WO2018139601A1

WO2018139601A1 - Method and kit for cryopreserving xenopus laevis egg extract, concentrated xenopus laevis egg extract, and method for analyzing cell cycle

Info

Publication number: WO2018139601A1
Application number: PCT/JP2018/002546
Authority: WO
Inventors: 勇太島本; 高木　潤
Original assignee: 大学共同利用機関法人情報・システム研究機構
Priority date: 2017-01-30
Filing date: 2018-01-26
Publication date: 2018-08-02
Also published as: JP7023519B2; JPWO2018139601A1

Abstract

A method for cryopreserving a Xenopus laevis egg extract, characterized by comprising a partition step for partitioning the Xenopus laevis egg extract into water and solid matters and a freezing step for separately freezing the water and the solid matters.

Description

Xenopus egg extract frozen storage method, cryopreservation kit, Xenopus egg concentrate extract, and cell cycle analysis method

The present invention relates to a method for cryopreserving Xenopus egg extract, a cryopreservation kit, a Xenopus egg concentrate extract, and a method for analyzing the cell cycle.
This application claims priority based on Japanese Patent Application No. 2017-014441 filed in Japan on January 30, 2017, the contents of which are incorporated herein by reference.

Xenopus egg extract is the only experimental system that can achieve a highly synchronized cell cycle in a cell-free system (extract without cell membrane). In this experimental system, by removing specific factors from the system using specific antibodies (immune elimination), factors necessary for cell cycle progression and control, and DNA replication and distribution control can be clearly identified.

In addition, research using single-cell yeast has become a powerful means in elucidating the cell cycle control mechanism, and genes involved in these processes have been identified one after another. However, the cell-free system using Xenopus egg extract is superior to yeast in the following points.
(1) Suitable for biochemical and physiological studies: For example, the cyclinB / Cdk1 complex, which is the substance of the M phase promoting factor MPF, is identified for the first time by a cell-free system using Xenopus egg extract. (For example, see Non-Patent Document 1). (2) Partial reconfiguration is possible in the test tube. (3) Unlike yeast, the frog is a multicellular animal and is a model closer to humans.
So far, the cell cycle control mechanism specific to multicellular animals has been clarified by a cell-free system using Xenopus egg extract.

However, Xenopus laevis egg extract must be prepared at the time of use because, when frozen, the components in the cytoplasm are damaged due to crystallization of water and lose its activity.

The present invention has been made in view of the above circumstances, and provides a method for cryopreservation of a Xenopus egg extract that can be stored for a long period of time and retains cytoplasmic activity, and a kit for cryopreservation.

As a result of intensive studies to achieve the above object, the present inventors have found that the present invention has been completed.

That is, the present invention includes the following aspects.
[1] A Xenopus egg extract comprising: a fractionation step of fractionating water and solids from the Xenopus egg extract; and a freezing step of separately freezing the water and solids. Frozen storage method.
[2] The method for cryopreserving a Xenopus egg extract according to [1], wherein a filtration membrane having a fractional molecular weight of 1000 or more is used in the fractionation step.
[3] The method for cryopreserving a Xenopus egg extract according to [1] or [2], wherein a filtration membrane having a molecular weight cut off of 10,000 or more is used in the fractionation step.
[4] The method according to any one of [1] to [3], wherein in the freezing step, the moisture and the solid content are frozen at a rate of not less than −0.5 ° C./min and not more than −1.5 ° C./min. Method for cryopreservation of Xenopus laevis egg extract.
[5] A kit for cryopreservation of Xenopus egg extract, comprising a centrifuge tube equipped with a filtration membrane for water fractionation.
[6] The kit for cryopreservation of Xenopus egg extract according to [5], wherein the molecular weight cut-off of the filtration membrane for water fraction is 1000 or more.
[7] The kit for cryopreservation of the Xenopus egg extract according to [5] or [6], wherein the fractional molecular weight of the filtration membrane for water fraction is 10,000 or more.
[8] The protein concentration is 83 mg / mL or more and 103 mg / mL or less, the content of the protein having a molecular weight of 20000 or more and 30000 or less is 20% by mass or less with respect to the total protein amount, and the molecular weight is 100000 or more. The Xenopus laevis egg concentrate extract, wherein the content of protein of 150,000 or less is 3% by mass or more with respect to the total protein amount.
[9] Thaw and mix the water and solid content of the Xenopus egg extract frozen and stored in the process of cryopreservation of the Xenopus egg extract according to any one of [1] to [4]. A cell cycle analysis method comprising using a reconstructed Xenopus egg extract.

According to the present invention, it is possible to provide a method for cryopreserving a Xenopus egg extract that can be stored for a long period of time and that retains cytoplasmic activity, and a kit for cryopreservation.

It is a schematic process drawing which shows one Embodiment of the cryopreservation method of the Xenopus egg extract of this invention. It is a schematic process drawing which shows one Embodiment of the reconstruction method of the Xenopus egg extract of this invention. A small amount of each extract (fresh extract, extract that had been subjected to cold thawing with an unfractionated fraction, and an extract reconstructed in Example 1) in a state where the cell cycle was stopped in the metaphase in Test Example 1 It is the fluorescence image which fluorescently stained the tube | pipe and the chromosome. Threshold values for each extract (fresh extract, extract that had been subjected to cold thawing with an unfractionated extract, and an extract reconstructed in Example 1) in a state where the cell cycle was stopped in the metaphase in Test Example 1 It is a graph which shows the ratio of the sperm DNA which has a microtubule signal exceeding. Bipolar in each extract (fresh extract, extract that has been subjected to cold thawing with an unfractionated fraction, and an extract reconstructed in Example 1) in a state where the cell cycle is stopped at the metaphase in Test Example 1 It is a graph which shows the ratio of the spindle which has a structure. Bipolar in each extract (fresh extract, extract that has been subjected to cold thawing with an unfractionated fraction, and an extract reconstructed in Example 1) in a state where the cell cycle is stopped at the metaphase in Test Example 1 It is a graph which shows the length of the spindle which has a structure. Bipolar in each extract (fresh extract, extract that has been subjected to cold thawing with an unfractionated fraction, and an extract reconstructed in Example 1) in a state where the cell cycle is stopped at the metaphase in Test Example 1 It is a graph which shows the width | variety of the spindle which has a structure. Bipolar in each extract (fresh extract, extract that has been subjected to cold thawing with an unfractionated fraction, and an extract reconstructed in Example 1) in a state where the cell cycle is stopped at the metaphase in Test Example 1 It is a graph which shows the total microtubule signal intensity | strength of the spindle which has a structure. It is a graph which shows the capacity | capacitance of the flow-through fraction (water | moisture fraction) obtained in Example 1 (fraction molecular weight 100k) and Example 2 (fraction molecular weight 10k) using the centrifugal filter apparatus of a different fraction molecular weight. . The threshold value in each extract (the fresh extract, the extract reconstructed in Example 1, and the extract reconstructed in Example 2) in a state in which the cell cycle is stopped in the metaphase in Test Example 2 It is a graph which shows the ratio of the sperm DNA which has the microtubule signal exceeded. Bipolar structure in each extract (fresh extract, extract reconstructed in Example 1, and extract reconstructed in Example 2) in a state in which the cell cycle is stopped at the metaphase in Test Example 2 It is a graph which shows the ratio of the spindle which has. Bipolar structure in each extract (fresh extract, extract reconstructed in Example 1, and extract reconstructed in Example 2) in a state in which the cell cycle is stopped at the metaphase in Test Example 2 It is a graph which shows the length of the spindle which has. Bipolar structure in each extract (fresh extract, extract reconstructed in Example 1, and extract reconstructed in Example 2) in a state in which the cell cycle is stopped at the metaphase in Test Example 2 It is a graph which shows the width | variety of the spindle which has. Bipolar structure in each extract (fresh extract, extract reconstructed in Example 1, and extract reconstructed in Example 2) in a state in which the cell cycle is stopped at the metaphase in Test Example 2 It is a graph which shows the total microtubule signal intensity | strength of the spindle which has a. It is a graph which shows the capacity | capacitance of the flow through fraction (water | moisture fraction) obtained in Example 1 (10 minutes), Example 3 (5 minutes), and Example 4 (20 minutes) which are different centrifugation time. . Each extract with a cell cycle stopped at the metaphase in Test Example 3 (fresh extract, extract reconstructed in Example 1, extract reconstructed in Example 3, and Example 4) It is a graph which shows the ratio of the sperm DNA which has the microtubule signal exceeding the threshold value in the (reconstructed extract). Each extract with a cell cycle stopped at the metaphase in Test Example 3 (fresh extract, extract reconstructed in Example 1, extract reconstructed in Example 3, and Example 4) It is a graph which shows the ratio of the spindle which has a bipolar structure in the reconstructed extract). Each extract with a cell cycle stopped at the metaphase in Test Example 3 (fresh extract, extract reconstructed in Example 1, extract reconstructed in Example 3, and Example 4) It is a graph which shows the length of the spindle which has a bipolar structure in the reconstructed extract). Each extract with a cell cycle stopped at the metaphase in Test Example 3 (fresh extract, extract reconstructed in Example 1, extract reconstructed in Example 3, and Example 4) It is a graph which shows the width | variety of the spindle which has a bipolar structure in the (reconstructed extract). Each extract with a cell cycle stopped at the metaphase in Test Example 3 (fresh extract, extract reconstructed in Example 1, extract reconstructed in Example 3, and Example 4) It is a graph which shows the total microtubule signal intensity | strength of the spindle which has a bipolar structure in the reconstructed extract).

Hereinafter, a mode for carrying out the present invention (hereinafter, sometimes simply referred to as “the present embodiment”).
) Will be described in detail. The following embodiment is an example for explaining the present invention, and is not intended to limit the present invention to the following contents. The present invention can be implemented with appropriate modifications within the scope of the gist thereof.

<Frozen preservation method of Xenopus egg extract>
In one embodiment, the present invention provides a Xenopus egg extraction comprising: a fractionation step of fractionating water and solids from Xenopus egg extract; and a freezing step of freezing the water and solids separately. A method for freezing and storing a liquid is provided.

The conventional Xenopus egg extract solution must be prepared at the time of use because, when frozen, the components in the cytoplasm are damaged due to crystallization of water and lose its activity.
On the other hand, the cryopreservation method of the Xenopus egg extract of the present embodiment is less affected by crystallization of water by fractionating moisture and solid content and storing it in a frozen state, and is a constituent component in the cytoplasm. Therefore, even if thawed frozen Xenopus egg extract is thawed, the cytoplasmic activity is not lost and can be used for analysis of the cell cycle and the like. Moreover, according to the method for cryopreserving the Xenopus egg extract of this embodiment, the Xenopus egg extract can be stored frozen for a long period of time (eg, about 3 months to 1 year).

In the present specification, “egg” used for Xenopus egg extract means a matured oocyte, that is, an unfertilized egg.
In this specification, “intracytoplasmic activity” refers to proteins such as enzymes contained in the cytoplasm, or organelles (eg, nucleus, mitochondria, Golgi apparatus, endoplasmic reticulum, lysosome, peroxisome, vacuole, ribosome) Etc.).
Each step of the method for cryopreserving the Xenopus egg extract of the present embodiment will be described in detail below with reference to the drawings.

[Fractionation process]
First, water and solid content are fractionated from Xenopus egg extract. The temperature condition in the fractionation step may be 0 ° C. or more and 16 ° C. or less (preferably about 4 ° C.).
The fractionation method is not particularly limited. For example, chromatography, immunoprecipitation method, separation method by change in solution composition, sucrose density gradient centrifugation method, vacuum drying method, centrifugation method using a filtration membrane, etc. Is mentioned. Among them, it is preferable to use a centrifugal separation method using a filtration membrane because water and solids can be fractionated very easily.
The conditions for centrifugation are, for example, 0 ° C. or more and 16 ° C. or less (preferably 4 ° C.), 10000 g or more and 20000 g or less, and may be 5 minutes or more and less than 20 minutes.

FIG. 1 is a schematic process diagram showing an embodiment of the method for cryopreserving Xenopus egg extract of the present invention. In FIG. 1, the fractionation method by the centrifugation method using a filtration membrane is illustrated.
The molecular weight cut off of the filtration membrane used is 1000 or more, preferably 3000 or more, more preferably 10,000 or more, and further preferably 100,000 or more.

The water fraction of the Xenopus egg extract obtained in the fractionation step has a protein concentration of 3.0 mg / mL or less (preferably 2.5 mg / mL, more preferably 2.0 mg / mL or less, more preferably 1. 8 mg / mL).
The content of the protein having a molecular weight of 20000 or more and 30000 or less in the water fraction is 10% by mass or more, preferably 15% by mass or more and 25% by mass or less, and 18% by mass with respect to the total protein amount. It is more preferable that the content is not less than 22% and not more than 22% by mass. On the other hand, the content of a protein having a molecular weight of 100,000 to 150,000 is 5% by mass or less, preferably 1% by mass to 5% by mass, and preferably 2% by mass to 4% by mass with respect to the total protein amount. The following is more preferable.

In addition, the solid fraction of the Xenopus egg extract obtained in the fractionation step (ie, Xenopus egg concentrate extract) preferably has a protein concentration of 83 mg / mL to 103 mg / mL, 86 mg / mL More preferably, it is not less than mL and not more than 100 mg / mL, more preferably not less than 88 mg / mL and not more than 98 mg / mL, and particularly preferably not less than 90 mg / mL and not more than 96 mg / mL.
Further, the content of the protein having a molecular weight of 20000 or more and 30000 or less in the solid fraction (ie, Xenopus laevis egg concentrate extract) is preferably 20% by mass or less based on the total protein amount, and 12% by mass. % To 20% by mass, more preferably 14% to 18% by mass. On the other hand, the content of a protein having a molecular weight of 100,000 or more and 150,000 or less is preferably 3% by mass or more, more preferably 3% by mass or more and 7% by mass or less, more preferably 4% by mass with respect to the total protein amount. More preferably, it is 6 mass% or less.
Here, as a method for measuring the protein concentration, for example, a method using an antibody antigen reaction (for example, ELISA method), a colorimetric method (for example, a bicinchoninic acid (BCA) method using a reaction between a protein and a reagent). , Bradford method, Raleigh method, Biuret method, etc.).
Examples of the method for measuring the molecular weight include SDS-PAGE, mass spectrometry (for example, MALDI-Tof MS, ESI-QTof, etc.), gel filtration column chromatography, and the like.

[Freezing process]
Next, the water and solid content of the obtained Xenopus egg extract are frozen separately.
As a freezing method, it is preferable to slowly freeze moisture and solid content. Specifically, using a programmable freezer, etc., at a rate of −0.5 ° C./min to −1.5 ° C./min (preferably −1 ° C./min) until it reaches −80 ° C. Frozen.
By freezing at a speed in the above range, damage to the components in the cytoplasm due to a rapid temperature change can be prevented.

The frozen water and solids retain the cytoplasmic activity even after a storage period of, for example, 1 day to 12 months, and reconstitute the Xenopus egg extract by thawing and mixing. Can be used for the desired analysis.

[Extract preparation process]
Moreover, in the frozen storage method of the Xenopus egg extract of this embodiment, the extract preparation process which prepares the Xenopus egg extract may be provided before the fractionation process.

For the preparation of Xenopus egg extract, see the known methods (eg, “Murray AW,“ Cell cycle extracts ”, Methods Cell Biol., Vol. 36, p581-605, 1991.) using Xenopus laevis. ), And may be prepared from an unfertilized egg whose cell cycle has been stopped by a mitogen.
Specifically, first, placental gonadotropin is injected into Xenopus to induce ovulation and obtain eggs. Next, the egg is immersed in MMR solution (100 mM NaCl, 2 mM KCl, 1 mM MgCl ₂ , 2 mM CaCl ₂ , 0.1 mM EDTA, 5 mM HEPES) or the like, and the jelly-like membrane is removed by enzyme treatment such as collagenase. Next, if necessary, the eggs are washed using an extraction buffer (containing 100 mM KCl, 0.1 mM CaCl ₂ , 1 mM MgCl ₂ , 5 mM EGTA, 50 mM sucrose, and 10 mM potassium HEPES, pH 7.7) or the like. And prepare the eggs.

Next, the egg is crushed and the cytoplasm of the egg is recovered, that is, an egg extract is prepared. At this time, in order to increase the stability of the obtained extract and the protein expression efficiency, additives such as cytokinesis inhibitors, protease inhibitors, and energy substances may be added to the extraction buffer.
The egg crushing method is not particularly limited, and examples thereof include a method using a homogenizer, an ultrasonic treatment method, and a centrifugal separation method. Among them, it is preferable to use a centrifugal separation method because a cytoplasmic fraction can be collected very easily.
The collected cytoplasmic fraction may be further filtered through a porous membrane or the like to remove cell debris and the like.
The egg extract used in the present embodiment can be prepared from an arbitrary number of eggs, but it is preferable to use a certain number or more of eggs in order to facilitate crushing treatment and addition of a stabilizer, and at least 100 More preferably, it is prepared from individual eggs.
The obtained egg extract is fresh, for example, within about 8 hours after preparation, preferably within about 3 hours in the fractionation step, thereby obtaining high cytoplasmic activity.

<Frozen storage kit for Xenopus egg extract>
In one embodiment, the present invention provides a kit for cryopreservation of Xenopus egg extract comprising a centrifuge tube with a water separation filtration membrane.

According to the kit for cryopreservation of Xenopus egg extract of the present embodiment, it is stored frozen for a long period (for example, about 3 months or more and about 1 year or less) while maintaining the activity in the cytoplasm contained in the Xenopus egg extract. be able to.

[Centrifuge tube]
The centrifuge tube in the present embodiment is composed of a filter cup and a collection tube, and the filter cup is a container that is provided with a filtration membrane for water fractionation and into which Xenopus egg extract is charged. The collection tube is a container for collecting the filtrate that has passed through the water fractionation filtration membrane provided in the filter cup during centrifugation.

The fractional molecular weight of the filtration membrane for water fractionation is preferably 1000 or more, more preferably 3000 or more, still more preferably 10,000 or more, and particularly preferably 100,000 or more.

Examples of the material for the water fraction filtration membrane include, but are not limited to, nonwoven fabrics such as polyester, polyethylene terephthalate, polyethersulfone, and polypropylene, porous materials such as polyurethane, and combinations thereof.

Examples of the shape of the centrifuge tube include, but are not limited to, a columnar shape, a weight shape, and the like.
Moreover, as a material of a centrifuge tube, a metal, glass, a ceramic, a synthetic polymer etc. are mentioned, for example, It is not limited to these.

[Others]
The kit for cryopreservation of Xenopus egg extract of this embodiment further comprises an MMR solution (100 mM NaCl, 2 mM KCl, 1 mM MgCl ₂ , 2 mM CaCl ₂ , 0.1 mM EDTA, 5 mM HEPES), placental gonadotropin, collagenase, extraction buffer (including 100 mM KCl, 0.1 mM CaCl ₂ , 1 mM MgCl ₂ , 5 mM EGTA, 50 mM sucrose, and 10 mM HEPES potassium, pH 7.7), etc. May be.

<Xenopus concentrated egg extract>
According to one embodiment, the present invention provides a protein concentration of 83 mg / mL or more and 103 mg / mL or less, and the content of a protein having a molecular weight of 20000 or more and 30000 or less is 20% by mass or less with respect to the total protein amount. A Xenopus laevis egg concentrate extract having a protein with a molecular weight of 100,000 to 150,000 is 3% by mass or more based on the total protein amount.

The Xenopus laevis concentrated egg extract of this embodiment is hardly affected by water crystallization even if it is stored frozen, and the components in the cytoplasm are retained. Therefore, the Xenopus egg concentrate extract of the present embodiment that has been stored frozen is thawed and mixed with the water fraction produced when the Xenopus egg concentrate extract is produced to reconstitute the Xenopus egg extract. Can be built. The reconstituted Xenopus egg extract retains cytoplasmic activity, studies DNA replication, transcription, transcript processing, translation, and modification of translated proteins, introduced foreign genes Expression analysis, cell cycle analysis, protein production by a cell-free protein synthesis system using the extract.

The Xenopus laevis egg concentrate extract of this embodiment has a protein concentration of 83 mg / mL or more and 103 mg / mL or less (preferably 86 mg / mL or more and 100 mg / mL or less, more preferably 88 mg / mL or more and 98 mg / mL or less, more preferably 90 mg / mL or more and 96 mg / mL or less).
In addition, in the Xenopus laevis egg concentrated extract of this embodiment, the content of the protein having a molecular weight of 20000 or more and 30000 or less is 20% by mass or less and 12% by mass or more and 20% by mass or less with respect to the total protein amount. It is preferable that it is 14 mass% or more and 18 mass% or less. On the other hand, the content of a protein having a molecular weight of 100000 to 150,000 is 3% by mass or more, preferably 3% by mass or more and 7% by mass or less, and preferably 4% by mass or more and 6% by mass with respect to the total protein amount. The following is more preferable.

Examples of the method for producing the concentrated Xenopus egg extract of the present embodiment include the method described in [Fractionation step] in <Method for cryopreserving Xenopus egg extract> described above.

<Cell cycle analysis method>
In one embodiment, the present invention relates to a Xenopus egg extract reconstituted by thawing and mixing the water and solid content of the Xenopus egg extract frozen and stored in the process of the method of cryopreserving Xenopus egg extract. A method for analyzing a cell cycle using a liquid is provided.

According to the cell cycle analysis method of the present embodiment, since it is not necessary to prepare the Xenopus egg extract at the time of use, a large amount of analysis can be performed easily and simultaneously.
Each step of the method for reconstructing Xenopus egg extract used in the present embodiment will be described in detail below with reference to the drawings.

[Thawing process]
First, the water content and solid content of the frozen Xenopus egg extract are stored in a frozen state. As a thawing method, it is preferable to thaw slowly. Specifically, the tube may be left on ice and thawed.
Such a thawing step can prevent damage to the components in the cytoplasm due to a rapid temperature change.

[Reconstruction process]
Next, the thawed Xenopus egg extract is reconstructed by mixing the thawed water and solids and incubating under conditions of 0 to 16 ° C. (preferably about 4 ° C.) and 5 to 60 minutes. To do.

The reconstituted Xenopus egg extract has a cytoplasmic activity comparable to that of fresh Xenopus egg extract (eg, Xenopus egg extract within about 8 hours, preferably within about 3 hours after preparation). Therefore, it can be used for cell cycle analysis.

The invention is illustrated by the following examples. However, these do not limit the scope of the present invention.

[Example 1]
1. Preparation of Xenopus egg extract (1) Xenopus egg extract is prepared using a known method (for example, “Murray AW,“ Cell cycle extracts ”, Methods Cell Biol., Vol. 36, p581-605, 1991. ", etc.)) and prepared from unfertilized eggs whose cell cycle was stopped by a cytostatic factor.
Specifically, Xenopus laevis was first injected with placental gonadotropin to induce ovulation and obtain eggs. Next, the eggs were immersed in MMR solution (100 mM NaCl, 2 mM KCl, 1 mM MgCl ₂ , 2 mM CaCl ₂ , 0.1 mM EDTA, 5 mM HEPES) or the like, and the jelly-like membrane was removed by reduction treatment with cysteine or the like. The eggs were then washed with extraction buffer (containing 100 mM KCl, 0.1 mM CaCl ₂ , 1 mM MgCl ₂ , 5 mM EGTA, 50 mM sucrose, and 10 mM HEPES potassium, pH 7.7).

(2) Next, the egg was crushed by a centrifugation method, and the cytoplasm of the egg was collected, that is, an egg extract was prepared. In order to increase the stability of the resulting extract and the protein expression efficiency, additives such as cytokinesis inhibitors, protease inhibitors, and energy substances were added to the extraction buffer. The protein concentration of the egg extract measured using the Bradford method was 70 mg / mL.

2. Frozen storage of Xenopus egg extract (1) Fractionation step Next, using a centrifugal filter device (UFC510024, molecular weight cut off 100 k, manufactured by Millipore), a cooling centrifuge (5424R, manufactured by Eppendorf) at 17,000 × g Under conditions of 4 ° C. and 10 minutes, 200 μL of the extract obtained in “1. Preparation of Xenopus laevis egg extract” was centrifuged. 150 μL of the concentrated extract (solid fraction) after centrifugation and 50 μL of the flow-through fraction (water fraction) were each transferred to a 1.5 mL test tube.
The protein concentration of the concentrated extract (solid fraction) measured using the Bradford method is 93 mg / mL, and the content of the protein having a molecular weight of 20000 or more and 30000 or less is 16 masses based on the total protein amount. The content of the protein having a molecular weight of 100,000 to 150,000 was 5% by mass with respect to the total protein amount.
The protein concentration of the flow-through fraction (water fraction) measured using the Bradford method is 1.8 mg / mL, and the content of the protein having a molecular weight of 20000 or more and 30000 or less is based on the total protein amount. The content of the protein having a molecular weight of 100000 to 150,000 was 3% by mass with respect to the total protein amount.

(2) Freezing step Next, a concentrated extract (solid fraction) and a flow were used under the condition of -1 ° C / min using a Chillette (registered trademark) 12 Portable Tube Cooler (manufactured by Denville Scientific). The through fraction (moisture fraction) was cryogenic frozen.

3. Reconstitution of Xenopus egg extract (1) Thawing step Next, the concentrated extract (solid fraction) and the flow-through fraction (water fraction) 7 days after frozen storage were left on ice and thawed. did.

(2) Reconstruction step Next, the thawed concentrated extract (solid fraction) and the flow-through fraction (water fraction) are mixed and incubated on ice for 20 minutes to reconstruct the Xenopus egg extract. did.

[Test Example 1]
1. Confirmation of spindle formation (1) Transition from interphase to metaphase metaphase Next, the Xenopus egg extract reconstructed in Example 1 was added to Xenopus sperm nuclei (final concentration: ~ 400 / μL) was added, and the phase was shifted from interphase to metaphase under a temperature condition of 16 ° C. In addition, as a control group, a fresh extract within about 8 hours after preparation and an extract subjected to cold thawing with an unfractionated fraction were also prepared, and the same operation as described above was performed to shift from the interphase to the mid-phase. .

(2) Staining of spindle microtubules Next, tetramethylrhodamine labeled tubulin (final concentration 500 nM) was added to each extract to stain spindle microtubules.

(3) Observation of spindles Next, 2 μL of each extract was dropped on a cover glass, and 4 μL of a buffer solution for fixation (125 mM Hepes, 2.5 mM EDTA, 2.5 M NaCl, 50 mM KCl, 25 mM MgCl ₂ , 1 μg) / ML DAPI, containing 10% formaldehyde and 60% glycerol), and placed an 18 mm cover slip. At 80 minutes after sperm addition, the nucleus was fixed, and at 90 minutes from the transition to metaphase, the metaphase spindle was fixed. 20x objective lens (Plan Apo, 0.75NA, Nikon), mercury lamp (Nikon), sCMOS camera (Neo sCMOS, Andor), and motorized XY stage (MS-2000, Applied Scientific Instrument) A fluorescence image was taken using an inverted epifluorescence microscope equipped with a). Also, large images of ˜10 mm × ˜11 mm were obtained using NIS-Elements software (version 4.50, manufactured by Nikon Corporation). The result is shown in FIG. 2A. In FIG. 2A, “Fresh” means a fresh extract within about 8 hours after preparation, “Non-filtered” means an extract that has been subjected to cold thawing in an unfractionated state, and “Filtered” "Means the extract reconstituted in Example 1. The length of the scale bar is 10 μm.

From FIG. 2A, in the extract reconstructed in Example 1, a metaphase spindle consisting of microtubules and chromosomes is formed as in the fresh extract within about 8 hours after preparation. It was confirmed. On the other hand, in the extract that had been subjected to cold thawing in an unfractionated form, no spindle formation was observed during metaphase.

(4) Middle spindle morphological analysis Next, DAPI-stained DNA signals are searched from the fluorescence images obtained by removing large aggregates of DNA signals, and using the image analysis pipeline (Grenfell et al., 2016). Spindle morphology and microtubule signal intensity around the DNA signal was analyzed. The results are shown in FIGS. In FIGS. 2B to F, “Fresh” means a fresh extract within about 8 hours after preparation, “Non-filtered” means an extract that has been subjected to cold thawing in an unfractionated state, “Filtered” means the extract reconstructed in Example 1.
Note that “mean ± SD” in FIGS. 2B and 2C is a value calculated from three independent test samples.
In addition, the box plots in FIGS. 2D to 2F were created using Origin 2016 (OriginLab).

From FIG. 2B, the percentage of sperm DNA with a microtubule signal that exceeded the threshold was very low in the unfractionated cold thawed extract (58% of the total sperm DNA tested, N = 229). ). On the other hand, the proportion of sperm DNA having a microtubule signal exceeding the threshold was determined by the fresh extract (89% of the total sperm DNA tested, N = 227) and the extract reconstituted in Example 1 (test 94% of the total sperm DNA, N = 177) was significantly higher.
This suggests that freeze-thaw reduces the activity of microtubule assemblies around the chromosome, but this reduction can be rescued by fractionation.

From FIG. 2C, about 53% of the spindles in the fresh extract were spindles having a bipolar structure (N = 201). On the other hand, in the extract that had been subjected to cold thawing without fractionation, the proportion of spindles having a bipolar structure was about 10% of the total spindles (N = 132).
On the other hand, in the extract reconstructed in Example 1, the ratio of spindles having a bipolar structure was approximately 69% of the total spindles (N = 166).

2D to F, the spindle length, width, and total microtubule signal intensity in the spindle having a bipolar structure are the same in the fresh extract and the extract reconstructed in Example 1. there were.
From the above, it was confirmed that the extract using the cryopreservation method of the present invention can form a highly active medium-term spindle assembly.

[Example 2]
1. Preparation of Xenopus egg extract Using the same method as in Example 1, Xenopus egg extract was prepared.

2. Frozen storage of Xenopus egg extract (1) Fractionation step Next, instead of the centrifugal filter device (UFC510024, fractional molecular weight 100k, manufactured by Millipore), the centrifugal filter device (UFC501024, fractional molecular weight 10k, manufactured by Millipore) Except that was used, centrifugation was performed using the same method as in (1) of “2. Xenopus egg extract frozen storage” in Example 1, and 160 μL of the concentrated extract (solid fraction) was obtained. A flow-through fraction (water fraction) of 40 μL was obtained.
The protein concentration of the concentrated extract (solid fraction) measured using the Bradford method is 72 mg / mL, and the content of the protein having a molecular weight of 20000 or more and 30000 or less is 16 masses relative to the total protein amount. The content of the protein having a molecular weight of 100,000 to 150,000 was 5% by mass with respect to the total protein amount.
Moreover, the protein concentration of the flow-through fraction (water fraction) measured using the Bradford method was less than 0.2 mg / mL.
FIG. 3A is a graph showing the volume of the flow-through fraction (water fraction) obtained in Example 1 and Example 2.
From FIG. 3A, the flow-through fraction (moisture fraction) obtained in Example 1 having a fractional molecular weight of 100 k is more flow-through fraction (water content) obtained in Example 2 having a fractional molecular weight of 10 k. There was more capacity than (fraction).

(2) Freezing step Next, using the same method as (2) in “2. Frozen storage of Xenopus egg extract” in Example 1, the concentrated extract (solid fraction) and the flow-through fraction ( The water fraction) was frozen.

3. Reconstitution of Xenopus egg extract (1) Thawing step Next, concentration was performed using the same method as in (1) of “3. Reconstruction of Xenopus egg extract” in Example 1 after 7 days of frozen storage. The extract (solid fraction) and the flow-through fraction (water fraction) were thawed.

(2) Reconstruction Step Next, the Xenopus egg extract was reconstructed using the same method as in (2) of “3. Reconstruction of Xenopus egg extract” in Example 1.

[Test Example 2]
1. Confirmation of spindle formation (1) Transition from interphase to metaphase Metastasis in Example 1 and Example 2 using the same method as in (1) of “1. Confirmation of spindle formation” in Test Example 1. The constructed extract was moved from interphase to metaphase. In addition, as a control group, a fresh extract within about 8 hours after preparation was prepared, and the same operation as described above was performed to shift from the interphase to the mid-phase.

(2) Staining of spindle microtubules Next, tetramethylrhodamine labeled tubulin (final) was added to each extract using the same method as in (2) of “1. Confirmation of spindle formation” in Test Example 1. The concentration of 500 nM) was added to stain the spindle microtubules.

(3) Mid-term spindle morphology analysis Next, the spindle morphology and microtubule signal intensity around the DNA signal were analyzed using the same method as in (4) of “1. Confirmation of spindle formation” in Test Example 1. did. The results are shown in FIGS. In FIG. 3B to F, “Fresh” means a fresh extract within about 8 hours after preparation, and “10k” is reconstructed in Example 2 using a centrifugal filter device with a molecular weight cut off of 10 k. "100k" means the extract reconstituted in Example 1 using a centrifugal filter device with a molecular weight cut off of 100k.
Note that “mean ± SD” in FIGS. 3B and 3C is a value calculated from two independent test samples.
In addition, the box plots in FIGS. 3D to 3F were created using Origin 2016 (OriginLab).

From FIG. 3B, the percentage of sperm DNA with a microtubule signal that exceeded the threshold was determined in fresh extract (N = 155), reconstructed extract in Example 2 (N = 211), and Example 1. The reconstructed extract (N = 169) was similar.
From FIG. 3C, the ratio of spindles having a bipolar structure is slightly lower in the extract (N = 190) reconstructed in Example 2 than in the fresh extract (N = 144). The extract liquid reconstructed in Example 1 (N = 149) was significantly higher.

Also, from FIGS. 3D to F, the spindle length, width, and total microtubule signal intensity in the spindle having a bipolar structure were reconstructed in the extract reconstructed in Example 1 and in Example 2. The same extract was equivalent.
From the above, in the case of using a centrifugal filter device having a fractional molecular weight of 10k, the proportion of spindles having a bipolar structure is lower than in the case of using a centrifugal filter device having a fractional molecular weight of 100k. It was confirmed that even a molecular weight can form a highly active metaphase spindle assembly.

[Example 3]
1. Preparation of Xenopus egg extract Using the same method as in Example 1, Xenopus egg extract was prepared.

2. Frozen storage of Xenopus egg extract (1) Fractionation step Next, (1. Frozen storage of Xenopus egg extract in Example 1) except that the centrifugation time was changed to 5 minutes instead of 10 minutes (1 Using the same method as in (2), centrifugation was performed to obtain 170 μL of a concentrated extract (solid fraction) and 30 μL of a flow-through fraction (water fraction).

[Example 4]
1. Preparation of Xenopus egg extract Using the same method as in Example 1, Xenopus egg extract was prepared.

2. Frozen storage of Xenopus egg extract (1) Fractionation step Next, (1. Frozen storage of Xenopus egg extract in Example 1) except that the centrifugation time was 20 minutes instead of 10 minutes (1 Using the same method as in (2), centrifugation was performed to obtain 140 μL of a concentrated extract (solid fraction) and 60 μL of a flow-through fraction (water fraction).
FIG. 3G is a graph showing the volume of the flow-through fraction (moisture fraction) obtained in Examples 1, 3 and 4.
From FIG. 3G, it became clear that the capacity | capacitance of a flow-through fraction (water | moisture fraction) increases as centrifugation time becomes long.

[Test Example 3]
1. Confirmation of spindle formation (1) Transition from interphase to metaphase stage Using the same method as in (1) of “1. Confirmation of spindle formation” in Test Example 1, Example 1, Example 3, and The extract reconstructed in Example 4 was transferred from the interphase to the metaphase.

(3) Mid-term spindle morphology analysis Next, the spindle morphology and microtubule signal intensity around the DNA signal were analyzed using the same method as in (4) of “1. Confirmation of spindle formation” in Test Example 1. did. The results are shown in FIGS. 3H to L. In FIG. 3H to L, “Centrifume time (centrifugation time) 5 min” means the extract reconstituted in Example 3 in which the fractionation step was performed with a centrifuge time of 5 minutes, and “Centrifuge time 10 min” It means the extract reconstituted in Example 1 where the fractionation process was performed at a centrifugation time of 10 minutes, and “Centrifuge time”
“20 min” means the extraction reconstructed in Example 4 in which the fractionation step was performed with a centrifugation time of 20 minutes.
Note that “mean ± SD” in FIGS. 3H and I is a value calculated from two independent test samples.
In addition, the box plots in FIGS. 3J to 3L were created using Origin 2016 (OriginLab).

From FIG. 3H, the percentage of sperm DNA having a microtubule signal exceeding the threshold is determined by the extract reconstructed in Example 1 (N = 134) and the extract reconstructed in Example 3 (N = 174). ) And the same level, and the extract reconstructed in Example 4 (N = 177) was slightly lower.
Further, from FIG. 3I, the ratio of spindles having a bipolar structure is compared with the extract (N = 155) reconstructed in Example 3, compared to the extract reconstructed in Example 1 (N = 126). ) Was slightly lower, and the extract reconstructed in Example 4 (N = 123) was significantly lower.

Also, from FIGS. 3J to 3L, the spindle length, width, and total microtubule signal intensity in the spindle having a bipolar structure were reconstructed in Example 3, the extract reconstructed in Example 1. And the extract reconstructed in Example 4 were equivalent.
From the above, it was confirmed that long-term centrifugation over 10 minutes reduced the activity of the extract and reduced the formation efficiency of the mid-term spindle.

According to the cryopreservation method and cryopreservation kit of the Xenopus egg extract of the present invention, while maintaining the cytoplasmic activity contained in the Xenopus egg extract, a long period (for example, about 3 months to 1 year) Can be stored frozen.
In addition, the Xenopus laevis concentrated egg extract of the present invention is hardly affected by crystallization of water even if it is stored frozen, and the components in the cytoplasm are retained. Therefore, the Xenopus egg concentrate extract of the present embodiment that has been stored frozen is thawed and mixed with the water fraction produced when the Xenopus egg concentrate extract is produced to reconstitute the Xenopus egg extract. Can be built. The reconstructed Xenopus egg extract retains cytoplasmic activity and can be used for cell cycle analysis and the like.

DESCRIPTION OF SYMBOLS 1 ... Xenopus egg extract, 2 ... Filter membrane for water fractionation, 3 ... Solid content, 3a ... Frozen solid content, 3b ... Thawed solid content, 4 ... Water, 4a ... Frozen water, 4b ... Thawed Water, 5 ... Reconstituted Xenopus egg extract, 10 ... Centrifuge tube.

Claims

A fractionation step of fractionating water and solids from Xenopus egg extract;
A freezing step of separately freezing the water and the solid content, and a method for freezing and storing the Xenopus egg extract.
The method for freezing and storing a Xenopus egg extract according to claim 1, wherein a filtration membrane having a fractional molecular weight of 1000 or more is used in the fractionation step.
The method for freezing and storing a Xenopus egg extract according to claim 1 or 2, wherein a filtration membrane having a molecular weight cut off of 10,000 or more is used in the fractionation step.
The Xenopus egg extraction according to any one of claims 1 to 3, wherein in the freezing step, the water and the solid content are frozen at a rate of -0.5 ° C / min or more and -1.5 ° C / min or less. Freezing storage method of liquid.
A cryopreservation kit for Xenopus laevis egg extract characterized by including a centrifuge tube equipped with a filtration membrane for moisture fractionation.
The kit for cryopreservation of Xenopus egg extract according to claim 5, wherein the molecular weight cut-off of the membrane for water fractionation is 1000 or more.
The kit for cryopreservation of the Xenopus laevis egg extract according to claim 5 or 6, wherein the molecular weight of the filtration membrane for water fractionation is 10,000 or more.
The protein concentration is 83 mg / mL or more and 103 mg / mL or less,
The content of a protein having a molecular weight of 20000 or more and 30000 or less is 20% by mass or less based on the total protein amount, and
The Xenopus laevis egg concentrate extract, wherein the content of a protein having a molecular weight of 100,000 or more and 150,000 or less is 3% by mass or more based on the total protein amount.
The water and solid content of the Xenopus egg extract frozen and stored in the process of the cryopreservation method of the Xenopus egg extract according to any one of claims 1 to 4 were thawed, mixed and reconstituted. A method for analyzing a cell cycle, comprising using an Xenopus egg extract.