Progatzky F, Sangha NJ, Yoshida N, McBrien M, Cheung J, Shia A, Scott J, Marchesi JR, Lamb JR, Bugeon L, Dallman MJ. Dietary cholesterol directly induces acute inflammasome-dependent intestinal inflammation. Nat Commun. 2014 Dec 23;5:5864. doi: 10.1038/ncomms6864.
This paper claims delivery of Morpholino to the intestinal epithelium of zebrafish by putting Morpholinos in the fish water around feeding time.
"As the knockdown efficiency of MO injected into single-cell stage embryos diminishes after 5 days post fertilization (dpf) ... we devised a novel delivery method by simply adding the MO to the fish water 24-h before and during the 6-h feeding period."
"First, we validated this new method by imaging MO uptake in the intestine ..."
"Second, analysis by flow cytometry following treatment with the FITC-conjugated ASC splice-blocking MO revealed an increase in FITC fluorescence in Cytokeratin+ epithelial cells but not in mCherry+ macrophages in Tg(fms:mCherry), or in dsRed+ neutrophils in Tg(lyz:dsRed) larvae ..."
"Finally, we confirmed the ability of this MO to significantly knock down ASC mRNA by performing reverse-transcription PCR (RT-PCR) on injected embryos (Supplementary Fig. 10) and quantitative RT-PCR (qRT-PCR) on sorted FITC+ cells"
"...we used a GFP MO (not tagged with FITC) to target GFP in Tg(ubi:EGFP) fish35 and found significantly decreased fluorescence intensity in intestinal cells (Supplementary Fig. 11)."
Protocol, figure 1 legend:
"Fig. 1. Schematic representation of the protocol used for experiments. Zebrafish larvae at 6 days post-fertilisation (dpf) were pre-treated with indicated drugs for 30 minutes (min) followed by feeding with ZM control diet or high cholesterol diet (HCD) for 6 hours (h) in the presence of drugs (top panel). Zebrafish larvae (6 dpf) were pre-treated with indicated morpholino oligonucleotides for 24 hours and followed by feeding with ZM control diet or HCD for 6 hours in the presence of morpholinos (bottom panel). The quantification of total cell numbers of indicated immune cells in the intestine were performed at 18 hours post feeding."
Add new comment