DNA methods

2.6.1. Cloning and construct preparation

Expression constructs

For expression in oocytes/embryos and in vitro translations, Flag-tagged Celf1, Tia1, Ptbp1 (VgRBP60) in pCS2+Flag vector were kindly provided by M. Claußen. The Celf1 open reading frame corresponds to the isoform Celf1b (NCBI accession no. Q6PF35.1) and the Tia1 open reading frame corresponds to the isoform Tia1a (NCBI accession no.

NP_001167497.1). The Dead end 1 in pCS2+Flag vector was kindly provided by K.

Koebernick.

For bacterial expression of recombinant proteins, Celf1 and Tia1 open reading frames were amplified from plasmid templates using oligonucleotides containing Nhe1 and Xho1 restriction sites (DO1-4, Table 2.3) and cloned into expression vector pET21a (Novagen).

Mutations in the Celf1 RRM domains or potential phosphorylation sites, as predicted by the Scansite motif scan program (Obenauer et al., 2003) and the NetPhos 2.0 Server (Blom et al., 1999), were created stepwise by site-directed mutagenesis using the QuikChange II Site-Directed Mutagenesis Kit (Agilent technologies) and oligonucleotides with base substitutions (DO97-102, DO114-125, Table 2.5) according to manufacturer's instructions.

The Celf1 oligomerization domain was deleted by whole plasmid amplifications of the Flag-Celf1 (MC401b) with non-overlapping 5' phosphorylated oligonucleotides flanking the deleted sequence in opposing directions (DO83/84, Table 2.5). For the Celf1 deletion constructs used for nuclear/cytoplasmic distribution studies, the GST sequence was amplified with EcoRI containing oligonucleotides (DO112/113, Table 2.3) from Flag-GST plasmid template (MC410) and ligated into EcoRI sites of Flag-Celf1 (MC401b). This Flag-GST-Celf1 plasmid (DO75) was used as template for whole plasmid amplifications with non-overlapping 5' phosphorylated oligonucleotides (Table 2.5) as described above. The N-terminal nuclear localization signal (NLS) was inserted into Flag-GST-Celf1 del119-236 by amplification of the whole plasmid with each oligonucleotide containing a fragment of the NLS (DO90/91, Table 2.5). Fragments with insertions or deletions were purified using Invisorb Fragment Cleanup kit (Invitek), re-ligated and transformed. All constructs described in this section are listed in Table 2.13.

Constructs for in vitro transcription of localization elements

For in vitro transcriptions of LEs, dnd1-LE, grip2-LE and ß-globin-3'UTR in pGEM-T easy (Promega) as well as gdf1-LE and velo1-LE in pBluescript KS+ (Stratagene) were kindly provided by M. Claußen. For detection ofdnd1-LE localization by WMISH, dnd1-LE in lacZ containing pBK-CMV was kindly provided by K. Horvay. The constructs are listed in Table 2.14.

Deletion fragments and site-directed mutagenesis of dnd1-LE

Deletion fragments of the dnd1-LE were amplified from plasmid template (MC 319) using forward oligonucleotides that contain a T7 sequence (Table 2.7). The 5'del1 fragment was cloned into pGEM-T easy and mutant versions of 5'del1 dnd1-LE were generated by site-directed mutagenesis using the QuikChange II Site-Directed Mutagenesis Kit (Agilent technologies) and oligonucleotides with base substitutions (Table 2.3) according to manufacturer's instructions. The constructs are listed in Table 2.15.

Reporter constructs for RNA stability analyses in Xenopus embryos

Wild-type and mutated dnd1-LE 5'del1 fragments were amplified with XhoI/NotI restriction sites from plasmid templates and cloned into mgfp-psp64 (provided by E. Raz, Institute of Cell Biology, Münster, Germany). Constructs are listed in Table 2.16

Control constructs for quantitative real-time PCR

Plasmid templates used as controls in quantitative real time PCR were amplified from oocyte cDNA and cloned into pGEM-T easy. The constructs are listed in Table 2.17.

Single Guide RNA (sgRNA) expression vectors

The customized sgRNA expression vectors were mainly constructed as described in Hwang et al. (2013). The pDR274 plasmid (Addgene) harboring a T7 promoter upstream of a partial guide RNA sequence was digested with BsaI. A pair of oligonucleotides containing the sgRNA target sequence and overhangs that are compatible with cloning into the BsaI-digestion sites were annealed as follows: 100µM of each oligo in a 50 µl volume containing 1x annealing buffer was heated to 95 °C and cooled (-1 °C per 30 sec) to 4 °C. 15µM of the annealed oligos were cloned into the pDR274 vector backbone. The genomic target sites in thecelf1gene and sequences of the oligonucleotides are listed in Table 2.10. The constructs are listed in Table 2.18.

Constructs for luciferase assays

The firefly luciferase ORF alone or with adjacentgdf1 translational control element (VTE) in pBK-CMV was kindly provided by M. Claußen. The firefly luciferase constructs containing

52 Materials and methods

5'del1 wild-type and mutant dnd1-LE fragments were cut out with BahmHI and NotI from DO28, DO29 and DO30 and cloned into BamHI/NotI sites of MC276. The renilla luciferase construct is described in Souopgui et al. (2008) and was kindly provided by J. Souobgui. His-MS2BP and MS2-PABP in pCS2+MT vector were kindly provided by M. Püschel. Celf1 and Tia1 were amplified from plasmid templates with oligonucleotides containing NheI and XhoI restriction sites (Table 2.3) and cloned into MS2-PCS2+MT. The firefly luciferase construct used for the MS2 tethering assay contains MS2 binding sites in its 3'UTR and was kindly provided by S. Koch. All constructs used for luciferase assays are listed in Table 2.19.

Constructs used to prepare in situ antisense probes

The lacZ dnd1-LE fragment in pGEM-T easy vector was kindly provided by M. Claußen. The zebrafish cyclin b1 and dazl fragments in pGEM-T were kindly provided by R. Dosch and the syntabulin fragment was amplified from oocyte cDNA and cloned into pGEM-T easy. These constructs are listed in Table 2.20.

2.6.2. Plasmid DNA isolation and purification

Plasmid DNA in analytical amounts (miniprep) was isolated using the illustraTM plasmidPrep MiniSpin Kit (GE Healthcare) and Plasmid DNA in preparative amounts (midiprep) was isolated using the Plasmid Midi Kit (Qiagen) according to manufacturer's instructions.

2.6.3. DNA restriction digestion

DNA was digested using restriction enzymes (Thermo Scientific) according to manufacturer's instructions

2.6.4. Agarose gel electrophoresis

DNA/RNA fragments were separated using standard agarose gel electrophoresis (Fisher and Dingman, 1971; Helling et al., 1974) in 1x TAE buffer and DNA/RNA was visualized using 0.5 µg/ml ethidium bromide (Sharp et al., 1973).

2.6.5. Polymerase chain reaction

DNA fragments were amplified by standard PCR reactions (Bartlett and Stirling, 2003) using DreamTaq polymerase (Thermo Scientific) or GoTaq polymerase (Promega) for analytical amplifications and High Fidelity PCR Enzyme Mix (Thermo Scientific) or PfuUltra HS II (Agilent Technologies) for amplifications with downstream cloning procedures according to manufacturer’s instructions.

2.6.6. DNA ligation

DNA fragments were ligated using T4 Ligase (Thermo Scientific) according to manufacturer’s instructions.

2.6.7. Transformation of bacteria

Chemical transformation of bacteria was performed according to Mandel and Higa (1970).

Cells were thawed and incubated with 100 ng of plasmid DNA or 5 µl of a ligation reaction for 30 min on ice. A heat shock was performed for 90 sec at 42 °C, and cells were incubated on ice for 2 min. Cells were then supplemented with LB medium, cultivated for 30 min at 37 °C with shaking and plated on LB-agar supplemented with antibiotics.

2.6.8. DNA sequencing

DNA sequencing was performed according to Sanger et al. (1977) using the Big DyeTMTerminator Kit (Applied Biosystems) according to manufacturer’s instructions and an ABI 3100 Automated Capillary DNA Sequencer (Applied Biosystems).

2.6.9. DNA extraction of zebrafish fin clips

DNA extraction of adult zebrafish tail fin clips was done by Proteinase K digestion. Fin clips were lysed in 100µl lysis buffer at 55 °C for 1 h. Proteinase K was inactivated at 95 °C for 10 min, and the reaction was cooled down to 4 °C and centrifuged at 3000 rpm for 5 min. The DNA solution was stored at -20 °C until use.

54 Materials and methods

2.6.10. Evaluation of mutation efficiency by T7 endonuclease 1 (T7E1) assay

The celf1 mutation efficiency in CRISPR/Cas treated zebrafish was estimated using the T7 Endonuclease I assay as described before (Reyon et al., 2012; Hwang et al., 2013). In brief, the genomic target region in celf1 (exon 4) was amplified with oligonucleotides designed to anneal approximately 200 bp up- and downstream of the expected restriction site (Table 2.11). 2 µl of gDNA from embryos or fin clips were applied in a 50 µl PCR reaction using GoTaq polymerase (Promega) according to manufacturer’s instructions. The PCR program was as followed: 2 min at 95 °C followed by 33 cycles of 30 sec at 95 °C, 30 sec at 52 °C and 30 sec at 72 °C and final 2 min at 72 °C. The PCR on fin clips was done in a two step nested PCR using the outer oligonucleotides DO181/182 (Table 2.11) in a first PCR reaction.

1 µl of a 1:3 dilution of this PCR product was used as template in a second PCR with the inner oligonucleotides DO171/172 (Table 2.11). PCR products of the second PCR were purified using the Invisorb Fragment Cleanup kit (Invitek) and eluted in 20µl of 10 mM Tris-HCl (pH 7.5). 200 ng of purified PCR product were then denatured and re-annealed in NEBuffer 2 (New England Biolabs, M0302) using a thermocycler and the following protocol:

95 °C for 5 min; 95–85 °C at −2 °C/s; 85–25 °C at −0.1 °C/s; hold at 4 °C. Hybridized PCR products were treated with 10 U of T7 Endonuclease I (New England Biolabs) at 37 °C for 1 h in a reaction volume of 20 µl and subsequently visualized on a 1 % agarose gel. Mutation efficiency was estimated by visual inspection of cleaved DNA fractions, and adult fish were divided into groups of individuals with high, intermediate, low and no detectable mutation rates.

2.6.11. Sequence confirmation of somatic celf1 gene mutations

Two PCR products of zebrafish showing high, intermediate, low or no mutation efficiency in the T7EI assay were chosen for confirmation via Sanger sequencing. PCR products corresponding to these fish were cloned into pGEM-T easy vector and transformed into XL1-blue cells. Plasmid DNA was isolated from multiple colonies of each transformation and sequenced using T7 oligonucleotides (Table 2.2).

2.6.12. Evaluation of germline mutation efficiency in female zebrafish

Female zebrafish were anesthetized in 0.02 % tricaine, and unfertilized eggs were obtained by slightly pressing the abdomen region. From each fish, 20 eggs were used for DNA extraction using the DNeasy Blood and Tissue Kit (Qiagen) according to manufacturer's instructions. To amplify the exon 4 region in celf1, approximately 200 ng of genomic DNA were used in a first 12.5µl PCR reaction using the outer oligonucleotides DO181/182 (Table 2.11), GoTaq polymerase (Promega) according to manufacturer’s instructions and the PCR program as follows: 2 min at 95 °C followed by 40 cycles of 30 sec at 95 °C, 30 sec at 50 °C and 30 sec at 72 °C and a final 2 min step at 72 °C. 1µl of a 1:3 dilution of this PCR product was used as template in a second PCR with the inner oligonucleotides DO171/172 (Table 2.11) using the PCR program described above. PCR products were purified using the Invisorb Fragment Cleanup kit (Invitek) and cloned into pGEM-T easy. From each PCR product, approximately 10 clones were sequenced.

2.6.13. Genotyping of zebrafish line sa11143

Genomic DNA was extracted from fin clips as described above. The target region in celf1 exon 10 was amplified as described for T7E1 assay using outer oligonucleotides DO186/187 (Table 2.12). The PCR product was diluted 1:3 and 1 µl was used as template in a second PCR reaction with the inner oligonucleotides DO188/184 and the forward oligonucleotide containing a T7 sequence (Table 2.12). PCR products were purified using Invisorb Fragment Cleanup kit (Invitek), eluted in 20 µl of 10 mM Tris-HCl pH 7.5 and sequenced using T7 oligonucleotides (Table 2.2). Heterozygous fish were identified by single nucleotide polymorphisms in the sequence chromatogram.