24/Sep/2010:
I scaned through the paper,
JCB
In this paper PI3Kdelta is required for membrane fission of TGN.
In my impression, exactly PI3K delta plays important roles in fission of membrane of TGN. So what? There are a lot of PI3K superfamily. Even though they discover new function of PI3Kdelta, is it new?
I don't think so.
However they do RNAi and rescue experiments.
Pretty reliable.
By the way, this paper's author is Tomohiko Taguchi sensei.
I met him and e-mailed several time.
トランスジェニックマウス。
ちなみに、トランスジェニックマウスという名称は幅広く使われる。
河野さんがやっているような、GCAMPを導入するのは、
普通のトランスジェニックマウスは、どこに目的のDNAが入っているかはわからない。
ねずみが50匹くらい生まれる。
そこから、ねずみの尻尾を切って、本当にきちんとできているかを確認しないといけない。
ノックインマウスは、作製するのにとても時間がかかる。
最低で３５万くらい。
RRCがやってくれて、うちらが、ES細胞にきちんと入っているかを確認する。
マウスと人でのデータが一致することは、結構少ないこともある。
ある人によっては、目的のタンパク質をノックアウトしたが、予期したフェノタイプじゃないものが発現されて、結局、何がおきているかわからないために、論文をかかなかったということもあるようだ。
暫く使わないときには、受精卵か精子で保存できる。
18 and 19/Aug/2010:
What I think about PIP3 FLIM probe improvement.
At first, It seems that PIP3 exists in the stedy state in spine in neuron judging from Jose's paper.
However my probe is not able to detect this PIP3 using LY29402 in part because my probe attached to PM and strong binding to PIP3 and LY cannot affect the probe binding to PIP3.
So I thought that point mutation of PH domain is gonna be good for me.
I gonna take same strategy with Dr. Yasuda, Ras FLIM probe.
I refered Lambright's paper in which they describe how much the binding ability of PH domain reduced by the keypoint mutation.
I focus on 2 mutation points.
One is 279. 279 is important to bind PIP3 but not directly binding.
So I can use this mutation.
And then I looked 280. 280 is important for binding to 1 position phosphate. Using this, I can reduce binding ability of PH domain to PIP3.
278 279 280
GTG(V)AAG(K)ACC(T)
I want to insert mutation as follows.
1. PIP3FLIM probe (K279A)
GTG(V)GCG(A)ACC(T)
2. PIP3FLIM probe (T280A)
GTG(V)AAG(K)GCC(A)
3. PIP3FLIM probe (K279A,T280A)
GTG(V)GCG(A)GCC(A)
279 is the point mutation which is not related to binding to PIP3,
but this make binding ability weak than normal PH domain.
280 is the point mutation which is related to 1 position of the inositol ring. So I expect that this mutation keeps binding specificity of PIP3 and PIP3, however make binding ability weak.
After that, I am gonna do the protein purification of PH domain with His tag.
21/July/2010:
Today is the talk of Malinow.
When checking his face in some photos, he looks older person.
But real Malinow I actually saw looks very cool.
He looks younger than Yasunori.
I do not know which is PI.
He introduced the paper which is published by Makino-san, who is tha postdoc in Malinow lab.
The result was something I wanted to do.
That is the something to found out the input pattern of spines in single neuron. I wanted to do with Camui probe. But he carried out it with SER-AMPAR. I did not know it is possible, because SEP-AMPAR has a very week fluorescence.
Whatever the case, the time flis quickly.
I need to do a lot as fast as possible....
20/July/2010:
Mandai-san talk,
He belongs to David D. Ginty lab, Johns hopkins university? and works on dorsal root ganglion (DRG).
technical term:
I am gonna make long story short.
phenocopy
afferent
you can appreciate---
15/July/2010:
Dan Ohtan Wang talk,
She was posdoc of Kelsey Martin in UCLA.
Her talk is something she published in science.
Science
Now she belongs to Akimitsu Okamoto Lab.
Martin is pupil of Eric Kandel, so She also studies aplysia.
keyword They used mRNA of sensorin and Dendra2, which is photoconvertible fluorescence. Dendra2 was flanked between mRNA.They applied serotonin to the postsynapses and observe the translocation of mRNA to the presynapses matched with postsyanpse.
最終更新：2010年09月24日 21:42
