Journal: Biology Open

Article Title: Establishing an auxin-inducible GFP nanobody-based acute protein knockdown system to mimic hypomorphic mutations during early medaka embryogenesis

doi: 10.1242/bio.062081

Figure Lengend Snippet: Endogenous GFP -tagging of pmm2 does not interfere with Pmm2 function in medaka. (A) Schematic representation of the medaka pmm2 gene locus. Endogenous GFP knock-in outline at the C-terminus of the pmm2 coding sequence via CRISPR/Cas9 (scissors) and biotinylated (red stop signs) donor template [coding sequence, red boxes; untranslated region (UTR), white boxes]. Single copy integration by homology directed repair (HDR) with the right homology flank (RH) and a non-homologous end-joining (NHEJ) event of the left homology flank (LH). Transcript analysis revealed correctly spliced pmm2-GFP mRNA. (B) Representative hatchling homozygous for pmm2-GFP shows ubiquitous GFP expression and proper development. (C) Biochemical Pmm2 enzyme activity assay highlights matching levels of wild-type (wt) and GFP-tagged Pmm2. Results from three independent experiments with lysates of pooled embryos ( n =25, 34 and 34). Mean±s.d. are shown, t -test, ns, P >0.05. (D) Western blot analysis of lysates of wild-type (wt), Pmm2-GFP heterozygous (het) and Pmm2-GFP homozygous (hom) hatchlings ( n =9 pooled hatchlings per lysate for each genotype), reveal the presence of Pmm2-GFP as a stable fusion protein using Pmm2 and GFP-specific antibodies; see absence of Pmm2 band in hom hatchlings (arrowhead), Gapdh was used as loading control. Scale bars: 500 µm. *, stop-codon.

Article Snippet: 10 μg pmm2-GFP gDNA and 200 pg of GFP control plasmid were digested with 10 U of BspHI (NEB) in combination with 20 U of HindIII (NEB) or 20 U of BsaI HF (NEB) at 37°C overnight.

Techniques: Knock-In, Sequencing, CRISPR, Non-Homologous End Joining, Expressing, Enzyme Activity Assay, Western Blot, Control

Journal: Biology Open

Article Title: Establishing an auxin-inducible GFP nanobody-based acute protein knockdown system to mimic hypomorphic mutations during early medaka embryogenesis

doi: 10.1242/bio.062081

Figure Lengend Snippet: TIR1 and mAID/GFP-nanobody degron system has strong basal activity in the absence of auxin in medaka. (A) Schematic representation of an mAID and GFP-nanobody based, auxin inducible degron system planned for acute degradation of Pmm2-GFP in medaka. Upon auxin (NAA) induction, the E3 ubiquitin ligase complex SCF (Skp1, Cul1, F-box protein TIR1) dimerizes with the mAID-GFP nanobody, resulting in ubiquitination and degradation of GFP-tagged proteins by the proteasome. (B) Validation of Pmm2-GFP degradation via western blot analysis of stage 23 pmm2-GFP embryos comparing uninjected to degron-injected and induced (50 µM NAA) at 6 h post fertilization (hpf; n =35 pooled embryos per condition). Gapdh was used as loading control. (C) Time-lapse imaging of uninjected homozygous pmm2-GFP embryos and degron-injected embryos in ERM or induced with 50 µM NAA. (D) Quantification of mean GFP fluorescence following baseline correction over 18 h. Mean±s.d. are shown, one-way ANOVA test with Tukey’s post-hoc multiple comparison was performed on endpoints (grey box), adjusted P -values shown, *** P ≤0.001, **** P ≤0.0001. Uninjected homozygous pmm2-GFP ( n =7), degron-injected in ERM ( n =8) or induced with 50 µM NAA ( n =25). Scale bar: 500 µm. hpi, hours post induction.

Article Snippet: 10 μg pmm2-GFP gDNA and 200 pg of GFP control plasmid were digested with 10 U of BspHI (NEB) in combination with 20 U of HindIII (NEB) or 20 U of BsaI HF (NEB) at 37°C overnight.

Techniques: Activity Assay, Ubiquitin Proteomics, Biomarker Discovery, Western Blot, Injection, Control, Imaging, Fluorescence, Comparison

Journal: Biology Open

Article Title: Establishing an auxin-inducible GFP nanobody-based acute protein knockdown system to mimic hypomorphic mutations during early medaka embryogenesis

doi: 10.1242/bio.062081

Figure Lengend Snippet: Combination of TIR1(F74G) with mAID/GFP nanobody as degron system enables acute and inducible knockdown of Pmm2-GFP in medaka. (A) Schematic representation of the TIR1(F74G) variant combined with the mAID/GFP-nanobody (vhhGFP4) based degron ( ; ) to induce degradation of Pmm2-GFP in an auxin-analog (5-Ph-IAA) inducible manner in medaka. (B) Time-lapse imaging of uninjected pmm2-GFP , degron-injected pmm2-GFP embryos incubated in ERM or 5 µM 5-Ph-IAA at 6 h post fertilization (hpf). (C) Quantification of mean GFP fluorescence following baseline correction over 26 hpi. Mean±s.d. of triplicates shown, uninjected pmm2-gfp control ( n total=15; green), degron/mCherry injected, non-induced embryos ( n total=61; purple), induced ( n total=65; orange). (C′) Scatterplot of raw data at 26 hpi. Mean is shown as black line, one-way ANOVA test with Tukey’s post-hoc multiple comparison was performed, adjusted P -values shown, ns, P >0.05, **** P ≤0.0001. Scale bar: 500 µm. hpi, hours post induction; 5-Ph-IAA, auxin analog.

Article Snippet: 10 μg pmm2-GFP gDNA and 200 pg of GFP control plasmid were digested with 10 U of BspHI (NEB) in combination with 20 U of HindIII (NEB) or 20 U of BsaI HF (NEB) at 37°C overnight.

Techniques: Knockdown, Variant Assay, Imaging, Injection, Incubation, Fluorescence, Control, Comparison

Journal: Biology Open

Article Title: Establishing an auxin-inducible GFP nanobody-based acute protein knockdown system to mimic hypomorphic mutations during early medaka embryogenesis

doi: 10.1242/bio.062081

Figure Lengend Snippet: Recovery of Pmm2-GFP expression and enzyme activity. (A) Time-lapse imaging of uninjected pmm2-GFP and degron-injected pmm2-GFP embryos incubated in 5 µM 5-Ph-IAA (lower row) from 6 h post fertilization on. (B) Quantification of mean GFP fluorescence over 143 hpi. Mean±s.d. shown, uninjected pmm2-gfp control ( n =7), degron/mCherry injected, induced ( n =18). (B′) Scatterplot of individual mean GFP levels at 31 hpi and 69 hpi. Mean is shown as black line, t -test, ns, P >0.05, * P ≤0.05. (C) Pmm2 enzyme activity assay comparing control (injected, non-induced; grey) and degron-injected and induced (5 µM 5-Ph-AA) embryos (orange). Three independent experiments with lysates of n =25, 26 and 34 pooled embryos for 31 hpi. Two independent experiments with lysates of pooled embryos in ERM ( n =19 and 23) and in 5-Ph-IAA ( n =12 and 32) for 69 hpi. Mean±s.d. are shown, t -test, ** P ≤0.01. (D) Differential GFP levels of uninjected and injected and depletion induced specimens from B plotted and correlated with measured Pmm2-GFP enzyme activity at 31 and 69 hpi from C. Estimated window of Pmm2-GFP activity below 50% indicated (dashed grey line). Scale bars: 500 µm. hpi, hours post induction; 5-Ph-IAA, auxin analog.

Article Snippet: 10 μg pmm2-GFP gDNA and 200 pg of GFP control plasmid were digested with 10 U of BspHI (NEB) in combination with 20 U of HindIII (NEB) or 20 U of BsaI HF (NEB) at 37°C overnight.

Techniques: Expressing, Activity Assay, Imaging, Injection, Incubation, Fluorescence, Control, Enzyme Activity Assay