help inform the hypertension research community of the variety of acceptable models available for study and will enhance accessibility to unique strains. To ensure that well characterized genetic rat models are preserved, both panels recommended the banking of frozen embryos for maintaining unique rat strains. Cryopreservation of rat embryos has been selected as the method for preserving valuable hypertensive strains because it is cheaper, more efficient, and safer than maintaining live colonies of rats. Technologies for the cryopreservation of embryos are already available for many species, and it should not be difficult to modify established procedures for cryopreservation of mouse embryos to apply to the rat. Genetic rat models of high blood pressure and their normotensive controls provide many opportunities for studying physiological and biochemical mechanisms of blood pressure regulation. These models have helped in the identification of numerous abnormalities in blood pressure homeostasis and have led to a clearer understanding of the normal mechanisms controlling blood pressure. The spontaneously hypertensive rat (SHR) is among the most extensively used animal model for studying human essential hypertension because there are many similarities in the hypertensive disease process in the SHR and in human essential hypertensives. These include multiple genetic components of the disease; blood pressure that increases with age; myocardial, vascular, cerebral, and renal lesions; hemodynamic changes; greater severity of the disease in the male; and responsiveness to antihypertensive agents. In spite of the wide use of SHRs and their Wistar-Kyoto (WKY) normotensive controls in research, recent studies indicate that genetic variability limits the utility of this model, particularly in molecular genetic studies. For example, WKY rats exhibit considerable heterogeneity when obtained from different sources. This heterogeneity is observed both in phenotype, e.g., differences in growth rates and blood pressures, and in genotype, e.g., variable DNA restriction fragment length polymorphisms. In fact, separate reports from a number of independent laboratories have provided molecular evidence of genetic heterogeneity in rats obtained commercially in the United States, England, Germany, and Japan. Although a strong interest exists among investigators to study the contribution of specific genes to aberrations in blood pressure control, the application of genetic linkage, transgenic, or gene targeting technologies to the field of hypertension has been limited, in part due to the absence of clear evidence that animals from the colonies of different commercial vendors are phenotypically and genotypically identical. Although a relatively small number of investigators have thus far studied the genetic determinants of blood pressure variability, many important questions regarding the genetic basis of the disease remain, and for such questions to be answered, appropriately inbred rats of fixed genotype are crucial. A system for banking specific hypertensive rat strains and appropriate normotensive controls will be of enormous utility to investigators studying hypertension, as well as other cardiovascular diseases affected by blood pressure status, such as cardiac hypertrophy, kidney failure, and stroke. Researchers currently spend considerable sums of money to produce and maintain strains of rats through conventional breeding programs and modern genetic approaches. Because a large amount of time and money are devoted to producing unique strains, methods of preserving animals other than through housing and further breeding must be considered. Cryopreservation of embryos has a number of advantages over maintenance of live breeding colonies. This technology will reduce maintenance costs and can safeguard valuable animals produced through expensive and time-consuming molecular genetic techniques. Cryopreservation can also safeguard against damage to animal housing facilities, disease, and genetic contamination. While methodologies have been developed for the cryopreservation of embryos from several mammalian species (e.g., mice, rabbits, sheep, goats, cattle, horses, cats, and humans), it is only recently that techniques have been established for the cryopreservation of rat embryos. Although it is still necessary to optimize superovulatory techniques to prepare female rats for fertilization and to take precautions to maintain genetic integrity when using cryopreservation, the state of knowledge is such that freezing of rat embryos can be undertaken without further significant financial investment in technology development. The immediate need for this type of repository activity has been emphasized in two reports issued by the National Research Council, both of which point to the necessity of preserving diverse animal species. The NHLBI will maintain records of rat strains whose embryos are cryopreserved through this program and will publish and distribute a listing of such strains periodically. NHLBI staff will facilitate interaction among investigators who wish to use strains cryopreserved through this program. However, it will be the responsibility of individual investigators to establish agreements for usage and collaboration. APPLICATION PROCEDURES Applications are to be submitted on the grant application form PHS 398 (rev. 9/91) and will be accepted on the following two application deadlines each year: March 8 and November 8, starting with March 8, 1993. Application kits are available at most institutional business offices and may be obtained from the Office of Grants Inquiries, Division of Research Grants, Westwood Building, Room 449, National Institutes of Health, Bethesda, MD 20892, telephone number 301-496-7441. The title and number of the announcement must be typed in Section 2a on the face page of the application. The completed original supplemental application and five legible copies must be sent or delivered directly to: Chief, Centers and Special Projects Section Review Branch, Division of Extramural Affairs National Heart, Lung, and Blood Institute Westwood Building, Room 553 Bethesda, MD 20892 DO NOT SEND APPLICATION TO THE DIVISION OF RESEARCH GRANTS. REVIEW PROCEDURES All applications submitted in response to this Program Announcement will be reviewed for responsiveness to the objectives of this program. If the supplemental application is judged to be unresponsive, the applicant will be contacted and given the opportunity to withdraw the application for revision and resubmission at a future date. The Review Branch, Division of Extramural Affairs, NHLBI, in consultation with program staff, will convene a panel of consultants to assist in the evaluation of the merit of the supplemental applications. The role of this peer review group will be to assess the uniqueness of the genotype and phenotype of the strain to be cryopreserved and to evaluate overall strategies for cryopreservation, revitalization, and handling of requests for embryos. Following this initial technical review, the application will undergo a second level of review by NHLBI program staff. APPLICATION GUIDELINES The strain will need to be genotypically and phenotypically characterized in a standard manner so that uniqueness can be judged by an independent panel of consultants. The original source of the rats, as well as their breeding history, should be included along with a clear phenotypic and genotypic characterization as described below. The overall phenotypic description of rat strains could potentially include a number of physiological and biochemical parameters. Careful documentation of arterial blood pressure is a necessity. The method of measurement (i.e., tail cuff, direct measurement with indwelling arterial catheter, anesthetized or unanesthetized, and any other specifics that could affect quantitation of blood pressure) must be described. A minimum of three separate measurements over a period of one week under identical conditions will generally be expected. Inclusion of mean arterial pressure, systolic and diastolic pressures, the standard deviation of the mean, and the range of pressures for each rat is suggested. The age and weight of the rats at the time of blood pressure determination, pertinent dietary information (e.g., protein, carbohydrate, fat, and mineral content), and gender-related differences in blood pressure should also be discussed. Other phenotypic variables that have bearing on blood pressure regulation should be described. Careful documentation of strain-specific dysfunctions in blood vessel, renal, or neuronal function, or in the hormones and neurotransmitters known to influence arterial blood pressure, would be appropriate phenotypic information to discuss. Variations in strain responses to environmental stressors, such as salt-sensitivity or sensitivity to psychologic stressors (e.g., noises, fear, performance of tasks), should be considered for inclusion. In addition, documentation of a strain-specific propensity to develop end-organ damage, such as cerebrovascular disease, atherosclerosis, cardiac hypertrophy, or renal failure, should be addressed in detail. Lastly, well documented genetic sensitivities to known cardiovascular risk factors, such as obesity, insulin-sensitivity, and alterations of lipid metabolism, should be part of the phenotypic characterizations. The genetic background of all strains to be considered for embryonic cryopreservation should be clearly described. To the extent possible, a complete genealogy should be provided for all strains and the nature of the genetic lesion should be documented by molecular analysis. For strains in which the genetic lesion has not been characterized at the molecular level, a thorough description of the non-molecular methods used for genotype assignment must be provided. For inbred strains, molecular evidence of genetic homogeneity should be documented by DNA fingerprint analysis and analysis of a panel of loci known to be highly polymorphic in the species of interest. Support for disease models maintained on non-inbred genetic backgrounds will be considered only if the reasons for using non-inbred lines are clearly justified. The supplemental application should also include a well described experimental protocol to assess periodically the genetic viability of frozen rat embryos as a function of time. In particular, quality control protocols for damage to DNA should be discussed in the application. The following budgetary requests would be appropriate for the supplement applications: salaries for key staff, which would include a cryobiologist and an individual with experience in endocrinological and surgical preparation of rats for embryo excision; limited supplies; and equipment for cryopreservation (e.g., a liquid nitrogen carboy). Additional funds may be requested for small research components related to the program. Potential applicants should discuss budgets with program staff prior to submission. Current estimates are that approximately $10,000 to $20,000 in direct costs during the first year will be sufficient to undertake the activities outlined above for a single strain. It is recognized that costs will vary among laboratories. Preservation of approximately 1,000 embryos per strain will be recommended to cover a certain percentage of failed revitalizations. It is expected that supplement award recipients will recover costs for revitalization of embryos from investigators requesting rats of a particular strain. Such cost recovery would be considered program income and would be treated according to applicable PHS policies. For further information contact Ms. Jane Davis at the address listed under INQUIRIES. AWARD CRITERIA The following criteria will be considered in making funding decisions: the uniqueness of the rat strain and the quality of the cryopreservation protocols as determined through the review; the availability of funds; and program balance among the strains selected for preservation. INQUIRIES Written and telephone inquiries are encouraged. The opportunity to clarify any issues or questions from potential applicants is welcome. Direct inquiries regarding scientific aspects of this program to: Paul A. Velletri, Ph.D. Hypertension and Kidney Diseases Branch Division of Heart and Vascular Diseases National Heart, Lung, and Blood Institute Federal Building, Room 4C10 Bethesda, MD 20892 Telephone: (301) 496-1857 FAX: (301) 402-2044 Direct inquiries regarding fiscal matters relating to this program to: Ms. Jane Davis Chief, Blood Diseases and Resources Grants Management Section Grants Operation Branch Division of Extramural Affairs National Heart, Lung, and Blood Institute Westwood Building, Room 4A15B Bethesda, MD 20892 Telephone: (301) 496-7257 FAX: (301) 402-1200 AUTHORITY AND LEGISLATION This program is described in the Catalog of Federal Domestic Assistance No. 93.837. Awards are made under authorization of the Public Health Service Act, Title IV, Part A (Public Law 78-410, as amended by Public Law 99-158, 42 USC 241 and 285) and administered under PHS grants policies and Federal Regulations 42 CFR 52 and 45 CFR Part 74. This program is not subject to the intergovernment review requirements of Executive Order 12372 or Health Systems Agency review. .
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