A High-Throughput Method for Identifying N-Ethyl-N-Nitrosourea (ENU)-Induced Point Mutations in Zebrafish

Draper, Bruce W.; McCallum, Claire M.; Stout, Jennifer L.; Slade, Ann J.; Moens, Cecilia B.

doi:10.1016/s0091-679x(04)77005-3

Cited by 99 publications

(112 citation statements)

References 22 publications

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“…Thus, optimization of cooling rates, specific extenders, containers, and cryoprotectants are important, and this process is usually dictated by the interaction of these parameters. Previous studies of zebrafish sperm were not able to quantify the cooling rate because the freezing was performed by placement of cryovials or capillary tubes on crushed dry ice [5,7,8]. In the present study, the effect of cooling rate with three cryoprotectants was tested using a programmable freezer and standardized plastic straws.…”

Section: Discussionmentioning

confidence: 92%

“…The first protocol was published more than 20 y ago [5], uses 10% methanol as cryoprotectant. This protocol was later adapted and used by genetics laboratories for recovery of specific strains and lines [6,7]; the fertilization level of frozenthawed sperm was reported to be 28 to 36%. In addition to methanol, N, N-dimethyl acetamide (DMA, 10%) was an effective cryoprotectant for zebrafish sperm in another study (fertilization level of thawed sperm was 9 to 14% [8]).…”

Section: Introductionmentioning

confidence: 99%

“…All three procedures were complicated, and steps such as freezing rate were not quantified, making it difficult to obtain consistent results in other laboratories [8]. Also, these procedures relied on the use of a specific brand of powdered milk in the extender solution [5,7] which is not readily available worldwide, and also impedes the observation of sperm motility and morphology, especially after thawing. Therefore, further investigation was needed to simplify and standardize sperm cryopreservation, and to identify reproducible procedures and containers to provide consistent cooling and thawing profiles, as well as reliable labeling of samples.…”

Section: Introductionmentioning

confidence: 99%

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Development of a simplified and standardized protocol with potential for high-throughput for sperm cryopreservation in zebrafish Danio rerio

et al. 2007

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Sperm cryopreservation offers potential for long-term storage of genetic resources. However, the current protocols for zebrafish Danio rerio are cumbersome and poorly reproducible. Our objective was to facilitate adoption of cryopreservation by streamlining methods from sperm collection through thawing and use. First, sperm activation was evaluated, and motility was completely inhibited when osmolality of the extender was ≥ 295 to 300 mOsmol/kg. To evaluate cryoprotectant toxicity, sperm were incubated with dimethyl sulfoxide (DMSO), N, N-dimethyl acetamide (DMA), methanol, or glycerol at 5, 10, and 15% concentrations. Based on motility, DMSO, DMA, and methanol (≤ 10%) were less toxic; therefore, sperm were cryopreserved using these cryoprotectants at cooling rates of 10 and 20 °C/min. The highest motility (mean ± SD) (35 ± 23%; P ≤ 0.0001) and fertility (13 ± 8%; P ≤ 0.001) in thawed sperm were obtained with the combination of 8% methanol and a cooling rate of 10 °C/min. Further evaluations of 8% methanol and 10 °C/min were performed with males from populations with high (2.05 ± 0.24) and low (1.18 ± 0.12) body condition (P = 0.0001). Motility of thawed sperm from the two populations was 38 ± 16% and 78 ± 10% (P = 0.0001), and fertilization was 6 ± 6% and 33 ± 20% (P = 0.0001). These values were positively related with body condition factor. Overall, this study simplified and standardized sperm cryopreservation, and established a protocol using French straws as a freezing container and an extender without powdered milk. This protocol can be readily adapted for high-throughput application using automated equipment, and motility and fertility comparable to previous reports were obtained. Male variability and sperm quality remain important considerations for future work, especially in mutant and inbred lines.

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Section: Discussionmentioning

confidence: 92%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Development of a simplified and standardized protocol with potential for high-throughput for sperm cryopreservation in zebrafish Danio rerio

et al. 2007

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“…For example, TILLING organisms with large introns, such as zebrafish, requires short exon targets, leading to the development of alternative protocols for zebrafish TILLING 12,13 . In addition, TILLING can be performed using alternative readout platforms including slab gel and capillary systems 7 (http://www.jicgenomelab.co.uk/services/ mutation-detection/development.html).…”

Section: Overview Of Tillingmentioning

confidence: 99%

“…A less expensive alternative would be alcohol precipitation 12 . When testing options, consider the time required, the percent of sample recovery and the material cost of the assay.…”

Section: | Spin the Plate For 2 Min At 1300g At Room Temperature (Amentioning

confidence: 99%

A protocol for TILLING and Ecotilling in plants and animals

et al. 2006

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We describe Targeting-Induced Local Lesions IN Genomes (TILLING), a reverse-genetic strategy for the discovery and mapping of induced mutations. TILLING is suitable for essentially any organism that can be mutagenized. The TILLING procedure has also been adapted for the discovery and cataloguing of natural polymorphisms, a method called Ecotilling. To discover nucleotide changes within a particular gene, PCR is performed with gene-specific primers that are end-labeled with fluorescent molecules. After PCR, samples are denatured and annealed to form heteroduplexes between polymorphic DNA strands. Mismatched base pairs in these heteroduplexes are cleaved by digestion with a single-strand specific nuclease. The resulting products are size-fractionated using denaturing polyacrylamide gel electrophoresis and visualized by fluorescence detection. The migration of cleaved products indicates the approximate location of nucleotide polymorphisms. Throughput is increased and costs are reduced by sample pooling, multi-well liquid handling and automated gel band mapping. Once genomic DNA samples have been obtained, pooled and arrayed, thousands of samples can be screened daily.

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