Kyoto university medical facultys “MY COURSE PROGRAM”

Kyoto university medical faculty’s “MY COURSE PROGRAM”
Takehiro Okada 岡田武大
Kyoto Univ. Medical Faculty
Introduction
“MY COURSE PROGRAM” is a curriculum for 4th-grade students in Kyoto university
medical faculty. All students in medical faculty study some experiments and do researches
in laboratories.
I stayed in Brighton, England, for two and a half months, and studied many things in
Dr.Hochegger’s
lab
(http://www.sussex.ac.uk/lifesci/hocheggerlab/index)
of
Genome
Damage and Stability Centre in Sussex University. The Main theme of My boss in England
is “cell cycle”.
The theme has three subjects,
1) Control of centrosome separation
2) Chemical genetic analysis of mitotic kinases
3) Proteomic analysis of mitotic kinases targets and interactors
My supervisor, Nisha Peter, has researched the second subject. I did some experiments
and helped the research.
My Purposes
The biggest purpose of “my course program” is to learn experiments and methods of
medical research. The lab where I studied is a lab researching cell biology. So I could learn
basic medical experiments, such as DNA cloning, electrophoresis, tissue culture, protein
purification, western blotting, genomic DNA purification and Southern blotting. I wanted to
learn these experiments in English. So I had chosen to learn experiments in Brighton. This
stay in Europe made my English skills far better and I could know European cultures and
peoples. Getting accustomed to foreign countries is my another purpose. Surely, this
experience will help me to do master course abroad in the future.
Purposes of the lab
As I wrote in the introduction, the main purpose of the lab is understanding cell cycle.
They are investigating how mitotic kinases coordinate accurate chromosome segregation
and cell division by using microscopy and proteomic analysis in genetic and biochemical
way. Minor mistakes in the control of chromosome segregation can lead to loss of entire
chromosomes and cause genome instability and cancer. Cells undergo dramatic changes
as they enter mitosis, such as centrosome separation, nuclear envelope breakdown,
chromosome condensation, and mitotic spindle formation. Understanding the precise
molecular mechanisms of these cellular rearrangements is an important undertaking in cell
biology. These efforts are critical to help us understand how things can go wrong in
chromosome segregation in cancer cells and how we can exploit these processes to target
cancer cells.
Purposes of my supervisor
My supervisor, Nisha Peter, studies about chemical genetic analysis of mitotic kinases.
Her research is about the kinase, Greatwall. Greatwall kinase controls mitosis. But how
Greatwall is regulated is poorly understood. She has been doing many experiments to know
how Greatwall works in cell mitosis.
Experiments
I did experiments to help my supervisor, Nisha. At the beginning of the program, I
learned these experiments from Nisha and Hilary, another supervisor, then I did by myself.
DNA cloning and electrophoresis
First, I learned some easy experiments such as DNA cloning and electrophoresis. In
basic cell biology experiments, we need to get plenty amount of DNA and check whether the
DNA is correct. So we do DNA cloning and electrophoresis.
I used QIAGEN plasmid DNA cloning kit. It helps us to get DNA from E.coli easily. For
the first time, I made a mistake and I could not get DNA pellet. It was because I didn’t shake
tubes after adding isopropanol. This is only for plasmid DNA. We have another way to get
genomic DNA.
After getting plasmid DNA, we cut the DNA by restriction enzymes. Then we run the
restricted DNA into agarose gel to check that we have the correct DNA. Gel running is for
around 20 minutes.
Tissue culture
We have used DT40 cells in Genome Damage and Stability Centre. DT40 cells are
from chicken. The cell cycle is eight hours, so we can get many cells in a short time. We do
transfection on DT40 cells. Because cell cycle is short, transfection is easier. When we do
the tissue culture work, we use a special hood. It is kept sterile. DT40 cells are easily
damaged by bacteria. So We should pay attention to keep it clean.
I learned two ways of transfection. One is a chemical method and another is a
electroporation. The chemical method is to make DNA micelle in a transfection reagent and
get them through cell’s membrane. Electroporation is done by using a electroporator.
Electronic stimuli make pores on cell membrane and DNA go through the pores into the cell
centre. Transfection is a very important experiment in cell biology.
Protein purification and western blotting
In the research, we wanted to get ensa, a special protein. So we did protein purification.
We got many bacterial cells with gst-ensa protein by transformation. After overnight culture,
we started the experiment. Destroying cell membrane, we could get a solution with protein
and DNA. Then we used special beads to separate gst-ensa protein. The beads can
combine only with gst, so we could get only the protein. For the first time, I couldn’t get much
protein. I guess it was because the solution buffer was made wrongly. Next time I could
succeed in protein purification.
After getting the protein, we should check whether the protein is correct. We made a gel
and run protein and ladder into the gel to check their molecular weight.
Genomic DNA purification and Southern blotting
To know about Greatwall kinase, we made many cells with Greatwall DNA by
transfection and got genomic DNA by genomic DNA purification. The way of genomic DNA
purification is not difficult. It is similar to the protocol of plasmid DNA cloning. Genomic DNA
is more fragile than plasmid DNA. We should be careful about genomic DNA storage.
Southern blotting is the way to identify genomic DNA. We can recognize specific
sequences. After cutting DNA by restriction enzyme, we ran the DNA into gel. We get the
DNA we needed from the gel and did Southern blotting.
Result
I did many experiments and got some good data. Especially in protein purification, I
could get plenty amount of protein. I’m very proud of being able to helping my supervisor. I
made some mistakes during my stay. The reasons were making wrong solution, using
wrong solution, taking a tube for another tube, and so on. I will be careful about these
mistakes and concentrate on the research in the future.
感想
マイコースプログラムを海外ですることは少し不安もありましたが、基礎実験を英語で
学んでみたいという思いから、武田先生に紹介していただき、サセックス大学のゲノムセ
ンターのヘルフリッド・ホヘガー先生のラボで学ばせていただきました。初めのころは英
語の聞き取りもままならず、何度も聞き返してラボのみなさんに迷惑をかけてしまいまし
たが、2 週間ほど経つと慣れてきて、実験に集中できるようになって行きました。いろいろ
な基礎実験をやらせていただいて、とてもいい勉強になったと思います。今回のマイコー
スプログラムでは、PhD のニーシャの研究のお手伝いのような働きをしていたため、自分
でテーマを決めての実験ということはできませんでしたが、ラボの教授やポスドクの方々
の研究テーマや実験などをミーティングなどで聞くことで、どのような目的を持ってどの
ような実験をするのか、などということも考えられるようになったように思えます。
ヨーロッパでの生活も新しいことの連続でした。ホームステイ先のご夫婦はインド系イ
ギリス人で、暮らしや文化はインドのもので、食事のカレーはとてもおいしかったです。
毎週のように旅行に行き、平日はラボで実験をするという暮らしはとてもハードでしたが、
本当に充実した日々でした。
英語は 2 ヶ月半ではそれほど上達しませんでしたが、会話で困らないくらいには使える
ようになれました。もうすっかり忘れてしまって使えなくなっているような気がしますが、
次に海外暮らしをするときの自信につながると思います。将来大学院を選ぶ際に、海外を
選択肢に含めることができるように、という、マイコースで海外を選んだ目的の１つも個
人的にはしっかり達成できました。
貴重な経験を将来に生かせるように勉強していきたいと思いました。お世話になったす
べての方々、ありがとうございました。

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