SUPPLEMENTS FOR EMBRYONIC CRYOPRESERVATION OF RATS IN HYPERTENSION
RESEARCH

NIH Guide, Volume 26, Number 37, November 7, 1997

PA NUMBER:  PAR-98-009 (inactive per NOT-HL-00-003)

P.T.

National Heart, Lung, and Blood Institute

Application Receipt Dates:  March 9 and November 9 each year
beginning with March 9, 1998

PURPOSE

The objective of this supplement program is to preserve embryos
from unique rat strains that have well-defined phenotypic and
genotypic characteristics and that are employed in hypertension
research.  This is a rerelease of a program announcement that was
originally published in the NIH Guide for Grants and Contracts
(Vol.  21, No. 38, Oct. 23, 1992).  Funds will be provided to
investigators with active regular research project (R01), Method to
Extend Research in Time (MERIT) (R37), and program project (P01)
grants from the National Heart, Lung, and Blood Institute (NHLBI)
for the cryopreservation of embryos for future revitalization and
use.

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 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 Summary Report:  Stock No. 017-001-00473-1) through the
Superintendent of Documents, Government Printing Office,
Washington, DC 20402-9325 (telephone 202-512-1800).

ELIGIBILITY REQUIREMENTS

Supplement 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 program 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), MERIT (R37),
and program project (P01) grants.

The rat model to be cryopreserved must already be in use in the
parent grant at the time the supplement application is submitted.

MECHANISM OF SUPPORT

Supplements will be made for active R01, R37, and P01 grants as
described under ELIGIBILITY REQUIREMENTS.

RESEARCH OBJECTIVES

Summary

This supplement program will allow unique and valuable rat 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.  In addition, this
effort is necessary to avoid unwanted genetic heterogeneity in
particular strains.  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 female rats via
superovulation protocols or other methods; determination of the
exact number of 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 originated in deliberations by the
NHLBI Arteriosclerosis, Hypertension, and Lipid Metabolism Advisory
Committee (AHLMAC) in 1990 and two separate expert panels of
scientists that were convened in 1991 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, and 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 compromise the scientific
value of many studies and can lead to wasteful and inefficient
experiments that render inter-laboratory comparisons difficult.

It was pointed out that preservation efforts will enhance the
accessibility of valuable models that can be shared by the research
community so that direct comparisons of results from different
laboratories can be made.  To ensure that well characterized rat
models are preserved, both panels recommended the banking of the
frozen embryos.  It was also noted that the use of genetically
altered rats will certainly increase over time and the
cryopreservation of these embryos will be an efficient way of
keeping valuable strains.

A system for banking specific hypertensive rat models and
appropriate normotensive controls will be of enormous utility to
investigators studying hypertension, as well as other
cardiovascular disorders affected by blood pressure status, such as
cardiac hypertrophy, kidney failure, vascular remodeling, and
stroke.  Researchers currently spend considerable sums of money to
produce and maintain special strains of rats through conventional
breeding programs and modern genetic approaches.  Because a large
amount of time and money is devoted to producing unique models,
methods of preserving animals other than through housing and
further breeding must be considered.

Cryopreservation of rat embryos is an excellent method for
preserving valuable hypertensive models that are not in current
use, because it is cheaper, more efficient, and safer than
maintaining live colonies of rats.  The cost of cryopreserving a
rat embryo in an individual laboratory (including breeding of the
females before harvesting the embryos) is equal to the cost of
maintaining a minimal rat colony for just two years.  The cost of
maintenance of the frozen embryos is nominal.  Therefore, the
potential cost savings to the investigator and the National Heart,
Lung, and Blood Institute is in the amount of labor, space,
equipment, biochemical reagents, and other support needed to breed
and maintain the animals, for periods longer than two years.  More
importantly, it ensures against loss of valuable models due to
genetic drift, diseases and other catastrophes.  Reestablishing a
lost strain whose embryos had never been cryopreserved can be
extremely costly, and sometimes virtually impossible.

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), only recently have
techniques been established for the cryopreservation of rat
embryos.  Rat embryo cryopreservation has been more difficult,
particularly in the synchronization of the estrous cycle of the
female rats to prepare them for fertilization.  The superovulation
method is not always effective and needs to be optimized for each
genotype.  Other methods, such as the use of analogs of
gonadotropin-releasing hormone, are being investigated.

In recent years, there has been a dramatic increase in the types
and the use of genetically altered rats for the investigation of
physiological and biochemical mechanisms of blood pressure
regulation.  There has been a corresponding increase in the number
of grants that utilize rats.  At present, unique rat models are
usually developed and kept in individual research laboratories, and
the need is greater than ever before for the preservation of these
valuable animals.

Cryopreservation of embryos has a number of advantages over the
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,
against damage to animal housing facilities, disease, genetic
drift, and contamination.

Although it is still necessary to optimize 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 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

Potential applicants are strongly encouraged to discuss their
interest in applying for a supplemental award for cryopreservation
with NHLBI staff prior to submission.  This communication is
essential not only to help the NHLBI plan for review loads but also
to ascertain that the necessary funds are available if an award can
be made.

Applications are to be submitted on the grant application form PHS
398 (rev. 5/95) and will be accepted on the following two
application deadlines each year:  March 9 and November 9, starting
with March 9, 1998.  Application kits are available at most
institutional offices of sponsored research and may be obtained
from the Division of Extramural Outreach and Information Resources,
National Institutes of Health, 6701 Rockledge Drive, MSC-7910,
Bethesda, MD 20892-7910, telephone 301-710-0267, fax 301-480-0525,
email [email protected]).

The title and number of the program announcement must be typed in
Section 2 on the face page of the application.  The completed
original supplemental application and three legible copies must be
sent or delivered to:

CENTER FOR SCIENTIFIC REVIEW (formerly Division of Research Grants)
NATIONAL INSTITUTES OF HEALTH
6701 ROCKLEDGE DRIVE, ROOM 1040 - MSC 7710
BETHESDA, MD  20892-7710
BETHESDA, MD  20817 (for express/courier service)

At the same time two additional copies of the application must be
sent to:

Chief, Review Branch
Division of Extramural Affairs
National Heart, Lung, and Blood Institute
6701 Rockledge Drive, Suite 7220
Bethesda, MD  20892-7924

REVIEW CONSIDERATIONS

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
rat strain to be cryopreserved and to evaluate overall strategies
for cryopreservation, revitalization, and handling of requests for
embryos from interested scientists.  Following this initial
technical review, the application will undergo a second level of
review by NHLBI program staff.

Application Guidelines

The rat 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 should 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, telemetry monitoring, 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.  Strain variations in 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,
renal failure, or variations in drug responses, should be addressed
in detail. Lastly, well documented genetic susceptibilities to
known cardiovascular risk factors, such as obesity, insulin-
sensitivity, and alterations in lipid metabolism, should be part of
the phenotypic characterizations.

The genetic background of all rat strains to be considered for
embryonic cryopreservation should be clearly described. To the
extent possible, a complete genealogy should be provided for each
strain.  A genetic profile must also be included.  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 strain 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 supplement 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 possible damage to DNA over time are
to be discussed in the application.

The following budgetary requests would be appropriate for the
supplement applications:  salaries for key staff, which might
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., liquid nitrogen storage tank, controlled rate freezer,
microscope).  Funds may also be requested for small research
components clearly related to the optimization of protocols for
cryopreservation. Potential applicants should discuss budgets with
program staff prior to submission.

Current estimates are that approximately $20,000 to $40,000 in
direct costs during the first year, including new equipment, will
be sufficient to undertake the activities outlined above for a
single strain.  After the first year, the supplement will cover
recurrent costs such as salary and limited supplies through the
remaining years of the parent grant, with the usual 3% yearly
escalation.  It is recognized that costs will vary among
laboratories. Preservation of approximately 1,000 embryos per
strain is 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 on budgetary
matters, contact Ms. Jane Davis at the address listed under
INQUIRIES.  Proposed funding levels are subject to change due to
budgetary, administrative, and/or scientific considerations.

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.  Applicants are
strongly encouraged to contact program staff to discuss a possible
submission of an application.  For further information on rat
embryo cryopreservation, such as names of cryopreservation experts,
possible training opportunities, or cryopreservation services
available to researchers, contact Dr. Barouch.

Direct inquiries regarding scientific aspects of this program to:

Dr. Winnie Barouch, Ph.D.
Division of Heart and Vascular Diseases
National Heart, Lung, and Blood Institute
6701 Rockledge Drive, Suite 10193
Bethesda, MD  20892-7956
Telephone:  (301) 435-0560
FAX:  (301) 480-2849
Email:  [email protected]

Direct inquiries regarding fiscal matters to:

Ms. Jane Davis
Division of Extramural Affairs
National Heart, Lung, and Blood Institute
6701 Rockledge Drive, Room 7174
Bethesda, MD  20892-7926
Telephone:  (301) 435-0166
FAX:  (301) 480-3310

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.

The PHS strongly encourages all grant and contract recipients to
provide a smoke-free workplace and promote the non-use of all
tobacco products.  In addition, Public Law 103-227, the Pro-
Children Act of 1994, prohibits smoking in certain facilities (or
in some cases, any portion of a facility) in which regular or
routine education, library, day care, health care or early
childhood development services are provided to children.  This is
consistent with the PHS mission to protect and advance the physical
and mental health of the American people.

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