Morpholino oligonucleotides (MOs) from Gene Tools (USA) were used (Supplementary Table 6 ). Specificity of KD by MO was confirmed in a slightly different manner in medaka and zebrafish. Since rescue of the phenotype by mRNA injection did not work effectively in zebrafish, three different types of MOs, translation blocking (TB), splicing blocking (SB) and 5’UTR MOs, were used and all were confirmed to induce a similar phenotype. In medaka, TB and SB MOs were used, and the phenotype was rescued by co-injecting corresponding mRNAs. To determine efficiency of KD, semi-quantitative RT-PCR was carried out using primers that distinguish defective splicing from normal forms of mRNA (Supplementary Table 5 ).
Morpholino oligonucleotides mos
Manufactured by Gene Tools
Sourced in United States
Morpholino oligonucleotides (MOs) are synthetic oligonucleotides that are used to modulate gene expression by binding to and blocking the translation or splicing of target mRNA. MOs are designed to be complementary to specific sequences within the mRNA, preventing the ribosome from accessing the translation start site or disrupting the normal splicing process.
Automatically generated - may contain errors
Lab products found in correlation
A1301, by Thermo Fisher Scientific (2 mentions)
Mmessage mmachine sp6 kit, by Thermo Fisher Scientific (2 mentions)
Mmessage mmachine sp6 transcription kit, by Thermo Fisher Scientific (1 mentions)
Control rna, by GenePharma (1 mentions)
Iodoacetic acid, by Merck Group (1 mentions)
Dansyl chloride, by Merck Group (1 mentions)
Methanol, by Thermo Fisher Scientific (1 mentions)
γ glutamyl glutamate, by Merck Group (1 mentions)
Glutathione (gsh), by Merck Group (1 mentions)
Perchloric acid, by Merck Group (1 mentions)
Mmessage machine, by Thermo Fisher Scientific (1 mentions)
Mmessage mmachine, by Thermo Fisher Scientific (1 mentions)
Pgem t easy, by Thermo Fisher Scientific (1 mentions)
Sp6 polymerase mmessage mmachine, by Thermo Fisher Scientific (1 mentions)
23 protocols using morpholino oligonucleotides mos
1
Morpholino Knockdown Validation Strategies
2
Zebrafish Embryo Manipulation and Imaging
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Embryos were injected at the one-cell stage with morpholino oligonucleotides (MOs; GeneTools) or 50 ng plasmid with 100ng tol2 mRNA. Ephrinb2a (efnb2a) translation blocking MO (5′- CGGTCAAATTCCGTTTCGCGGGA-3′)39 (link); Silent heart morpholino tnnt2a (5′-CATGTTTGCTCTGATCTGACACGCA-3′)40 (link). Scrambled MO was injected at similar concentrations as the targeted MO (5′-CCTCTTACCTCAGTTACAATTTATA-3′). Capped tol2 mRNA was synthesized from linearized pCS2+ constructs using the mMessage mMachine SP6 kit (Ambion, AM1340). Injected and un-injected control embryos were heat-shocked at 38 °C for 30 minutes. Embryos were treated with 50 μM DMH1 (#16679, Cayman Chemical), 40 μM DAPT (#13197, Cayman Chemical) dissolved in DMSO (1000X stock solution) and control embryos were treated with DMSO alone. Blood flow was inhibited by treating the embryos with 0.48 mg/ml (3x) Ethyl 3-aminobenzoate methanesulfonate (MS222; sigma E10521). Live embryos were soaked in 10 μg/ml (Thermofisher A1301) dissolved in E3 medium for 30 minutes and extensively washed with E3 medium afterwards and subsequently imaged.
3
Zebrafish Embryo Manipulation and Imaging
4
Zebrafish Aquaporin Knockdown using Morpholinos
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Morpholino oligonucleotides (MOs) were obtained from Gene Tools (Philomath, OR, USA). The sequence of MOs against aqp1a.1 was 5’-AAGCCTTGC TCT TCA GCT CGT TCA T-3’ which was shown to effectively suppress the expression of aqp1a.1 in zebrafish [16 (link)]. A standard control MO (5’-ATCCATCTTGTGTGTTAGAAAACTG-3’ ) was also used as a control. The control MO provided by Gene Tools had no target and no significant biological activity. An MO solution was prepared with sterile water and contained 0.1% phenol red as a visualizing indicator. The MO was microinjected into embryos at the 1~4-cell stage with an IM-300 microinjector system (Narishige Scientific Instrument Laboratory, Tokyo, Japan). In preliminary tests, embryos injected with 4 ng of the control MO showed no significant differences in survival rates, morphology, or H+ gradients compared to WT embryos. Embryos injected with 4 ng of the aqp1a.1 MO had a normal morphology and survival rate.
5
Transient and Heritable Zebrafish Genetic Manipulations
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Morpholino oligonucleotides (MOs) used for microinjection were ordered from Gene Tools. The MOs were microinjected into one-cell-stage embryos as previously described (Nasevicius and Ekker, 2000 (link)). Transient tprb transgene construct within Tol2 vectors (40 pg) was microinjected into one-cell-stage embryos with Tol2 transposase mRNA (50 pg). CRISPR-Cas9–mediated generation of zebrafish mutants (tprb–3+1 and vhl) were performed as previously described (Xiao et al., 2013 (link)). The gRNAs (50 pg) and Cas9 protein (500 pg) were co-microinjected into one-cell-stage embryos.
6
Snai2 Knockdown and Overexpression
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Embryos were injected at the one-cell stage with morpholino oligonucleotides (MOs, GeneTools) and/or mRNA. Antisense MOs were used at the following concentrations: 10 ng snai2 splice-block MO (SB MO)[15 (link),44 (link)] and 0.75 ng snai2 5’UTR MO (UTR MO). Capped mRNA was synthesized from linearized pCS2+ constructs using the mMessage mMachine SP6 Transcription Kit (ambion, AM1340), according to manufacturer’s recommendations. Full length snai2 mRNA was injected into embryos at 150 ng/μl.
7
Knockdown of Mdka in Zebrafish Embryos
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Morpholino oligonucleotides (MOs; Gene Tools, LLC, Cowallis, OR, USA) were used to knockdown Mdka in embryos. Previously described and validated protocols for the ATG-targeted mdka MOs were used (see [44 (link)]). Embryos were injected at the 1-cell stage with 3ng of Mdka MO (5’-CCGCATTTTGTTTTCTGTGTCGAAA-3’) or mismatch control (Mdka MM, 5’-CCGgATTTTcTTTTCTcTcTgGAAA-3’) diluted in 1x Danieau buffer [45 (link)], and analyzed at 48 hours post fertilization (hpf).
8
Zebrafish Genetic Knockdown Techniques
9
Zebrafish miRNA-133a Modulation Assay
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Sexually mature zebrafish were reared in a constant temperature environment at 28 °C. One male and two female zebrafish were placed in separate tanks one day in advance. After 12 h, the dividers were removed to fertilize the males and females. The fertilized eggs were collected after 30 min. Morpholino oligonucleotides (MOs) were purchased from Gene Tools (Philomath, OR, USA). The MOs sequences used were as follows, miR-133a MO: 5′-TGATTTGGTTCCATTTTACCAGCTT-3′, control MO: 5′-CCTCTTACCTCAGTTACAATTTATA-3′. MOs were stored in RNase-free water diluted to a concentration of 1 mmol/L and diluted to the appropriate concentration before use22 (link). MOs were injected into the yolk of zebrafish at the single-cell stage using a glass microinjection needle (0.07 mm inner diameter at the tip). The injection volume was 1.5–15 nL. The spontaneous tail curl rate of zebrafish embryos was measured at 24 h post-injection, and embryo death and hatching were observed and recorded every 24 h. At 120 hpf, zebrafish brains were collected. 10–15 zebrafish brains were combined to form an independent sample.
10
Zebrafish Embryo Microinjection with MOs
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Morpholino oligonucleotides (MOs) were purchased from Gene Tools (Philomath, OR). Lyophilized MOs were dissolved in nuclease-free water. Zebrafish embryos were microinjected at the one-cell stage with 8 ng miR-132 MO, 8 ng miR-132 loop MO, 0.5 ng Cdh5 MO, 0.5 - 1 ng eef2k MO, 4 ng nSMase2 MO, or equivalent control MO. In miR-132 rescue experiments, 8 ng miR-132 MO was co-injected with 20 nM miR-132 RNA or control RNA (Genepharma). The sequences of MOs or RNAs used are as follows.
miR-132 MO: 5′-AGCGACCATGGCTGTAGACTGTTAC-3′
miR-132 loop MO: 5′-GGCTGTAGACTGTTACCAAAAATTC-3′
Cdh5 MO: 5′-TACAAGACCGTCTACCTTTCCAATC-3′
nSMase2 MO: 5′-CCACCTGCACCTGCACAAAACAACA-3′
eef2k MO: 5′-AGCTCCTCTTCAGCCATGATGCCCC-3′
control MO: 5′-CCTCTTACCTCAGTTACAATTTATA-3′
miR-132 RNA: 5′-UAACAGUCUACAGCCAUGGUCG-3′
control RNA: 5′-UUGUACUACACAAAAGUACUG-3′
miR-132 MO: 5′-AGCGACCATGGCTGTAGACTGTTAC-3′
miR-132 loop MO: 5′-GGCTGTAGACTGTTACCAAAAATTC-3′
Cdh5 MO: 5′-TACAAGACCGTCTACCTTTCCAATC-3′
nSMase2 MO: 5′-CCACCTGCACCTGCACAAAACAACA-3′
eef2k MO: 5′-AGCTCCTCTTCAGCCATGATGCCCC-3′
control MO: 5′-CCTCTTACCTCAGTTACAATTTATA-3′
miR-132 RNA: 5′-UAACAGUCUACAGCCAUGGUCG-3′
control RNA: 5′-UUGUACUACACAAAAGUACUG-3′
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