NIH GUIDE, Volume 21, Number 38, October 23, 1992

PA NUMBER:  PA-93-010

P.T. 34


  Preservation of Organs/Tissue 


  Biological Resources 

National Heart Lung and Blood Institute


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.


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).


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)



Administrative supplements will be made for active R01 and P01 grants




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.


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



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


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



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.


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


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


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.


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.


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


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


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



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