In utero Intraventrikulär direktinsprutning och elektroporation… (Translated to Swedish)

Cite this Article: In utero Intraventrikulär direktinsprutning och elektroporation av E15 musembryon

Walantus, W., Castaneda, D., Elias, L., Kriegstein, A. In Utero Intraventricular Injection and Electroporation of E15 Mouse Embryos. J. Vis. Exp. (6), e239, doi:10.3791/239 (2007).

Abstract: In utero Intraventrikulär direktinsprutning och elektroporation av E15 musembryon

In utero in vivo injektion och elektroporation av embryonala musen hjärnbarken ger ett kraftfullt verktyg för manipulering av enskilda stamceller kantar väggarna i den laterala ventrikeln. Denna teknik är nu allmänt används för att studera processer som corticogenesis av över-uttrycka sig eller knackar ner gener och observera effekter på cellulär proliferation, migration och differentiering. I jämförelse med traditionella knockout strategier, ger in utero elektroporering ett snabbt sätt att manipulera en befolkning av celler under en viss tidsmässig fönster. I den här videon protokollet redogör vi för den experimentella metoden för att förbereda möss för operation, utsätta livmodern hornen genom laporatomy, injicera DNA i den laterala ventriklarna av det utvecklande embryot, electroporating DNA i stamceller som kantar laterala väggen, och ta hand om djur efter operationen . Vårt laboratorium använder detta protokoll för operationer på E13-E16 möss, är det dock oftast utförs på E15, vilket visas i denna video.

Disclosures: In utero Intraventrikulär direktinsprutning och elektroporation av E15 musembryon

Ask the Author: In utero Intraventrikulär direktinsprutning och elektroporation av E15 musembryon

16 Comments

Great work – nice demo.

A few additional pieces of information might make it even more useful to researchers.Specifically the character of the pulse applied (voltage, pulse duration, wave form, etc.), the time at which the results have been analyzed (time pre-, postnatal?), and the typical transfection results seen with respect to both embryos (20-50-100% of electroporated embryos express target in neurons/glia?), and the normal levels of transfection observed (10’s, 100’s of neurons transfected, 90% of transfected cell macroglia, etc.). Such information would significantly help characterize and compare the nature of the results obtained among groups, assisting to determine optimal protocols.

Posted by: AnonymousApril 10, 2008, 10:17 AM

It is a very good demonstration. I still have some questions:
(1) you showed electroporation of one embryo. Can you electroporate all 8-10 embryos from the same female? Do you see that the multiple electroporations in one female will affect the efficency? Is it possible to electroporate different DNA into different embryos of the same female?
(2) What is your success rate?
(3) Do pups born normally? How long have you seen the pups survive?
(4) Napagene (Japan) is selling an electroporator CUY21 that seems more popular now than BTX. Have you seen any difference?

Thank you

Posted by: Xinyu ZhaoJuly 26, 2009, 12:11 PM

Dear Xinyu,

In my experience, you can electroporate all of the embryos without incident, however many researchers report that its best to avoid the embryos most proximal to the uterus and believe this practice improves survival.
We routinely electroporate multiple experimental variables or controls within the same dam.
There are many factors that may effect embryo survival and electroporation efficacy and it will take some practice to become proficient with this procedure.
The most critical points in my opinion are to handle the uterus as gently as possible and to ensure that it remains moist while out of the peritoneum. Also, the injection pipette should be sharp enough to pass through the uterus and into the ventricle without resistance. If your needle passes through the uterus with a “pop”, it’s not sharp enough. We expect a reabsorption rate of 10- 20% over the course of a year, with rats generally faring better than mice.
Pups should be born normally and survive well. I have no experience with the CUY21.

-William Walantus

2.1

Posted by: AnonymousJuly 31, 2009, 2:20 AM

In your experience, which strain of mice is the best for maximal pup survival and maturation.

Posted by: Varda lev-RamAugust 28, 2009, 5:20 PM

We use Swiss Webster exclusively. Inbred strains like C57BL6 with smaller liter sizes tend to be more problematic. -William

3.1

Posted by: AnonymousOctober 23, 2009, 8:17 PM

How much volts and ms do you use to electroporate a mouse in utero? You mentionned it for the rat but I haven't seen it for mice.

Thank you!

Posted by: Julie CardinOctober 22, 2009, 3:31 PM

Depending on the age of the embryos 5 pulses, 40-50volts for 50ms, with 1 sec interval. -William

4.1

Posted by: AnonymousOctober 23, 2009, 8:11 PM

Dear William Walantus:
where can I download this video?
Thanks!

Posted by: yang k.February 3, 2010, 10:13 AM

You should be able to see the video for this article at www.jove.com/index/Details.stp?ID=239. However, if you have trouble viewing it or would like to download it, please contact us at support@jove.com.

5.1

Posted by: AnonymousFebruary 3, 2010, 12:00 PM

This demo was very helpful thank you. I have been using your method with SW mice and a pCAG plasmid for over a year with an ~80% electroporation efficiency. Recently efficiency dropped off dramatically over several experiments to 0%. I purchased new platinum BTX electrodes and efficiency returned to normal for 2 experiments before dropping back down to 0% again over several sessions. The paddles produce bubbles when pulsed and the BTX electroporator still produces a square pulse on an oscilloscope. Have you experienced a rapid reduction in efficiency like this? I suspect the paddles, but I might be missing something.
Thank You

Posted by: Ben BartelleMay 23, 2010, 11:23 PM

A problem with the paddles is the most likely, however is it possible that anything has changed (vendor, prep. routine, base stock, etc) with respect your plasmid prep? We have seen that minor changes in the purity or prep procedures can substantially impact tranfection efficiency. Also is the pulse routine as given above (5 pulses, 40-50volts for 50ms, with 1 sec interpulse interval)?

6.1

Posted by: JeffMay 25, 2010, 10:37 AM

I have problem while injecting DNA to the lateral ventricle. I could see the DNA spread other hemisphere and also in midline... why it happens?

Posted by: NishaMay 13, 2011, 2:38 AM

Nisha, what age embryos are you electroporating? If they are E12 or younger you may want to inject less than the 1ul indicated in this protocol. We routinely see dye diffuse into both lateral entricles, but for our experiments this is not usually a problem. -William Walantus

Posted by: William May 13, 2011, 1:27 PM

Hi Thanks for your comments. we always use E15 age embryos for injecting.and we usually inject 1uL or more.
-Nisha

Posted by: NishaMay 13, 2011, 9:43 PM

Thanks for uploading this demonstration. It is a fantastic job. I am going to present this paper in my University. I am doing my MSc in Neuroscience from university of Calcutta,India.

Posted by: Prosenijt PalMay 25, 2011, 5:54 PM

I would like to know the preferable size of the electrode using mice hippocampus electroporation. we tried 3mm diameter electrode CUY21 (33v,5pulses). but no good expression, and we also tried CUY21 with diameter 7mm , but survival of pups are very less. Thanks

Posted by: NishaMay 30, 2011, 1:44 AM

Have you ever tried injecting E12 embryos. If so what parameters you used for elctroporation?

Posted by: NishaJune 23, 2011, 12:18 AM

Hi Nisha,
Dr. Nicholas Gaiano's group has demonstrated this at E9.5 in the mouse forebrain. You may access this article here: http://www.jove.com/details.php?id=2047. - JoVE Editorial Staff

12.1

Posted by: Nandita S.June 23, 2011, 10:05 AM

what is the concentration of the antimycotic?

Posted by: alexOctober 12, 2011, 4:59 AM

-Nisha, we routinely electroporate E12 Mouse embryos. The embryos are a bit more difficult to visualize but otherwise unremarkable.
-Alex, we no longer use AA for rodent surgeries. If you've seen complications with postoperative infection in the paritoneum or at the suture site be sure that your surgical tools have been autoclaved one day before surgery, use a bead sterilizer between surgeries, use surgical (sterile) gloves, clean the skin as shown before incision and be very careful that feces, bedding, fur etc. does not come in contact with the uterus or enter the paritoneum during surgery. -William Walantus

Posted by: William WalantusOctober 12, 2011, 2:00 PM

Hi William,
Thank you for this great movie. I have watched it many times and it is still useful for me. I am trying to inject E15 embryos with a virus for gene therapy and I am using your same technique for in utero surgery but I am still having some problems with embryo survival (Mouse strain Black 6). Do you have any advice?
And another question, if I may, I was wondering if you use Henry Schein size 6 absorbent sutures or silk sutures?
Thank you in advance,
-Reina

Posted by: ReinaOctober 24, 2011, 6:36 PM

Hi William,

Nice demonstration! I am wondering where can I purchase a rotating grinding stone to sharpen the glass pipette.

Thank you so much!

Zhiguang

Posted by: zhiguang g.April 17, 2013, 11:42 AM

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Date Published	7/19/2007
JoVE Section	General
JoVE Issue	6
Comments	16 Comments
View Count	Loading... (Details…)
DOI	doi:10.3791/239

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In utero Intraventrikulär direktinsprutning och elektroporation av E15 musembryon

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