RNAi in Zebrafish - vector for zebrafish knock-down (Mar/26/2009 )

Hi,
I am looking for a shRNA vector which can be used in zebrafish. We want to inject the vectors into the zygote to induce a silencing effect in all cells of the organism lasting until adult. Does anyone know such a vector?

We do not want to use Morpholinos as we would like to see effects in the adult fish.

Has anyone used the gene gun as delivery method in zebrafish zygote.

Thanks a lot,
Frank

Hi Frank,

A Vivo-Morpholino can be administered to a grown fish. While a Morpholino cannot be expressed in a fish as an siRNA can, you can still use Morpholinos for later stage knockdowns as long as a delivery moiety is attached to the oligo so that it can enter cells from the blood.

Best wishes,

- Jon

Hi Jon,

thanks. We think this might be problematic, as our gene of interest is exclusively expressed in the brain. Do the vivo-morpholinos cross the blood-brain-barrier? Do you experience with that? How much would you inject and how would you apply the vivo-morpholino? Just by injection of a defined amount into the abdomen?

Thanks and best wishes,
Frank

Jon Moulton on Mar 30 2009, 03:49 PM said:

Frank Gerlach on Apr 1 2009, 08:34 AM said:

You're right Frank, that is a tough problem. We know from mouse studies that the Vivo-Morpholinos don't appreciably cross the blood-brain barrier after intravenous injection. In mice, this will likely be solved by intracerebroventricular infusion, but I've never heard of cerebroventricular cannulation of a zebrafish. Could oligo be injected into the brain? Folks have been discussing that approach with us for mouse studies. I think that would be quite a challenge in a zebrafish! So far, no work has been published describing brain knockdowns with Vivo-Morpholinos. I've not addressed your dose questions because clearly there is a bigger roadblock, but here's a start -- in general we look for about 12 mg/kg dose, perhaps this should be scaled to the organ mass for brain injection. Following are a few descriptions of bare Morpholinos administered to rodent brains, but you'd have some experimental hurdles to cross before getting a Morpholino knockdown in Danio. Sorry, I wish the news was better.

Mice and cerebroventricular administration:

Kurokawa M. Effects of nestin-antisense treatment in mice on memory-related performance. Int J Neurosci. 2008 May;118(5):693-704.

Oh-I S, Shimizu H, Satoh T, Okada S, Adachi S, Inoue K, Eguchi H, Yamamoto M, Imaki T, Hashimoto K, Tsuchiya T, Monden T, Horiguchi K, Yamada M, Mori M. Identification of nesfatin-1 as a satiety molecule in the hypothalamus. Nature. 2006 Oct 12;443(7112):709-12. Epub 2006 Oct 1.

Oh-I S, Shimizu H, Sato T, Uehara Y, Okada S, Mori M. Molecular mechanisms associated with leptin resistance: n-3 polyunsaturated fatty acids induce alterations in the tight junction of the brain. Cell Metab. 2005 May;1(5):331-41.

Oops, I forgot the recent one!

Mice and cerebroventricular administration:

Sekiguchi M, Zushida K, Yoshida M, Maekawa M, Kamichi S, Yoshida M, Sahara Y, Yuasa S, Takeda S, Wada K. A deficit of brain dystrophin impairs specific amygdala GABAergic transmission and enhances defensive behaviour in mice. Brain. 2009 Jan;132(Pt 1):124-35. Epub 2008 Oct 16.