[Zbrafish] Re: Zbrafish Digest, Vol 30, Issue 4

Wilson Clements via zbrafish%40net.bio.net (by wclements from ucsd.edu)
Fri Nov 9 13:18:05 EST 2007


Dear Tony and Wei,

There is a link to an excellent protocol with good detail (from the  
Mullins lab) at the following website.  This protocol might help you  
troubleshoot your injections.

http://www.courses.mbl.edu/zebrafish/faculty/mullins/

Personally, I don't dilute my mRNA into 0.1M KCl, but rather just  
inject it in straight DEPC water.  Also, I use E3 media (from the  
zebrafish book, Westerfield et al.) to recover the injected embryos  
in.   I have never tried raising embryos in Danieau's buffer, but it  
seems pretty high salt:  could it be causing them to exogastrulate?

Injection trauma is also hard on the embryos.  How are you pulling  
your needles?  Are they very small bore?  What volume are you  
injecting?  A 1nl drop should be maybe a 1/5 or 1/6 the volume of the  
single blastomere at the 1-cell stage.  Large needles and large  
injection volumes will definitely kill the embryos (not to mention  
deliver way too much nucleic acid).

Have you considered that it might not be an injection problem, but a  
scope problem?  Are you using the right fluorescence filters?  Is the  
lamp working?  Do you have a positive control?

Best,
Wilson
------------------------------------------------------------------------ 
--------------------
Wilson Clements, Ph.D.

wclements from ucsd.edu

Dept. of Biology
Section of Cell and Developmental Biology
University of California at San Diego
9500 Gilman Dr.
Natural Sciences Building 6105
La Jolla, CA 92093-0380

TEL    (858) 534-6955
LAB    (858) 822-4658
FAX    (858) 822-5740


On Nov 9, 2007, at 9:04 AM, zbrafish-request from oat.bio.indiana.edu wrote:

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>    1. Help Help with microinjecting RNA (wei xia)
>    2. Help with microinjected mRNA (wei xia)
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> ----------------------------------------------------------------------
>
> Message: 1
> Date: Thu, 8 Nov 2007 13:33:30 -0500
> From: wei xia <xiaw from umbi.umd.edu>
> Subject: [Zbrafish] Help Help with microinjecting RNA
> To: zbrafish from magpie.bio.indiana.edu
> Message-ID: <4BEB7629-6442-4A04-A836-5AB1B8DB8985 from umbi.umd.edu>
> Content-Type: text/plain; charset=US-ASCII; delsp=yes; format=flowed
>
> Can you please forward my request to the author of this post? Cause I
> have the same problem that he had met and I really can't figure it
> out now.
> Thank you very much for your help.
> Wei
>
>
>
> ------------------------------
>
> Message: 2
> Date: Thu, 8 Nov 2007 13:46:50 -0500
> From: wei xia <xiaw from umbi.umd.edu>
> Subject: [Zbrafish] Help with microinjected mRNA
> To: zbrafish from magpie.bio.indiana.edu
> Message-ID: <38355AF6-DC46-4D94-BD31-6D0A0B773062 from umbi.umd.edu>
> Content-Type: text/plain; charset="us-ascii"
>
> Hi Tony,
> Sorry for the bothering to other peoples. I have the same problem you
> had before. I injected in vitro transcribed capped GFP mRNA (Ambio
> mMessage Kit) into zebrafish 2-cell-stage embryos but I got no
> expression from it. Instead when  I injected the plasmid templates
> which I used for the transcription I got strong fluorescence. I saw
> Bruce's reply and he said " 24hpf embryo should light up like a
> christmas tree." But why I couldn't!
> How could you solve your problems?
> Any suggestions are very very great appreciated!
> Wei
>
> Following is your post.
> Hi all,
>>
>> I am new to microinjection of zebrafish embryos and I need help.
>>
>> Briefly, I have injected 200pg of capped EGFP-coding RNA and the
>> embryos show no expression. In addition, their yolk kind of leaks
> out/
>> burst between 4hpf and 10hpf, thus killing the embryo.While
> uninjected
>> ones look fine.
>>
>> To help with understanding the problem, I have detailed my protocol
>> here.
>>
>> Capped RNA Synthesis
>>
>> I linearized pSP64TNE-EGFP at the Sal I site, Phenyl-Chloroform
>> extracted the linearized template and then transcribe capped RNA with
>> SP6 promoter using the Ambion mMessege Kit. The yield comes in at an
>> acceptable 21ug with A260/280 at 1.94 and a A260/230 at 2.36. Running
>> 200ng of the capped RNA in a 1% agarose gel gives me a sharp single
>> band.
>>
>> Microinjection
>>
>> I diluted the capped RNA to 50ng/ul in 0.1M KCl and 0.05% PhenolRed.
>> 4nl(200pg) was injected into each embryo, the embryos are recovered
>> into 30% Danieau's Solution, and keep at 28.5C incubator.
>>
>> Any idea what can I be doing wrong here?
>>
>> Thanks a million for reading.
>>
>> Tony
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