Fertilization and manipulation of Xenopus laevis embryos and microinjection of mRNA or morpholino oligo (MO) were carried out as described previously [10 (link)]. Embryos were staged according to the criteria of Nieuwkoop and Faber [12 ]. Antisense MOs for Xl_nemp1 (nemp1MOs) [10 (link)] or Xl_ran (ranMO) were obtained from Gene Tools LLC. ranMO is complementary to the sequence encompassing the translation start sites of both homoeologs of ran (Xl_ran-a: NM_001086713 and Xl_ran-b: NM_001135075) (5’-CTTGAGGTTCTCCTTGGGCTGCCAT-3’). Standard control MO (stdMO; Gene Tools LLC) was used as negative control. MOs were dissolved in water and heated at 65 °C for 10 min before use. mRNAs or MOs were injected into a dorsoanimal blastomere at the 4 cell stage, in which the injected area was fated to the anterior neural plate. FITC-dextran (50 ng/embryo) was used as a tracer.
Stdmo
Manufactured by Gene Tools
Sourced in United States
The StdMO is a laboratory equipment designed for standard molecular biology operations. It provides essential functionalities for common procedures in a molecular biology laboratory setting.
Automatically generated - may contain errors
4 protocols using stdmo
1
Xenopus Embryo Manipulation and Microinjection
2
Zebrafish lpin1 Knockdown Assay
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Antisense MO oligomers of zebrafish lpin1, a splicing-donor MO (lpin1 MO) and standard control MO (STD-MO) were obtained from Genetools, LLC (USA). The nucleotide sequences of the MOs were given in Table S1 . MOs were resuspended in 1 X Danieau solution to a concentration of 2 mM stock solution before further dilutions into the required concentrations. 0.2 pmol lpin1 MO, 1.2 pmol STD-MO, 200 pg human wt or mutant LPIN1 mRNA was injected into each embryo at the 1-cell stage, respectively, using PLI-100A Plus Pico Injector (Harvard, USA).
3
Zebrafish Nexmifa Knockdown and Rescue
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The nexmifa splice-blocking Morpholino (MO) and the standard control MO (Std MO) were synthesized by Gene Tools. The sequences are: 5′-AAAATGGTAGGAGTTATAAATGAGT-3′ and 5′-CCTCTTACCTCAGTTACAATTTATA-3, respectively. MOs were diluted to 0.3 mM in RNase-free water, injected into one-cell stage embryos, and then raised in E3 medium at 28.5°C to generate nexmifa knockdown embryos (morphants). To perform rescue experiments, we generated nexmifa mRNA, efna5b mRNA, and sema6ba mRNA in vitro. Briefly, we cloned zebrafish nexmifa, efna5b, and sema6ba separately into PCS2+ vectors. Next, we linearized plasmids, then in vitro synthesized mRNA using the mMESSAGE mMACHIN Kit (Ambion, Austin, Texas, United States) according to manufacturer’s instructions. Finally, we purified Capped mRNAs using the RNeasy Mini Kit (Qiagen, Hilden, Germany). MOs or mRNAs were injected into the yolk of one cell stage embryos using borosilicate glass capillaries (Sarasota, Florida, United States) and a PV830 pneumatic picopump (Sarasota, Florida, United States).
4
Morpholino-mediated Knockdown of Zebrafish Trdn
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For loss-of-function experiments, two antisense morpholinos (MOs, Gene Tools, Philomath, OR, USA) targeting trdn translational start site (ATG-MO) and exon 1—intron 1 junction (splice-MO) were designed to knockdown the zebrafish trdn transcript. MO sequences were trdn ATG-MO 5′-TCCATCTCTCTCATGCACTAACAGG-3′ and trdn splice-MO 5′-ATGAAGTTCACAGTACCTTCCATCT-3′. MOs were injected into 1- to 2-cell stage embryos at the concentrations of 0.3, 0.6 and 1.2 pmol/embryo in 1× Danieau buffer (pH 7.6). When co-injected, they were used at the concentration of 0.6 pmol/embryo each. Identical amounts of a standard MO (std-MO, Gene Tools), targeting human HBB (5′-CCTCTTACCTCAGTTACAATTTATA-3′), was used as a control for all injections.
For rescue experiments, human wild type or L56P mutant TRISK 32 full-length mRNA were injected (25 pg/embryo). mRNAs were in vitro transcribed by using the mMESSAGE mMACHINE T7 kit (Thermo Fisher Scientific, Waltham, MA, United States) from pcDNA3.1 +-TRISK32 WT or pcDNA3.1 +-TRISK32 L56P plasmids (Eurofins Genomics Srl, Vimodrone, Milan, Italy).
For rescue experiments, human wild type or L56P mutant TRISK 32 full-length mRNA were injected (25 pg/embryo). mRNAs were in vitro transcribed by using the mMESSAGE mMACHINE T7 kit (Thermo Fisher Scientific, Waltham, MA, United States) from pcDNA3.1 +-TRISK32 WT or pcDNA3.1 +-TRISK32 L56P plasmids (Eurofins Genomics Srl, Vimodrone, Milan, Italy).
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