The morpholino (MO) antisense oligonucleotide MO-Tbx5a (5′-
GAAAGGTGTCTTCACTGTCCGCCAT-3′) was purchased from Gene Tools (Philomatch, OR, USA). Human miR-30a, mir-9 and a negative control were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Zebrafish were raised under standard conditions at 28.5 °C. Each 1-2-cell stage embryo was injected with a constant injection of 5 ng MO, 100 pg TBX5 mRNA and 100 pg miRNAs using a microinjector (Narishige, Japan). Twelve hours post-injection, the dead embryos were removed, leaving only viable embryos that were used for further analysis. Consistent with the previously published studies [18 (link), 55 (link)], all live embryos were divided into the four categories according to their heart morphologies. At 48-h post fertilization (hpf), images were acquired with an Olympus stereomicroscope microscope or Leica TCS-SP5 LSM confocal microscope. For confocal imaging analysis of zebrafish embryos, they were anesthetized with egg water/0.16 mgml−1 tricaine/1% 1- phenyl-2-thiourea (Sigma-Aldrich, St Louis, MO, USA) and embedded in 0.6% low melting agarose. Confocal imaging analysis was performed using Imaris software. Two transgenic zebrafish lines: Tg(vmhc:eGFP) and Tg(vmhc:mCherry-NTR) were used as described in previous work [55 (link)]. Whole-embryo microRNA sensor assay in zebrafish was carried out as described previously [56 (link)].
GAAAGGTGTCTTCACTGTCCGCCAT-3′) was purchased from Gene Tools (Philomatch, OR, USA). Human miR-30a, mir-9 and a negative control were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Zebrafish were raised under standard conditions at 28.5 °C. Each 1-2-cell stage embryo was injected with a constant injection of 5 ng MO, 100 pg TBX5 mRNA and 100 pg miRNAs using a microinjector (Narishige, Japan). Twelve hours post-injection, the dead embryos were removed, leaving only viable embryos that were used for further analysis. Consistent with the previously published studies [18 (link), 55 (link)], all live embryos were divided into the four categories according to their heart morphologies. At 48-h post fertilization (hpf), images were acquired with an Olympus stereomicroscope microscope or Leica TCS-SP5 LSM confocal microscope. For confocal imaging analysis of zebrafish embryos, they were anesthetized with egg water/0.16 mgml−1 tricaine/1% 1- phenyl-2-thiourea (Sigma-Aldrich, St Louis, MO, USA) and embedded in 0.6% low melting agarose. Confocal imaging analysis was performed using Imaris software. Two transgenic zebrafish lines: Tg(vmhc:eGFP) and Tg(vmhc:mCherry-NTR) were used as described in previous work [55 (link)]. Whole-embryo microRNA sensor assay in zebrafish was carried out as described previously [56 (link)].
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