MUTAGENESIS SCREENS/PHENOTYPING TOOLS FOR ZEBRAFISH

Release Date:  February 16, 2000

RFA:  HD-00-004

Trans-NIH Zebrafish Coordinating Committee
National Institute of Child Health and Human Development 
National Cancer Institute
National Center for Research Resources
National Eye Institute
National Heart, Lung, and Blood Institute
National Human Genome Research Institute
National Institute on Alcohol Abuse and Alcoholism
National Institute of Arthritis and Musculoskeletal and Skin Diseases
National Institute on Deafness and Other Communication Disorders
National Institute of Dental and Craniofacial Research
National Institute of Diabetes and Digestive and Kidney Diseases
National Institute on Drug Abuse
National Institute of Environmental Health Sciences
National Institute of General Medical Sciences
National Institute of Mental Health
National Institute of Neurological Disorders and Stroke

Letter of Intent Receipt Date:  April 16, 2000
Application Receipt Date:  May 19, 2000

THIS RFA USES THE “MODULAR GRANT” AND “JUST-IN-TIME” CONCEPTS.  IT INCLUDES 
DETAILED MODIFICATIONS TO STANDARD APPLICATION INSTRUCTIONS THAT MUST BE USED 
WHEN PREPARING APPLICATIONS IN RESPONSE TO THIS RFA.

PURPOSE

The purpose of this Request for Applications (RFA) is to encourage research 
designed to exploit the power of mutagenesis screening in zebrafish in order 
to detect and characterize genes, pathways, and phenotypes of interest in 
development, behavior, organ formation, disease processes.  Applications that 
propose to advance the technologies associated with such phenotyping also are 
welcome.

This RFA is the result of an NIH initiative with participation of the 
Institutes listed above, working though the Trans-NIH Zebrafish Coordinating 
Committee (ZFCC), under the co-chairmanship of the National Institute of Child 
Health and Human Development (NICHD) and the National Institute of Diabetes 
and Digestive and Kidney Diseases (NIDDK). Since its formation in 1998, the 
committee has played an active role as an advocate for the zebrafish as an 
important model for development and disease research.

The following components of the National Institutes of Health (NIH) are co-
sponsors of this solicitation:  National Institute of Child Health and Human 
Development (NICHD), National Cancer Institute (NCI), National Center for 
Research Resources (NCRR), National Eye Institute (NEI), National Heart, Lung 
and Blood Institute (NHLBI), National Human Genome Research Institute (NHGRI), 
National Institute on Alcohol Abuse and Alcoholism (NIAAA), National Institute 
of Arthritis and Musculoskeletal and Skin Diseases (NIAMS), National Institute 
on Deafness and Other Communication Disorders (NIDCD), National Institute of 
Dental and Craniofacial Research (NIDCR), National Institute of Diabetes and 
Digestive and Kidney Diseases (NIDDK), National Institute on Drug Abuse 
(NIDA), National Institute of Environmental Health Sciences (NIEHS), National 
Institute of General Medical Sciences (NIGMS), National Institute of Mental 
Health (NIMH), and the National Institute of Neurological Disorders and Stroke 
(NINDS).   

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 is related to one or more of 
the priority areas.  Potential applicants may obtain  "Healthy People 2010" at 
http://odphp.osophs.dhhs.gov/pubs/hp2000.

ELIGIBILITY REQUIREMENTS

Applications may be submitted by domestic and foreign 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.  Racial/ethnic minority individuals, 
women, and persons with disabilities are encouraged to apply as Principal 
Investigators (PI).

MECHANISM OF SUPPORT

This RFA will use the NIH research project grant (R01) award mechanism.  
Responsibility for the planning, direction, and execution of the proposed 
project will be solely that of the applicant. The total project period for an 
application submitted in response to this RFA may not exceed five years.  The 
anticipated award date is April 1, 2001.

This RFA is a one-time solicitation.  Future new or competing continuation 
applications will compete with all investigator-initiated applications and be 
referred and reviewed according to the customary peer review procedures. 

Although this RFA is the result of a trans-NIH initiative, awards will be made 
through the Institute whose mission is most closely related to the proposed 
work.  Through the Trans-NIH Zebrafish Coordinating Committee, each 
Institute/Center (IC) will share with the other participating ICs, findings of 
any research supported as a result of this RFA. All investigators funded under 
this initiative will be expected to work together cooperatively so that the 
information learned and the models developed will be of maximum usefulness to 
the community.

FUNDS AVAILABLE

The participating ICs intend to commit approximately $4.5 million in total 
costs (direct plus Facilities and Administrative [F & A] costs) in FY 2001 to 
fund 8 to 10 new grants in response to this RFA.  An applicant may request a 
project period of up to five years and a budget for direct costs of up to 
$250,000 per year, including F & A costs on consortium arrangements.  Because 
the nature and scope of the research proposed may vary, it is anticipated that 
the size of awards also will vary.  Although the financial plans of the 
participating ICs provide support for this program, awards pursuant to this 
RFA are contingent upon the availability of funds and the receipt of a 
sufficient number of meritorious applications. 

RESEARCH OBJECTIVES

Background

The trans-NIH Zebrafish Coordinating Committee (ZFCC) continues to play an 
active role as an advocate for the zebrafish model.  The committee’s initial 
efforts resulted in RFA DK-98-006, entitled “Genomic Resources for the 
Zebrafish,” and continued with a Program Announcement PA-98-074, entitled ”The 
Zebrafish as an Animal Model for Development and Disease Research.”  On May 
10-11, 1999, the ZFCC sponsored a workshop entitled “Genomic and Genetic Tools 
for the Zebrafish.”  At this workshop, which expanded upon the “Non-mammalian 
Models Workshop” held on February 16-17, 1999, zebrafish researchers were 
asked to help prioritize the short- and long-term needs of the community.  One 
result of the workshop was the recommendation that more genetic screens in the 
zebrafish need to be supported by the NIH.  The current RFA addresses, in 
part, this and some of the other recommendations made by workshop 
participants. This RFA is designed to complement and continue NIH efforts to 
support the zebrafish as an important and timely animal model for research.

In the past decade, mutational analyses in the non-vertebrate genetic models 
of the worm (Caenorhabditis elegans) and the fruitfly (Drosophila 
melanogaster) have contributed significantly to our understanding of early 
developmental pathways.  For example, these studies have led to the discovery 
of genes encoding signals, components of signaling systems, enzymes, and 
transcriptional regulators that act during embryonic development.  These genes 
often operate in complex cascades to regulate pattern formation, cell fate, 
and specification, as well as later events such as development of the eye, 
heart, and other organs.  While these invertebrate systems have revealed much 
information and shown that numerous aspects of development are highly 
conserved among invertebrates and vertebrates, many features of patterning and 
morphogenesis of the vertebrate embryo are distinct and thus cannot be studied 
in invertebrates.  The vertebrate embryo has many characteristics not found in 
invertebrates, including a substantially different body plan and greater 
complexity of the nervous system, as well the presence of organs that have no 
clear counterparts in simple invertebrates.  Thus, a complete understanding of 
human development will require experimentation in vertebrate model organisms. 
 The study of mutations that affect development has been possible in the 
mouse, but the mouse embryo is not accessible in utero throughout much of its 
development.  Consequently, mutational studies in this species have been 
limited largely to defects in postnatal maturation.  While reverse genetics 
(e.g., gene knockouts) have been useful in the mouse model, the substantial 
cost of maintaining large mouse colonies has limited the applicability of 
forward genetic approaches on a large scale.  These limitations will have a 
profound impact on the rapid discovery of genes important in vertebrate 
development, and relevant to human disease.

As a vertebrate, the zebrafish, Danio rerio, is more closely related to humans 
than are yeast, worms or flies.  It has a number of advantageous features as a 
model organism for study of vertebrate development, disease, and biological 
pathways.  Many features of zebrafish development have been characterized, 
including early embryonic patterning, early development of the nervous system, 
and aspects of cell fate and lineage determination.  The embryos are easily 
obtainable in large numbers and accessible throughout development, they are 
transparent, and undergo rapid organogenesis, making them very amenable for 
developing phenotypic screens.  In live embryos, the same specific cell or 
even cellular processes can, in many cases, be identified from individual to 
individual, affording a high level of precision in characterizing the effect 
of a developmental, environmental or genetic perturbation.  The use of 
zebrafish to study vertebrate development, disease, and pathways of interest 
has been validated further by the demonstration that many of its genes show a 
high degree of structural and functional similarity to their human homologues. 
 The most powerful and unique feature of the zebrafish is that it is a 
vertebrate model organism that has proven that large-scale forward mutagenesis 
screens can be performed with relative ease.

The purpose of this initiative is to provide support for zebrafish mutagenesis 
and phenotypic screening efforts.  To date, screens have focused exclusively 
on phenotypes in embryonic development, despite interest in the zebrafish 
research community to do so, phenotypic screens in adult fish have not yet 
been possible, because of the general difficulty in obtaining funding for 
mutagenesis and screening research.  Phenotypic screens of post-embryonic fish 
will be needed to identify alterations in sensory and motor systems function, 
learning and memory, or behavioral responses to environmental toxins and 
pharmaceutical agents.  Two large-scale screens have been performed, and the 
transparent embryos have been screened for defects in overall embryonic 
pattern morphogenesis or organ formation. 

As reported in the December1996 issue of “Development,” screens have 
identified a substantial number of mutations that affect the formation of 
organ systems, including defects in the nervous system, skeletal muscle, 
craniofacial region, kidney and endocrine organs, cardiovascular and 
gastrointestinal systems, and the sensory cells of lateral line systems that 
are important to auditory and vestibular function.  In the last three years, 
many of the mutations responsible for these phenotypes have been mapped, and 
the genes cloned and characterized.  Investigators in the zebrafish community 
have only begun to exploit the power of mutagenesis screening to detect genes 
essential for organogenesis and associated developmental physiology, and they 
have yet to attempt screens for mutations affecting nervous system function 
(other than its development) and behavior.  Saturation mutagenesis of the 
zebrafish genome will be an indispensable means of achieving a complete 
understanding of the development, physiology, and behavior of this vertebrate. 
 With the advent of new, promising insertional mutagenesis strategies for the 
zebrafish, it is more pressing than ever to develop a strong support base for 
creative, intensive mutagenesis screens.

In the past year, significant progress has been made in the characterization 
of the zebrafish genome, which will facilitate the identification by 
positional cloning of zebrafish genes responsible for specific phenotypes.  
The Trans-NIH Initiative on Genomic Resources for the Zebrafish, that was co-
supported by 13 NIH Institutes and Centers and that funded five investigators 
beginning in the fall of 1998, has contributed significantly to this progress. 
 To date, grantees funded under this initiative are increasing the resolution 
of zebrafish physical maps using microsatellite markers, CA repeat markers, 
and conformational polymorphisms, they have completed and published a new 
radiation hybrid map that covers at least 88 percent of the zebrafish genome 
[an invaluable tool for mapping expressed sequence tags (ESTs), cloned genes, 
and candidate genes for specific mutations], they have generated approximately 
20 thousand new ESTs, and they have begun to determine a genetic map for the 
zebrafish by construction of a complete deletion panel.    	

Research Scope

The objective of this RFA is to broaden the range, power, and utility of 
screens for new mutants of zebrafish.  It will, therefore, support proposals 
for development of improved or novel methods for mutagenesis screens, as well 
as proposals for the actual execution of such screens.  Methodology developed 
and data and mutants generated as a result of this RFA are expected to be made 
widely available to the research community.  Applicants must include as part 
of their application a plan for disseminating these resources, adequacy of 
this plan will be considered in the review of applications responding to this 
RFA (see below).

Objectives to be addressed in applications submitted in response to this RFA 
include, but are not limited, to the following:

o Development and/or application of novel phenotypic screens for mutants.  
These screens may be based on observation of alterations in morphology, 
physiology, or behavior.

o Development and/or application of novel methods of mutagenesis (e.g., 
insertional).

o Genetic screens focusing on identifying mutations that affect the structure 
and function of specific tissue/organ systems.

o Screens focusing on identifying novel developmental genes and pathways, 
including those mediating sensitivity or resistance to environmental 
teratogens. 

o Screens to analyze the genetic basis of adult phenotypes including behavior, 
aging, organ disease, cancer, and responses to environmental toxins and drugs.

o Screens to detect altered gene expression patterns, as a tool to identify 
components of genetic pathways or those altered by environmental agents. 

o Sensitized screens, using strains carrying a known mutation, in order to 
identify extragenic suppressors or enhancers of that mutation.

In addition, all applications are expected to address the following:

o A proposed sharing plan to insure that mutant zebrafish, sperm, embryos, 
genetic and phenotypic screens, mutagenesis protocols, and genetic and 
phenotypic data for all mutant strains are widely available to the scientific 
community. 

o A proposed plan addressing if, or how, the PI and grantee institution will 
exercise their intellectual property rights regarding patentable research 
resources, such as mutant fish, sperm, embryos, genetic and phenotypic 
screens, mutagenesis protocols, and genetic and phenotypic data for all mutant 
strains produced in projects funded under this RFA (see SPECIAL REQUIREMENTS, 
below). 

Interests of Participating Institutes and Centers

The participating NIH ICs have provided a brief outline of their interests as 
they relate to the goals of this RFA.  These brief mission statements are 
intended to give some understanding of the breadth of the biomedical areas of 
interest in development of this model.

NCI:  Generation and study of zebrafish models to identify and place genes in 
functional pathways that affect growth and development, in particular, 
genes/pathways that, when altered, result in uncontrolled or cancerous growth. 
 Identification of key sites within these pathways that could be exploited for 
cancer therapeutic discovery purposes.

NCRR:  The NCRR supports research projects that broaden the utility of the 
zebrafish model for cross-cutting biomedical research that is not encompassed 
within a single NIH Institute or Center.  Interests include, but are not 
limited to, development of new methods for mutagenesis and/or phenotypic 
characterization that would be of use in research on a wide range of diseases 
or organs, particularly if these methods could be applied to other animal 
models as well as the zebrafish.

NEI:  Fundamental mechanisms underlying all aspects of eye development, 
function and disease, including development of the retina and lens, optic 
nerve axon guidance and the neural circuitry producing eye movements and 
oculomotor behaviors.
 
NHGRI:  Proposals for the development of high throughput, widely applicable 
technologies or methodologies to examine gene function on a genomic scale.  
This could include initial development of high throughput or large-scale 
methods for examining gene expression, development of tools for comprehensive 
mutational analysis, or genome-scale identification of regulatory regions. 

NHLBI:  Cellular and molecular functions of the mutant genes in development as 
models for human cardiovascular, blood, and pulmonary disorders, and circadian 
mechanisms regulating rest/activity cycles. Genetic basis of disorders of 
cardiovascular development and function, developmental aspects of endothelial 
dysfunction as the basis for systemic and pulmonary vascular disorders, 
developmental defects in hematopoiesis and relationship to disorders of the 
hematopoietic system, genetic basis of angiogenesis and vasculogenesis, effect 
of mutations on subsequent organ development leading to such disorders as 
arrhythmia, cardiac hypertrophy, dilated cardiomyopathy, heart failure, lung 
hypoplasia and bronchopulmonary dysplasia, the genetic basis, regulation, and 
role of biological clock mechanisms in development and circadian behavior. 

NIAAA:  Mechanistic studies of ethanol-induced teratogenesis, behavioral 
impairments, and organ damage.

NIAMS:  Mutations that have the potential to illuminate the development and 
function of the vertebrate musculoskeletal system and skin.  The 
musculoskeletal system includes muscle, bone, articulated joints, cartilage, 
tendon, and ligament.  Priority will be given to the establishment of 
collaborations between investigators with expertise in the zebrafish and 
investigators with expertise in the musculoskeletal systems and skin of 
mammals and humans.

NICHD:  Identification, cloning, and characterization of the genes important 
in normal development as well as those mutant genes that cause developmental 
defects.  Elucidation of the cellular, biochemical, molecular, and genetic 
mechanisms underlying normal and defective development.  This includes, but is 
not limited to, the study of general mechanisms of pattern formation and cell 
lineage, neural crest development, cell specification, differentiation, 
migration, and fate in early development of many organs/systems such as limb, 
nervous system, immune system, and heart.

NIDA:  Identification of mechanisms underlying tolerance, sensitization, and 
addiction to drugs of abuse such as nicotine, amphetamine, cocaine, opiates, 
barbiturates, and hallucinogens. Identification of genetic suppressors and 
enhancers of the teratological effects of drugs of abuse on behavior and the 
nervous system.  Processes involved in the development of brain regions
mediating the hedonic properties of drugs of abuse.

NIDCD:  Identification and cloning of genes involved in the normal and 
disordered development of hearing, balance, smell, and taste sensory systems. 
 Elucidation of the cellular, molecular, and biochemical mechanisms governing 
the proliferative, plastic, and regenerative capacities of these sensory cells 
and tissues.

NIDDK:  Research on diabetes, particularly studies on pancreatic beta cell 
function and development, obesity and mechanisms underlying satiety, other 
endocrine, and metabolic diseases, hematologic disorders, and diseases of the 
digestive system, liver, kidney, and urinary tract.  Studies aiming to clarify 
the cellular and molecular events that dictate tissue and organ formation in 
all these systems are considered of relevance.  These studies could include, 
but need not be limited to, studies to develop cell lines from any of the 
tissues or organs of interest, studies to characterize normal or abnormal 
function of tissues or organs of interest, methods to screen and identify 
additional mutations in these systems, studies to define the molecular 
mechanisms that dictate cell-specific gene expression in relevant cell types. 
   

NIDCR:  All aspects of normal and abnormal craniofacial development, including 
genetics, complex origins of craniofacial disorders, cell lineages and 
differentiation, cell signaling and gene regulation, embryonic patterning, 
imaging, biomimetics, and new technologies for high-throughput genetic and 
protein screens.

NIEHS:  Studies to examine the mechanism whereby environmental factors/agents 
alter any aspect of development.  This includes the screening for mutants that 
ameliorate the toxicity of environmental agents, and the subsequent 
identification and characterization of the genes and pathways involved in 
their action.  Characterization of the interactions among genetics, 
environmental agents, and time during development that lead to structural or 
functional abnormalities.  Development of a mechanistically based model for 
testing environmental agents for developmental toxicity.

NIGMS:  Basic biomedical research that addresses fundamental biological 
mechanisms such as those that underlie gene regulation, chromosome 
organization and mechanics, cell growth and differentiation, pattern 
formation, sex determination, morphogenesis, cell cycle control, behavior, the 
genetics of complex traits, and the application of mathematical models to 
complex biological systems.

NIMH:  Investigations that examine molecular, cellular, and biochemical bases 
of genetic mutations affecting neurogenesis, biological rhythms, learning, 
memory, and other cognitive functions and behaviors of the nervous system. 
These studies include, but are not limited to, development of screening 
methods for such mutations, identification, isolation, mapping, and functional 
analyses of the genes underlying mutations.

NINDS:  Research on the development, normal function, and diseases of the 
nervous system.  This research might include the use of mutants to understand 
the mechanisms controlling the following processes:  neurogenesis, nervous 
system patterning, cell lineage, cell migration, programmed cell death, axon 
pathfinding and regeneration, myelination, and motor and sensory function.  

The areas of interest listed above are not presented in any order of priority, 
they are only examples of areas of research to consider.  Applications 
representing areas of interest to more than one Institute or Center will be 
assigned to multiple Institutes or Centers for funding consideration.  
Applicants are encouraged to propose work in other areas that are related to 
the objectives and scope of this RFA.

SPECIAL REQUIREMENTS

Restricted availability of unique research resources, upon which further 
studies are dependent, can impede the advancement of research and delivery of 
medical care. The sharing of biomaterials, data, and software in a timely 
manner, on the other hand, has been an essential element in the rapid progress 
that has been made in the genetic analysis of mammalian genomes. NIH policy 
requires that investigators make unique research resources readily available 
for research purposes to qualified individuals within the scientific community 
when they have been published [NIH Grants Policy Statement 
(http://grants.nih.gov/grants/policy/nihgps, Principles and Guidelines for 
Recipients of NIH Research Grants and Contracts on Obtaining and Disseminating 
Biomedical Research Resources:  Final Notice, December 1999 
(http://www.ott.nih.gov/policy/rt_guide_final.html)].  Biomaterials (pathogen-free 
mutant animals, preserved sperm and embryos) and other patentable research 
resources (e.g., genetic and phenotypic screens, mutagenesis protocols, and 
genetic and phenotypic data for all mutant strains) produced in projects 
funded by this RFA are expected to be made available and distributed to the 
broader scientific community.

The NIH is interested in ensuring that the research resources developed 
through this RFA become readily available to the research community for 
further research, development, and application, in the expectation that this 
will lead to products and knowledge of benefit to the public. For this reason, 
NIH is concerned that patents on mutant fish, embryos, and sperm, genetic and 
phenotypic screens, mutagenesis protocols, and genetic and phenotypic data for 
all mutant strains and other research resources might have a chilling effect 
on the future development of products and information that may improve the 
public health.  At the same time, NIH recognizes the rights of grantees to 
elect and retain title to subject inventions developed under Federal funding 
under the provision of the Bayh-Dole Act.

For applications submitted in response to this RFA, there are two special 
requirements regarding research resources produced in the proposed project: 

(1) Applicants are required to include in their applications a specific plan by 
which they will
share research resources with the wider scientific community. 

(2) Applicants are required to include a plan addressing if, or how, they will 
exercise their
intellectual property rights while making available to the broader scientific 
community patentable research resources (e.g., mutant fish, embryos and sperm, 
genetic and phenotypic screens, mutagenesis protocols, and genetic and 
phenotypic data for all mutant strains).  

Applicants are encouraged to discuss their proposed plans for addressing these 
requirements with their institutional offices of technology transfer.  Each of 
the two requirements are discussed in detail below. 

Plan to Share Research Resources 

To address the joint interests of the government in the availability of, and 
access to, the results of publicly funded research, NIH requires applicants 
who respond to this RFA to propose detailed plans for sharing the research 
resources generated through the grant. It is expected that the resources to be 
shared include all materials developed in projects funded under the RFA, 
including but not limited to, the following:  mutant fish, embryos and sperm, 
genetic and phenotypic screens, mutagenesis protocols, and genetic and 
phenotypic data for all mutant strains.  For this purpose, it is NIH’s opinion 
that dissemination of such data and materials via individual laboratories and 
Web sites is not sufficient, as it would force interested investigators to 
search several different data collections to make use of the results of this 
initiative. It is preferable that data, protocols, technologies, and 
biomaterials generated in grants funded under this RFA should be placed in 
common, public repositories and databases that are widely accessible by 
investigators in the scientific community. 

The National Resource for Zebrafish at the University of Oregon is being 
established as a focal point for sharing of resources among investigators 
using zebrafish.  Currently, the Resource is capable of accepting frozen sperm 
and some live fish stocks.  It is expected to be fully operational, with 
expanded capacity to accept live fish stocks, by mid-2000.  In addition to 
serving as a stock center, the Resource operates a comprehensive database, 
ZFIN.  Plans to share materials generated by projects under this RFA through 
the National Resource for Zebrafish should include evidence/documentation of 
coordination with investigators at the Resource.  Plans to deposit fish at the 
Resource should state whether this will be done as frozen sperm or live fish 
stocks.  For frozen sperm, adequate personnel and funding must be requested to 
produce, cryopreserve, and ship the sperm according to Resource specifications 
and using appropriate shipping containers available from the Resource.  For 
live fish stocks, it must be clear that the Resource is prepared to accept the 
numbers of stocks to be generated or agrees to handle the demands of producing 
and freezing sperm from live stocks deposited.  Plans to deposit reagents 
should likewise be coordinated.  Plans for informatics should be coordinated 
with the Resource to ensure that necessary hardware/software requirements can 
be accommodated for automatic deposit of data finalized by individual 
laboratories to ZFIN on a periodic basis.  A reasonable time frame for 
periodic deposition of mutants, sperm, reagents and data should be specified 
in the application and will be considered during the review of the plan for 
sharing.

It is expected that the investigator’s data and biomaterials sharing plan will 
include the following elements: 

(1) establishment of and access to a comprehensive database containing 
detailed results from genetic and phenotypic screens, mutagenesis protocols, 
and genetic and phenotypic data for all mutant strains, (2) access to mutants 
identified through genetic and phenotypic screens, for example, through the 
National Resource for Zebrafish, (3) access to mutant fish, embryos, oocytes 
and sperm for these mutants, for example, through the National Resource for 
Zebrafish, (4) access to methods for genetic and phenotypic screens and 
mutagenesis protocols, not currently available to the wider scientific 
community, and (5) access to mutants with unusual phenotypes, not of interest 
to investigators associated with this grant. 

The scientific review group will evaluate the adequacy of the proposed plan 
for sharing and data access. Comments on the plan and any concerns will be 
presented in an administrative note in the summary statement.  The adequacy of 
the plan will be considered by NIH program staff on the Trans-NIH Zebrafish 
Coordinating Committee and will be important in determining whether the grant 
shall be awarded. The sharing plan as approved, after negotiation with the 
applicant when necessary, will be a condition of the award.  Evaluation of 
non-competing continuation applications will include assessment of the 
effectiveness of research resource release.

Intellectual Property Rights 

NIH is interested in ensuring that the research resources developed through 
this RFA become readily available to the research community. 
 
With regard to patentable research results, such as mutants identified through 
genetic and phenotypic screens, embryos, oocytes and sperm for these mutants, 
genetic and phenotypic screens, mutagenesis protocols, and genetic and 
phenotypic data for all mutant strains and methodologies, the NIH requires 
applicants who respond to this RFA to develop and propose a plan addressing 
if, or how, they will exercise their intellectual property rights while making 
available to the broader scientific community research resources produced in 
projects funded under this RFA. This is expected to include an elaboration of 
the applicant’s anticipated plans to generate, or not generate, patents and/or 
exclusive or non-exclusive licensing of biomaterials and other patentable 
subject matter created in projects funded under this RFA.  This plan is also 
expected to include disclosure of any pre-existing agreements involving 
intellectual property rights, including options to for-profit research 
sponsors that are associated with biomaterials and data that may be generated. 
The requirement for this plan is in addition to the requirement for the plan 
for sharing and disseminating research resources described in the previous 
section.  

The scientific review group will evaluate the adequacy of the proposed plan 
for handling intellectual property rights.  Comments on the plan and any 
concerns will be presented in an administrative note in the summary statement. 
 The adequacy of the proposed plan will be considered by NIH program staff on 
the Trans-NIH Zebrafish Coordinating Committee in determining whether the 
grant shall be awarded. The plan as approved, after negotiation with the 
applicant when necessary, will be a condition of the award.  Evaluation of 
non-competing continuation applications will include assessment of the 
awardee’s adherence to the proposed plan.

Applicants also are reminded that the grantee institution is required to 
disclose each subject invention to NIH within two months after the inventor 
discloses it in writing to grantee institutional personnel responsible for 
patent matters. The awarding Institute reserves the right to monitor awardee 
activity in this area to ascertain if patents or patent applications on 
mutants identified through genetic and phenotypic screens, embryos and sperm 
for these mutants, genetic and phenotypic screens, mutagenesis protocols, and 
genetic and phenotypic data for all mutant strains or other patentable subject 
matter are adversely affecting the goals of this RFA.

Principles and guidelines for recipients of NIH research grants on obtaining 
and disseminating biomedical research resources can be found at: 
http://www.ott.nih.gov/policy/rt_guide_final.html.

Post-Award Management

During the course of the award period, the Principal Investigators may be 
invited to meet with NIH staff to review and share scientific progress.  Other 
scientists external to and knowledgeable about these studies also may be 
invited to participate.  Application budget requests should include travel 
funds for the Principal Investigator to attend annual meetings in the 
metropolitan Washington, D.C. area.

LETTER OF INTENT

Prospective applicants are asked to submit a letter of intent that includes a 
descriptive title of the overall proposed research, the name, address and 
telephone number of the Principal Investigator, the identities of other key 
personnel and participating institutions, and the number and title of this 
RFA.  Although the letter of intent is not required, is not binding, and does 
not enter into the review of subsequent applications, the information that it 
contains allows NIH staff to estimate the potential review workload and to 
avoid conflict of interest in the review.  

The letter of intent is to be sent to Dr. Deborah Henken at the address listed 
under INQUIRIES by April 16, 2000.

APPLICATION PROCEDURES

The research grant application form PHS 398 (rev. 4/98) is to be used in 
applying for these  grants.  These forms are available at most institutional 
offices of sponsored research, on the Internet at 
http://grants.nih.gov/grants/funding/phs398/phs398.html, and from the Division 
of Extramural Outreach and Information Resources, National Institutes of 
Health, 6701 Rockledge Drive, MSC 7910, Bethesda, MD 20892-7910, telephone 
301-435-0714, E-mail: Grantsinfo@nih.gov.

Application Instructions

Specific application instructions have been modified to reflect “MODULAR 
GRANT” and “JUST-IN-TIME” streamlining efforts being examined by the NIH.  
Complete and detailed instructions and information on Modular Grant 
applications can be found at 
http://grants.nih.gov/grants/funding/modular/modular.htm.

The modular grant concept establishes specific modules in which direct costs 
may be requested as well as a maximum level for requested budgets.  Only 
limited budgetary information is required under this approach.  The 
just-in-time concept allows applicants to submit certain information only when 
there is a possibility for an award.  It is anticipated that these changes 
will reduce the administrative burden for the applicants, reviewers and 
Institute staff. 

Modular Grant applications will request direct costs in $25,000 modules, up to 
a total direct cost request of $250,000 per year. (Applications that request 
more than $250,000 direct costs in any year must follow the traditional PHS 
398 application instructions.)  The total direct costs must be requested in 
accordance with the program guidelines and the modifications made to the 
standard PHS 398 application instructions described below:

o FACE PAGE: Items 7a and 7b should be completed, indicating Direct Costs (in 
$25,000 increments up to a maximum of $250,000) and Total Costs [Modular Total 
Direct plus Facilities and Administrative  (F&A) costs] for the initial budget 
period.  Items 8a and 8b should be completed indicating the Direct and Total 
Costs for the entire proposed period of support.

o DETAILED BUDGET FOR THE INITIAL BUDGET PERIOD:  Do not complete Form Page 4 
of the PHS 398.  It is not required and will not be accepted with the 
application.

o BUDGET FOR THE ENTIRE PROPOSED PERIOD OF SUPPORT:  Do not complete the 
categorical budget table on Form Page 5 of the PHS 398.  It is not required 
and will not be accepted with the application.

o NARRATIVE BUDGET JUSTIFICATION:  Prepare a Modular Grant Budget Narrative 
page. (See http://grants.nih.gov/grants/funding/modular/modular.htm for sample 
pages.)  At the top of the page, enter the total direct costs requested for 
each year.  This is not a Form Page.

Under Personnel, list key project personnel, including their names, percent of 
effort, and roles on the project.  No individual salary information should be 
provided.  However, the applicant should use the NIH appropriation language 
salary cap and the NIH policy for graduate student compensation in developing 
the budget request.

For Consortium/Contractual costs, provide an estimate of total costs (direct 
plus F & A) for each year, each rounded to the nearest $1,000.  List the 
individuals/organizations with whom consortium or contractual arrangements 
have been made, the percent effort of key personnel, and the role on the 
project.  Indicate whether the collaborating institution is foreign or 
domestic.  The total cost for a consortium/contractual arrangement is included 
in the overall requested modular direct cost amount.  Include the Letter of 
Intent to establish a consortium.

Provide an additional narrative budget justification for any variation in the 
number of modules requested.

o BIOGRAPHICAL SKETCH:  The Biographical Sketch provides information used by  
reviewers in the assessment of each individual"s qualifications for a specific 
role in the proposed project, as well as to evaluate the overall 
qualifications of the research team.  A biographical sketch is required for 
all key personnel, following the instructions below.  No more than three pages 
may be used for each person.  A sample biographical sketch may be viewed at: 
http://grants.nih.gov/grants/funding/modular/modular.htm.

- Complete the educational block at the top of the form page,
- List position(s) and any honors,
- Provide information, including overall goals and responsibilities, on 
research projects ongoing or completed during the last three years,
- List selected peer-reviewed publications, with full citations.

o CHECKLIST:  This page should be completed and submitted with the 
application.  If the F&A rate agreement has been established, indicate the 
type of agreement and the date.  All appropriate exclusions must be applied in 
the calculation of the F&A costs for the initial budget period and all future 
budget years.

o The applicant should provide the name and phone number of the individual to 
contact concerning fiscal and administrative issues if additional information 
is necessary following the initial review.

Submission Instructions

The RFA label available in the PHS 398 (rev. 4/98) application form must be 
stapled to the bottom of the face page of the application and must display the 
RFA number HD-00-004.  A sample RFA label is available at 
http://grants.nih.gov/grants/funding/phs398/label-bk.pdf.  Please note this is 
in the pdf format.  Failure to use this label could result in delayed 
processing of the application such that it may not reach the review committee 
in time for review.  In addition, the RFA title and number must be typed on 
line 2 of the face page of the application form and the YES box must be 
marked.

Submit a signed, typewritten original of the application, including the 
Checklist, and five signed photocopies, in one package to:

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

Applications must be received by May 19, 2000.  If an application is received 
after that date, it will be returned to the applicant without review.  

The CSR will not accept any application in response to this RFA that is 
essentially the same as one currently pending initial review, unless the 
applicant withdraws the pending application.  The CSR will not accept any 
application that is essentially the same as one already reviewed.  This does 
not preclude the submission of substantial revisions of applications already 
reviewed, but such applications must include an introduction addressing the 
previous critique.

REVIEW CONSIDERATIONS

Upon receipt, applications will be reviewed for completeness and adherence to 
the Application Instructions above by CSR and for responsiveness by the Trans-
NIH Zebrafish Coordinating Committee.  Incomplete and/or non-responsive 
applications will be returned to the applicant without further consideration. 
 Applications that are complete and responsive to the RFA will be evaluated 
for scientific and technical merit by an appropriate peer review group 
convened by the CSR in accordance with the review criteria stated below.  As 
part of the initial merit review, all  applications will receive a written 
critique and undergo a process in which only those applications deemed to have 
the highest scientific merit, generally the top half of the applications under 
review, will be discussed, assigned a priority score, and receive a second 
level review by the appropriate National Advisory Council or Board.

Review Criteria

The goals of NIH-supported research are to advance our understanding of 
biological systems, improve the control of disease, and enhance health.  In 
their written comments, reviewers will be asked to discuss the following 
aspects of the application in order to judge the likelihood that the proposed 
research will have a substantial impact on the pursuit of these goals.  Each 
of these criteria will be addressed and considered in assigning the overall 
score, weighting them as appropriate for each application.  Note that the 
application does not need to be strong in all categories to be judged likely 
to have a major scientific impact and thus deserve a high priority score.  For 
example, an investigator may propose to carry out important work that by its 
nature is not innovative, but is essential to move a field forward.

1. Significance:  Does this study address an important problem?  If the aims 
of the application are achieved, how will scientific knowledge be advanced?  
What will be the effect of these studies on the concepts or methods that drive 
this field?

2. Approach:  Are the conceptual framework, design, methods, and analyses 
adequately developed, well-integrated, and appropriate to the aims of the 
project?  Does the applicant acknowledge potential problem areas and consider 
alternative tactics? 

3. Innovation:  Does the project employ novel concepts, approaches or methods? 
 Are the aims original and innovative?  Does the project challenge existing 
paradigms or develop new methodologies or technologies?

4. Investigator:  Is the investigator appropriately trained and well suited to 
carry out this work?  Is the work proposed appropriate to the experience level 
of the Principal Investigator and other researchers (if any)?

5. Environment:  Does the scientific environment in which the work will be 
done contribute to the probability of success?  Do the proposed experiments 
take advantage of unique features of the scientific environment or employ 
useful collaborative arrangements?  Is there evidence of institutional 
support?

In addition, applications responding to this RFA will be assessed with respect 
to the following criteria.  This evaluation will be presented in an 
administrative note in the summary statement, and will not factor into the 
numerical score:

o Exportability and accessibility:  What is the likelihood that the mutants 
and phenotypic information generated in the project will be made widely 
available in a timely fashion to the scientific community?  Are state-of-the-
art procedures employed to ensure the distribution of pathogen-free mutant 
strains, embryos, and/or sperm?  What is the likelihood that other patentable 
research results will be widely available for the scientific community, given 
the proposed plan to exercise (or not to exercise) intellectual property 
rights regarding mutants identified through genetic and phenotypic screens, 
embryos and sperm for these mutants, genetic and phenotypic screens, 
mutagenesis protocols, and genetic and phenotypic data for all mutant strains 
and methodologies?

o In addition, the plan to share research resources and the plan to exercise 
(or not exercise) intellectual property rights regarding patentable research 
resources will be judged for appropriateness.  

In addition to the above criteria, in accordance with NIH policy, all 
applications also will be reviewed with respect to the following:

o The reasonableness of the proposed budget and duration in relation to the 
proposed research.

o The adequacy of the proposed protection of human, animals, or the 
environment, to the extent they may be adversely affected by the project 
proposed in the application.

SCHEDULE

Letter of Intent Receipt Date:    April 16, 2000
Application Receipt Date:         May 19, 2000
Peer Review Date:                 October 2000
Advisory Council Review:          January 2001
Earliest Anticipated Start Date:  April 2001

AWARD CRITERIA

Factors that will be used to make award decisions are as follows:

o Scientific and technical merit of the proposed project as determined by peer 
review,
o Cost effectiveness of the proposed strategy,
o Adequacy of plans to make widely available to the research community all 
research resources developed during this project,
o Adequacy of plans to exercise (or not exercise) intellectual property rights 
while permitting wide availability to the research community of patentable 
research resources (mutants identified through genetic and phenotypic screens, 
embryos and sperm for these mutants, genetic and phenotypic screens, 
mutagenesis protocols, and genetic and phenotypic data for all mutant strains 
and methodologies) developed during this project,
o Program priorities and program balance,
o Availability of funds.

INQUIRIES

Potential applicants are strongly encouraged to contact program staff with any 
questions regarding the responsiveness of their proposed project to the goals 
of this RFA.

Written and telephone inquiries concerning this RFA are encouraged.  The 
opportunity to clarify any issues or questions from potential applicants is 
welcome.

A complete listing of contacts for both programmatic and fiscal/administrative 
inquiries may be found at: 
http://www.nichd.nih.gov/rfa/hd-00-004/hd-00-004.htm.

AUTHORITY AND REGULATIONS

This program is described in the Catalog of Federal Domestic Assistance Nos. 
93.865, 93.396, 93.306, 93.867, 93.839, 93.172, 93.273, 93.846, 93.173, 
93.121, 93.847, 93.848, 93.849, 93.279, 93.113, 93.862, 93.242, 93.853.  
Awards are made under authorization of Sections 301 and 405 of the Public 
Health Service Act, as amended (42 USC 241 and 284) and administered under NIH 
grants policies and Federal Regulations 42 CFR 52 and 45 CFR Parts 74 and 92. 
 This program is not subject to the intergovernmental 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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