8415 Rapid Generation Of An In Vivo Loss-Of-Function Genetic Model Of sdhb Zebrafish: Functional And Metabolic Characterization
Bibliographic record
Abstract
Abstract Disclosure: S. Parisien-La Salle: None. F. Nobilleau: None. J. Lamontagne: None. S. Éric: None. I. Bourdeau: None. Introduction: Animal research has been limited in pheochromocytomas and paragangliomas (PPGLs). Recently, Dona et al., described a sdhb mutated zebrafish model by introducing a 13 bp frameshift mutation at the sdhb exon1—intron1 boundary into the zebrafish sdhb gene that recapitulated features of PPGLs. Objective: To generate a new model of mutated sdhb zebrafish to further investigate the underlying metabolic and biochemical perturbations in vivo. Methods: We took advantage of the genetic accessibility of zebrafish embryo to generate loss-of-function model for sdhb using CRISPR/Cas9 technology. Three guide RNAs were designed and microinjected in one-cell stage zebrafish embryos to target the coding sequence of zebrafish sdhb gene. In order to control for the stress brought on by injections, we injected a group of zebrafish with Cas9 endonuclease as a control. At five days post fertilization, mutant and control zebrafish were flash frozen and sent for liquid chromatography mass spectrometry measurements of Krebs cycle metabolites and normetanephrines/metanephrines. All results were normalized to the control groups consisting of wild-type or Cas9 injected larvae. Sdhb expression was analyzed by RT-qPCR in sdhb mutants and wild-type zebrafish larvae. We also monitored survival and heart rate in sdhb mutant zebrafish, compared to wild-type and Cas9. Results: Firstly, we confirmed the significant reduction of sdhb expression in CRISPR-injected larvae compared to wild-type larvae at 5 dpf (0.0613 vs 1.000 p<0.0001). Our metabolic panel showed that succinate was up to 26 times more elevated in sdhb mutants than in wild-type fish extracts (sdhb: 26.51 p<0.0001). Lactate (sdhb: 5.27 p<0.0001), leucine (sdhb: 2.88 p<0.0001), arginine (sdhb: 1.50 p=0.0034) and HMG-CoA (sdhb: 1.36 p=0.0033) were also higher in sdhb mutants. Whereas aspartate (sdhb: 0.244 p<0.0001) and oxidized gluthiatone (sdhb: 0.64 p=0.0011) were significantly lower in sdhb mutants than in wild-types. In regards to functionality, normetanephrine and metanephrine levels were at least four folds higher in sdhb mutants than in Cas9 injected larvaes (4.06 p<0.0001 and 3.42 p<0.0001 respectively). Interestingly, this difference was more noticeable in the fish bathing medium than in larvae tissue extracts, suggesting an increased hormonal excretion (normetanephrines: 4.81 p<0.0001 and metanephrines: 9.40 p<0.0001). Finally, sdhb mutated zebrafish presented with a higher heart rate (148 vs 133 p<0.0001) and reduced lifespan (p<0.0001) when compared to the wild-type/Cas9 group. Conclusion: These findings confirm that the zebrafish is an ideal model for studying PPGLs and their associated genetic defects. Our approach allows the in vivo assessment of a large metabolic panel, opening avenues on new potential clinical metabolic biomarkers in a loss-of-function of the SDHB gene. Ref: Dona M, et al. Endocr Relat Cancer. 2021 Presentation: 6/1/2024
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".