Xuecai Ge —BIO-PROTOCOL

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Andrea Puhar The Laboratory for Molecular Infection Medicine Sweden (MIMS); Umeå Centre for Microbial Research (UCMR); Department of Molecular Biology, Umea University, Sweden
44 protocols

Antoine de Morree Stanford university
33 protocols

Emmanuelle Berret Ecole Polytechnique Fédérale de Lausanne
9 protocols

Hong-guang Xia Zhejiang University
7 protocols

Reviewer

Xuecai Ge

University of California, Merced

Research focus

Neuroscience

2 Author merit
Reviewer merit
Editor merit

Education

Ph.D. in Neurobiology, Harvard University, 2009

Current position

She is a postdoc fellow in the Department of Developmental Biology at Stanford. Her research focuses on Hedgehog signaling in embryonic development and the associated diseases.

Publications

Ge, X., Milenkovic, L., Suyama, K., Hartl, T., Purzner, T., Winans, A., Meyer, T. and Scott, M. P. (2015). Phosphodiesterase 4D acts downstream of Neuropilin to control Hedgehog signal transduction and the growth of medulloblastoma. Elife 4: e07068.
Frank, C. L., Ge, X., Xie, Z., Zhou, Y. and Tsai, L.-H. (2010). Control of activating transcription factor 4 (ATF4) persistence by multisite phosphorylation impacts cell cycle progression and neurogenesis. J Biol Chem 285(43): 33324-33337.
de Anda, F. C., Meletis, K., Ge, X., Rei, D. and Tsai, L.-H. (2010). Centrosome motility is essential for initial axon formation in the neocortex. J Neurosci 30(31): 10391-10406.
Singh, K. K., Ge, X., Mao, Y., Drane, L., Meletis, K., Samuels, B. A. and Tsai, L.-H. (2010). Dixdc1 is a critical regulator of DISC1 and embryonic cortical development. Neuron 67(1): 33-48.
Marin, O., Valiente, M., Ge, X. and Tsai, L. H. (2010). Guiding neuronal cell migrations. Cold Spring Harb Perspect Biol 2(2): a001834.
Ge, X., Frank, C. L., Calderon de Anda, F. and Tsai, L.-H. (2010). Hook3 interacts with PCM1 to regulate pericentriolar material assembly and the timing of neurogenesis. Neuron 65(2): 191-203.
Mao, Y., Ge, X., Frank, C. L., Madison, J. M., Koehler, A. N., Doud, M. K., Tassa, C., Berry, E. M., Soda, T., Singh, K. K., Biechele, T., Petryshen, T. L., Moon, R. T., Haggarty, S. J. and Tsai, L. H. (2009). Disrupted in schizophrenia 1 regulates neuronal progenitor proliferation via modulation of GSK3beta/beta-catenin signaling. Cell 136(6): 1017-1031.
Rawson, J. M., Dimitroff, B., Johnson, K. G., Rawson, J. M., Ge, X., Van Vactor, D. and Selleck, S. B. (2005). The heparan sulfate proteoglycans dally-like and syndecan have distinct functions in axon guidance and visual-system assembly in drosophila. Curr Biol 15(9): 833-838.
Ge, X., Hannan, F., Xie, Z., Feng, C., Tully, T., Zhou, H., Xie, Z. and Zhong, Y. (2004). Notch signaling in Drosophila long-term memory formation. Proc Natl Acad Sci U S A 101(27): 10172-10176.

2 Protocols published

In utero Electroporation of Mouse Embryo Brains

Author: Xuecai Ge, date: 07/20/2012, view: 20992, Q&A: 1

This is a non-invasive technique to introduce transgenes into developing brains. In this technique, DNA is injected into the lateral ventricle of the embryonic brains, and is incorporated into the cells through electroporation. Embryos then continue ...

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Cardin Julie

IRCM

Hi,
1) Is there a relation between the efficiency for the plasmid gene expression with the voltage?
At high voltage I get more death, obviously.

2) Is there a relation beteween the efficiency for the plasmid gene expression with the concentration of the DNA plasmid injected?

Thank you very much,
Best regards
Julie Cardin
Research Assistant

1/16/2014 3:09:23 PM Reply

Xuecai Ge

University of California, Merced

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

2014-05-05 22:33:15

Cardin Julie

IRCM

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!

2014-04-30 12:41:13

Cardin Julie

IRCM

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

2014-04-30 12:36:27

Xuecai Ge

University of California, Merced

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.

2014-04-29 19:55:12

Cardin Julie

IRCM

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

2014-04-29 14:34:11

Xuecai Ge

University of California, Merced

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

2014-04-28 19:13:21

Cardin Julie

IRCM

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

2014-04-28 14:55:07

Xuecai Ge

University of California, Merced

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

2014-03-17 13:51:04

Cardin Julie

IRCM

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.

2014-03-04 13:31:22

Cardin Julie

IRCM

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

2014-03-04 13:28:58

Xuecai Ge

University of California, Merced

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

2014-01-17 14:27:48

Immunofluorescence Staining on Mouse Embryonic Brain Sections

Author: Xuecai Ge, date: 06/05/2012, view: 18538, Q&A: 0

This protocol comprises the entire process of immunofluorescence staining on mouse embryonic brains, starting from tissue preparation to mounting of the tissue sections.

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6 Protocols reviewed

Preparation of Cerebellum Granule Neurons from Mouse or Rat Pups and Evaluation of Clostridial Neurotoxin Activity and Their Inhibitors by Western Blot and Immunohistochemistry

Authors: Domenico Azarnia Tehran and Marco Pirazzini, date: 07/05/2018, view: 4425, Q&A: 0

Cerebellar Granule Neurons (CGN) from post-natal rodents have been widely used as a model to study neuronal development, physiology and pathology. CGN cultured in vitro maintain the same features displayed in vivo by mature ...

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Analysis of Myosin II Minifilament Orientation at Epithelial Zonula Adherens

Authors: Magdalene Michael, Xuan Liang and Guillermo A. Gomez, date: 12/05/2016, view: 5207, Q&A: 0

Non-muscle myosin II (NMII) form bipolar filaments, which bind F-actin to exert cellular contractility during physiological processes (Vicente-Manzanares et al., 2009). Using a combinatorial approach to fluorescently label both N- and ...

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39 Protocols edited

Preparation and Immunofluorescence Staining of the Trachea in Drosophila Larvae and Pupae

Author: Feng Chen, date: 05/05/2016, view: 12804, Q&A: 0

The Drosophila melanogaster trachea is a branched network of rigid chitin-lined tubes that ramify throughout the body and functions as the fly’s respiratory organ. Small openings at the ends of the tracheal tubes allow gas ...

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Isolation and Primary Cell Culture of Mouse Dorsal Root Ganglion Neurons

Authors: Theresa Heinrich, Christian A. Hübner and Ingo Kurth, date: 04/05/2016, view: 18657, Q&A: 0

We here provide a detailed protocol for the isolation and culture of primary mouse sensory neurons. The cell bodies of sensory afferent pseudounipolar neurons are located in dorsal root ganglia (DRGs) along the vertebral column. Dissected mouse DRGs ...

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