Hi, 1) Is there a relation between the efficiency for the...

Hi Julie,

Regarding the solvent for DNA: avoid TE (Tris-EDTA), because the EDTA is not good for the embryo. Use PBS because it has some salt and is conductive.

It is true that when you manipulate the late stage embryos, it will increase the survival rate. But then you have to think about which layer of neurons you want to label. Since the cortex is generated in an "inside-out" manner, Electroporating at E12 will most label deep layer neurons, and after E17 most labelled neurons will be at the superficial layer. It is not so absolute and there is usually a time window. Most people are interested in looking at Layer V neurons, and electroporating between E13-15 will be OK. But after E17, you won't label Layer V neurons, and the majority of them will be at layer II-III.

-Xuecai

When I was doing experiments in which I was electroporating at e12 and collecting embryos, around 66% of embryos survived. Now I need to keep the embryos electroporated until they are born P14. Most of them are born but many of them dies between P1 and P5.

Have you noticed a difference in survival when you want to keep animals older?

( I discussed with people who are doing chicks and they told me that they have a much higher mortality increase with age.)

Thank you!

Do you think it is better to resuspend your plasmid in TE, in water, PSB ...?

I used the DNA from endofree maxiprep as well. Never re-precipitate to purify further. I wold guess it won't make a big difference. DNA quality is important; but comparing to how gentle you treat your animal and what voltage you use, it is minor.

I read in many papers that it is important to use endotoxin free Maxi prep kit to make your DNA for mice IUE. I always do it this way. But I wonder if it required to precipitate the DNA after to purify more or if it is already good after the Endotoxinfre Maxi prep?

Thank you

Hi Julie,

I have only tried CMV and CAG. CAG works better. I have not tried other promoters. Sorry that I cannot help with this question.

Best,
Xuecai

Hi,
I always use pCAG as promotor for IUE in mice. Do you know if there is some other kind of promotor that work well in mice?
What about promotor GfalphaA,B,C , is it working well?

Thank you very much,
Julie

Hi Julie,

In my experiments, I sac the animal before the pups are born. In this situation, I usually did not electroporate the entire litter, and made a map of which pups are electroporated. This helped the survival rate a lot. I did help with some colleagues who need to have the pups born. In this case, I would electroporate the entire litter, and we had similar problems as your had. The most possible reason is that the female did not take care of the pups well after birth, due to the trauma she has had. My suggestion would be to find a foster Mom for the new born pups, i.e. give the pups to another female who just delivered the baby. This needs to be done in a timely manner (within 1-2 day after the foster Mom deliver her baby), so that the foster mother will not kill the new baby. Another suggestion is to eletroporate a lot of pregnant females. This is what I did.

I electroporated the neocortex at E15, using 35V, exactly as what this protocol describes. In my hand, higher than 45V killed the pups.

I did use some pain killer to the females, as required by the animal facility in my university. I used Buprenox (Buprenorphine). I guess other brand with similar component will work. I injected into muscles, but not belly, to avoid further irritation to the pups.

Hope this helps.

Xuecai

3) Do you use any drug after surgery to reduce inflammation and pain for the mother? I had been recommended to give a dose of Metcam. I also tried Carpofen but it seems that I got more death so I came back to Metcam.

Thanks a lot for your answers.

1) I am electroporating at 35V, 45V 60V and 80V many litters. I got some very nice results but it take me too many litters to get just one. Many embryos are born but most of them died at P2-P3 and I cannot find why? Any suggestion?

2) In which tissue do you use 35V at e15 ?

Thank you very much in advence.

Best regards,
Julie

Hi Julie,

Thanks for your questions.

1) Yes, there is a correlation between the transfection efficiency and the voltage. Higher voltage gives you higher transfection efficiency (larger areas transfected in the brain); but, as you mentioned, it also causes higher death rate. So you have to find the right balance. After many trials in my experiments, I finally got to 35V for E15, and increased 1-2V for E16, and used 40V for E17-E18. But after working with many of my colleagues, I found that the transfection efficiency and death rate can be affected slightly by how tight you hold the embryos with the electrodes. The tighter, the higher efficiency. Therefore the voltage I give here is a point to start with; you might have to adjust it based on your own experience.

2) Yes, higher DAN concentration is preferred. In my experiments, I used as high as possible. But too higher concentration makes the DNA solution viscous, and difficult to aspirate. The highest concentration I have used is 4ug/ul. Any concentration above 2ug/ul will give you satisfying results.

Hope this helps.

Xuecai