Generation of pyrroline-5-carboxylate reductase 1 (pycr1) gene knockout (KO) zebrafish by Transcription activator-like effector nuclease (TALEN) genome editing tool. (A) Scheme showed the TALEN left and right arm sequences used to target pycr1 gene on exon 4 in zebrafish (top panel). The Left and right arm and spacer sequences used for TALEN design were listed in the middle panel. The high-resolution melting assay (HRMA) of F0 fish were showed in the bottom panel. (B) Comparison of the Sanger sequencing results of F1 generation that carried potential mutations for pycr1 gene. (C) The stable pycr1 KO fish used in this study carrying a 2 nucleotide-deletion (delCT) results in a predicted truncate PYCR1 protein with 54 amino acids. (D) The predicted three-dimensional structure of PYCR1 protein for WT (+/+) and pycr1 KO (−/−) fish.

Morphologies of pycr1 gene knockout (KO) Zebrafish. Quantitative (A) and qualitative (B) detection of fluorescence apoptotic intensity within tail in the pycr1 KO fish embryos aged at 24 hour-post-fertilization (hpf) by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Data were presented with mean ± SEM and the significance was tested by one-way ANOVA; n = 13–15. The label above column with different letter means reaching significant difference with p < 0.05. (C) The detection of intestinal integrity in the pycr1 KO fish at 60 day-post-fertilization (dpf) by Smurf dye staining. (D) Detection of intestinal integrity in the WT and pycr1 KO fish at 180 dpf or natural aging WT fish aged at 600 dpf by Smurf dye staining. (E) The Principle Component Analysis (PCA) plot for morphometric analyses among the WT (+/+), heterozygotic (+/−), and homozygotic (−/−) pycr1 KO fish aged at 180 dpf. (F) The growth curves in body length of the WT and the pycr1 KO fish. (G) The growth curves in body weight of the WT and the pycr1 KO fish. (H) The mortality curves of WT and pycr1 KO fish. (I) The retina histological comparison between WT and pycr1 KO fish aged at 180 dpf. Data were presented with mean ± SEM and the significance was tested by t-test in (F–H), n = 44; * p < 0.05, ** p < 0.01, *** p < 0.005, and **** p < 0.001. (J) The quantitative comparison of retina cell density between WT and pycr1 KO fish. The data were presented with mean ± SEM and the significance was tested by t-test, n = 6; ** p < 0.01.

Comparison of behavioral endpoints in novel tank exposure test and predator avoidance test in wild type and pycr1 gene knockout (KO) zebrafish. (A) Average speed, (B) total distance traveled in the top area, (C) duration time in the top of the tank, (D) number of entries to the top, (E) latency to entry to the top, (F) freezing time to movement ratio, (G) and (H) locomotion trajectories of wild type (WT) fish before and after acclimation, respectively, (I) and (J) locomotion trajectories of the pycr1 KO fish before and after acclimation, respectively. (K) Average speed, (L) predator approaching time, (M) average distance to separator, (N) freezing time to movement time ratio, (O) swimming time to movement time ratio, (P) rapid movement time ratio of WT and pycr1 KO fish. The data for novel tank test (A–F) were expressed as the mean ± SEM and analyzed by unpaired t-test (WT n = 30; pycr1 KO n = 14; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). The data for the predator avoidance test (K–P) were expressed as the mean and analyzed by Mann–Whitney test (control n = 30; pycr1 KO n = 15; * p < 0.05; ** p < 0.01; **** p < 0.0001). (Q) and (R) locomotion trajectories the of WT and pycr1 KO fish, respectively.

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