- Title
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Generation of a transparent killifish line through multiplex CRISPR/Cas9-mediated gene inactivation
- Authors
- Krug, J., Perner, B., Albertz, C., Mörl, H., Hopfenmüller, V.L., Englert, C.
- Source
- Full text @ Elife
(A–C) Gene expression analysis of mitfa (A), ltk (B), and csf1ra (C) in the skin of male and female wild-type fish via quantitative real-time PCR. The expression of mitfa was only significantly up-regulated in male fish at the age of 6 weeks post-hatching (wph) (p: 0.048). For both sexes, the expression of ltk increased significantly over time (pmale, 2wph: 0.028, pmale, 3wph: 0.026, pmale, 6wph: 0.002; pfemale, 2wph: 0.002, pfemale, 3wph: 0.019, pfemale, 6wph: 0.033), whereas no significant differences were observed for csf1ra. Expression levels were normalized to the expression at 1 wph in the respective sex. Rpl13a was used as housekeeping gene (nmale_1wph = 3, nmale_2wph = 4, nmale_3wph = 4, nmale_6wph = 4; nfemale_1wph = 3, nfemale_2wph = 4, nfemale_3wph = 4, nfemale_6wph = 4). Relative gene expression was calculated using the ΔΔCT method (Pfaffl et al., 2002). Student’s or Welch’s t-tests were computed to determine significant changes in gene expression. Horizontal line represents median. Whiskers show min. to max. values. (D) Phenotypical analysis of F0 embryos revealed a reduction of melanophores in GFP-positive compared to GFP-negative embryos. (E,F) Male (E) and female (F) wild-type N. furzeri. (G–J) Males and females of the F0 generation, display a mosaic loss of body pigmentation. Note that almost completely transparent individuals can be observed in (H–J), allowing a view on inner organs (o: ovary, s: stomach, sb: swim bladder). (J) Microscopic analysis of a female F0 fish with a view on individual eggs within the ovary and blood vessels (cv: cardinal vein, e: egg). (K,K’) Male (K) and female (K’) fish at the age of 38 days post-hatching (dph) with the genotype mitfa-/-,ltk+/-,csf1ra-/- showed a lack of melanophores and xanthophores, whereas iridophores were present. (L,L’) A lack of melanophores and iridophores was observed in male (L) and female (L’) N. furzeri with the genotype mitfa-/-, ltk-/-, csf1ra+/-. Despite a homozygous mutation in ltk (ltk-/-) individual scales with iridophores were detected in fish of both sexes. (M,M’) The presence of homozygous mutations in all the three genes mitfa, ltk, and csf1ra resulted in the loss of body pigmentation in males (M) and females (M’) allowing a view on inner organs.
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(A) Expression of csf1ra was analyzed via RT-PCR using cDNA from FACS-sorted populations of lymphocytes, progenitors, and myeloid cells obtained from the whole kidney marrow (WKM) of a wild-type N. furzeri. Csf1ra was detected in all subpopulations, most strongly in myeloid cells. As a negative control, an RT-sample (no reverse transcriptase during cDNA synthesis) was used to exclude contaminations with genomic DNA. As loading control, rpl13a was used. (B) Comparison of cell numbers in the different subpopulations of the WKM of fish with the following genotypes: mitfa-/-, ltk-/-, csf1ra+/+ (n=2), mitfa-/-, ltk-/-, csf1ra+/- (n=4), and mitfa-/-, ltk-/-, csf1ra-/- (n=4). One-way ANOVA followed by Tukey’s post hoc test did not reveal any significant differences. Horizontal line represents median. Whiskers show min. to max. value. (C,C’) Male klara fish (C) showed an appearance of melanophores on fin appendages, which was not observed in females (C’). (D,D’) Occurrence of melanophores intensified with age resulting in black fins in male fish (D). In female klara animals (D’) black fins were not observed.
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(A) For the analysis of egg quantity, eggs were collected once per week for four weeks (coll. #1–4) from wild-type (n=3 tanks with one male and two females each) and klara (n=3 tanks with one male and two females each) fish. To assess egg quality, the number of alive eggs stored on coconut coir plates was determined one-week post-collection (wpc). Student's or Welch's t-tests were computed revealing no significant differences regarding egg quantity and quality. Error bars represent standard deviation. (B) Collected eggs obtained from breeding groups with a wild-type female and a klara female and either a wild-type male (group #1) or a klara male (group #2) were genotyped via HRMA. As a mating partner, male fish preferred wild-type females (group #1: 76.2%; group #2: 75.1%) over klara females (group #1: 23.8%; group #2: 24.9%). In the presence of a wild-type male and a klara male and either a wild-type female (group #3) or a klara female (group #4) the majority of analyzed eggs were fertilized by the wild-type male (group #3: 97.6%; group #4: 90.9%). (C) Within the same sex, wild-type and klara fish did not differ in size(nWT_male = 9 nWT_female = 9, nklara_male = 9, nklara_female = 8). Wild-type males were significantly heavier than klara males (p: 0.0009). Student’s or Welch’s t-tests were computed to determine differences in size or weight. Error bars represent standard deviation.
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(A) Despite the inactivation of three genes involved in pigmentation, the eye pigmentation did not differ between klara and wild-type fish. (B) Targeting the slc45a2 locus in wild-types resulted in a reduction of melanophores in the eye, but also on the whole body of injected embryos compared to wild-type controls. (C) A loss of melanophores in the retinal pigmented epithelium was observed in F0 fish upon a microinjection of the sgRNA targeting slc45a2 in klara embryos. A silver-pigmented ring around the eye was still present. (ctrl: control; dpi: days post-injection; dph: days post-hatching).
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(A) The cdkn1a locus in klara was targeted via CRISPR/Cas9 to insert a reporter construct consisting of a GFP and an NTR cassette, separated by P2A self-cleavage sites, allowing the detection and ablation of senescent cells. (B) A 5’-biotinylated, double-stranded donor template flanked by two ~0.9 kb flanking arms was used for the target-specific insertion. (C,D) Function of the reporter construct was tested by exposing embryos (cdkn1a+/+ and cdkn1aki/+) to a γ-irradiation dose of 10 Gy. Representative images taken before irradiation showed autofluorescence originating from the yolk, which was observed in embryos of both genotypes (C). 24 hr post-irradiation (hpi), the presence of GFP-positive cells was detected only in cdkn1aki/+ embryos (D). (E) Gene expression analysis of cdkn1a in embryos (klara;cdkn1a+/+ and klara;cdkn1aki/+) at 24 (n=12) and 48 hpi (n=12) compared to non-irradiated controls (n=6). Expression of cdkn1a increased significantly as revealed by Student's t-test (p24hpi: 0.011, p48hpi: 0.0004). (F,G) For the expression of GFP (F) and ntr (G) at 24 (n=8) and 48 hpi (n=6) compared to non-irradiated controls (n=2), klara;cdkn1a+/+ embryos were excluded due to the absence of the reporter construct. Expressions of GFP and ntr were significantly increased at 48 hpi (pGFP: 0.045, pntr: 0.006) compared to controls. For all qRT-PCRs, rpl13a was used as a housekeeping gene. Relative gene expression was calculated using the ΔΔCT method. Student’s t-tests were computed to determine significant changes in gene expression. Error bars represent standard deviation. (H) Detection of GFP-positive cells in the liver of a young (4 wph) and an old (30 wph) klara;cdkn1aki/ki fish. A higher number of GFP+ cells was detected in the old liver sample. Images are displayed as extended depth of focus projections. In the young liver tissue, small individual GFP-positive cells were detected, whereas in the old liver, a high prevalence of enlarged GFP-positive cells was identified (see zoom-in). (I) Analysis of kidney samples via flow cytometry from klara;cdkn1aki/ki fish at the age of 4.5 wph (nmale = 5; nfemale = 4), 9.5 wph (nmale = 5; nfemale = 5) and 20.5 wph (nmale = 4; nfemale = 5). A significantly higher proportion of GFP-positive cells was detected in 20.5 wph versus 4.5 wph (p: 0.0009) and 9.5 wph (p: 0.0084) fish. One-Way ANOVA followed by Tukey’s post hoc test was computed to analyze for differences in cell number. Horizontal line shows mean value. Error bars represent standard deviation. (J–M) Representative images from light sheet microscopy show the presence of GFP-positive cells in living klara;cdkn1aki/ki fish (K, M) at 4 dph (K) and 17 dph (M). The panel displays representative 3D reconstructions from stacks of 31 (J,L) and 372 (K,M) optical slices. Klara fish at the same ages (J,L), which do not have any GFP + cells, were used as controls. The image processing settings for the klara fish were adjusted to make the background fluorescence visible in order to create comparable image pairs. While a high number of GFP-positive cells was present in the dorsal fin area of klara;cdkn1aki/ki fish at the age of 17 dph (M), only one cell (marked by the white arrowhead) was detected in the dorsal developing fin of the younger klara;cdkn1aki/ki animal (K).
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