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Establishing an Integrated Platform for Diploid Germplasm Conservation in Zebrafish (Danio rerio)

Award Number: R24OD034058
ORGANIZATION: OFFICE OF THE DIRECTOR, NIH
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 01/15/2023
PERIOD OF PERFORMANCE END DATE: 11/30/2026

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Establishing an Integrated Platform for Diploid Germplasm Conservation in Zebrafish (Danio rerio) - PROJECT SUMMARY Rigor and reproducibility in zebrafish research are in jeopardy because current storage methods to preserve zebrafish germplasm are inadequate. This is especially problematic for the most frequently used reference strains such as AB, Tübingen (TU), and their hybrid derivatives SAT and NHGRI, which are used for genome editing applications that critically depend on the accuracy of sequence information. The original DNA sequences for the four reference strains were generated years ago in select individuals, and random genetic drift has gradually modified these genome sequences. Other animal models rely on cryopreservation of the embryo; however, we currently have no working protocols for zebrafish embryo cryopreservation. Despite recent optimization, sperm cryopreservation – the current method for germplasm conservation – preserves only male- derived genomes. Although the cryopreserved haploid paternal genome remains stable over time, genome changes accumulate in live stocks used to provide females. Thus, half the embryo's genome is the same as the original fish, and half is not. We will develop an integrated platform for diploid germplasm conservation in zebrafish for the four reference strains. This platform will help preserve the entire genome close to the published sequence information or new sequences when they become available. To accomplish this goal, our laboratory at Michigan State University (MSU) will collaborate with the Aquatic Germplasm and Genetic Resources Center (AGGRC) at Louisiana State University Agricultural Center (LSUAC) and the Zebrafish International Resource Center (ZIRC) at the University of Oregon. The new integrated platform for diploid germplasm conservation encompasses two key steps: 1) isolation, culture, and cryopreservation of diploid somatic cells, and 2) thawing of cells and using them to derive the original strain by somatic cell nuclear transfer (SCNT – cloning), which is a technique we have successfully applied to produce founder individuals. This method preserves the sequence of all alleles and genomes. The specific aims for this project are: Aim 1. Development of an integrated platform to isolate, culture, cryopreserve and genotype diploid cells from wild-type lines. Aim 2. Standardization of the zebrafish cloning procedure, including the generation of homozygous zebrafish lines. Aim 3. Promotion, dissemination, and training of resource center and laboratory personnel using the diploid somatic cell conservation method. At the end of this R24 proposal, the scientific community will have access to: 1) stable diploid genomes of the most used reference lines, which is not possible by sperm cryopreservation, increasing the rigor and reproducibility of experimental results within and between laboratories; and 2) homozygous zebrafish capable of enhancing studies in cell and organ transplantation, sex determination, and other approaches. We will also show that this new integrated platform will be capable of: 1) rescuing precious research lines on the brink of extinction when only adult animals of one sex remain alive, and 2) eliminating pathogens from contaminated lines.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2026 ( Subtotal = $568,416 )
2026	2026	MICHIGAN STATE UNIVERSITY	426 AUDITORIUM RD RM 360	EAST LANSING	MI	48824	INGHAM	USA	Research Infrastructure Programs	000	4	2/11/2026	NON-COMPETING CONTINUATION	$568,416
														Subtotal = $568,416

Issue Date FY: 2025 ( Subtotal = $749,214 )
2025	2025	MICHIGAN STATE UNIVERSITY	426 AUDITORIUM RD RM 2	EAST LANSING	MI	48824	INGHAM	USA	Research Infrastructure Programs	001	3	7/21/2025	NON-COMPETING CONTINUATION	$149,843
2025	2025	MICHIGAN STATE UNIVERSITY	426 AUDITORIUM RD RM 2	EAST LANSING	MI	48824	INGHAM	USA	Research Infrastructure Programs	000	3	1/24/2025	NON-COMPETING CONTINUATION	$599,371
														Subtotal = $749,214

Issue Date FY: 2024 ( Subtotal = $730,053 )
2024	2024	MICHIGAN STATE UNIVERSITY	426 AUDITORIUM RD RM 2	EAST LANSING	MI	48824	INGHAM	USA	Research Infrastructure Programs	001	2	5/14/2024	NON-COMPETING CONTINUATION	$146,011
2024	2024	MICHIGAN STATE UNIVERSITY	426 AUDITORIUM RD RM 2	EAST LANSING	MI	48824	INGHAM	USA	Research Infrastructure Programs	000	2	11/13/2023	NON-COMPETING CONTINUATION	$584,042
														Subtotal = $730,053

Issue Date FY: 2023 ( Subtotal = $1,026,325 )
2023	2023	MICHIGAN STATE UNIVERSITY	426 AUDITORIUM RD RM 2	EAST LANSING	MI	48824	INGHAM	USA	Research Infrastructure Programs	002	1	7/14/2023	SUPPLEMENT FOR EXPANSION	$272,462
2023	2023	MICHIGAN STATE UNIVERSITY	426 AUDITORIUM RD RM 2	EAST LANSING	MI	48824	INGHAM	USA	Research Infrastructure Programs	000	1	1/15/2023	NEW	$681,302
2023	2023	MICHIGAN STATE UNIVERSITY	426 AUDITORIUM RD RM 2	EAST LANSING	MI	48824	INGHAM	USA	Research Infrastructure Programs	001	1	2/22/2023	NEW	$72,561
														Subtotal = $1,026,325

Grand Total All Awards = $3,074,008

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Establishing an Integrated Platform for Diploid Germplasm Conservation in Zebrafish (Danio rerio)

Award Number: R24OD034058

ORGANIZATION: OFFICE OF THE DIRECTOR, NIH

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 01/15/2023

PERIOD OF PERFORMANCE END DATE: 11/30/2026

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