Computational protocol: Histone Demethylases KDM4A and KDM4C Regulate Differentiation of Embryonic Stem Cells to Endothelial Cells

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Protocol publication

[…] Zebrafish experiments were carried out according to protocols approved by Animal Care and Use Committee (University of Illinois at Chicago). The breeding colony of zebrafish (Danio rerio, Sheer strain, Dr. H. Tomasiewicz, University of Wisconsin) was maintained at standard conditions (28.5°C [pH 7.5]) and staged according to morphology (). The transgenic line Tg(fli1:EGFP)y1 (Zebrafish International Resource Center) was sequentially outcrossed to the Sheer fish to obtain optically transparent fish harboring fli1:EGFP transgene. Homozygous Sheer fish harboring fli1:EGFP were used to obtain stronger intensity of the fluorescent signal and a better acquisition of long time-lapse series. Kdm4A and Kdm4C orthologs are well conserved in vertebrates. The sequences of zebrafish and human proteins shared 47% and 50% identity, respectively. For Kdm4a and Kdm4c knockdown, the following custom translation-blocking MOs were designed and purchased, along with standard control MOs, from Gene Tools: 5′-GGCTGACCGAGTCCTTAACACTGAC-3′ (KDMab_ZF MO), 5′-CACTTGCCCCAACACCTGCCATCCT-3′ (KDMC_ZF MO), as well as a random control oligo 25-N and a GFP-positive control. MOs reconstituted in nuclease-free water were dissolved in nuclease-free water, and their concentrations were determined with NanoDrop. Fertilized eggs were collected after timed mating of adult zebrafish and injected at one- to four-cell stage with the indicated amounts of MO into the yolk immediately below the blastomeres using a Pneumatic PicoPump (World Precision Instruments). MOs were injected using 0.1% phenol red in 0.5- to 2-nl Danieau buffer, and the volume of the injected drop was estimated with a micrometer scale. The efficacy of a translation-blocking MO was first assessed by targeting GFP in a strain expressing the GFP gene under the control of the fli1 promoter. The efficacy of MOs was assessed by RT-PCR with superscript one-step RT-PCR system (Invitrogen) and using as template total RNA from either control, Kdm4A or Kdm4A MO injected embryos. RNA was extracted with Trizol from 24- and 48-hr pooled embryos and treated with DNase. Embryos were injected with 2-, 5-, or 10-ng MOs in a 1- to 2-nl volume. Injection of control MOs (Random control oligo and GFP positive control at 10 ng) verified that injections at this concentration did not induce MO-mediated toxicity, and the same or lower MO concentrations were utilized in all experiments. For imaging zebrafish vessels, live embryos were dechorionated manually with a micro tweezers, anesthetized using 0.016% tricaine (Sigma), and mounted in 1% low melting agarose. Zebrafish embryos were visualized with stereo zoom microscope (Discovery V8, Zeiss). For fluorescent microscopy, a GFP filter was used. For visualization of vasculature formation, zebrafish embryos (24–48 hpf) were captured by AxioCAm-HR Zeiss Digital Camera. ImageJ (http://rsb.info.nih.gov/ij/) was used for image processing. Images were analyzed with Metamorph software (Molecular Devices). […]

Pipeline specifications

Software tools ImageJ, MetaMorph
Application Microscopic phenotype analysis
Organisms Danio rerio, Mus musculus