JP2003088383A - Methods for collecting biomolecules from living cells - Google Patents

Abstract

(57) [Summary] [PROBLEMS] To provide means for collecting biomolecules such as RNA from living cells. A method for collecting biomolecules, comprising at least the following steps (1) and (2). (1) a step of piercing a needle capable of binding to a biomolecule to be collected into a living cell using a device capable of fine position control; (2) inserting the needle from a living cell using the device; Extraction process

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for collecting various biomolecules from a living cell and a method for identifying a gene expressed in a living cell using the method. The method of the present invention makes it possible to examine changes in gene expression and the like in one cell over time.

[0002]

2. Description of the Related Art One of the major trends in recent biological research is to investigate the individuality of individual biological samples. Even in the case of biopolymers such as proteins, it is not only to investigate the whole properties such as enzyme activity in a solution consisting of many molecules, but also to investigate the activity of a single molecule. being called.

In the field of cell biology, studies have been conducted to investigate the individuality of each cell. For example, a nerve cell in a certain area of the brain has conventionally been treated as a collection of cells of the same type. By investigating subtle differences in expression of receptors and the like of individual cells in these cell populations of the same type, it has been considered that more complicated functional analysis can be performed. One could call it single cell biology for single molecule biochemistry.

[0004] In order to observe changes in gene expression in one cell, it is indispensable to take out a small amount of biomolecule from the living cell and analyze it. Until now, comparisons have been made among multiple cells that seem to be in the same state, but it cannot be said to be a sufficient method for conducting one-cell biology for investigating subtle changes between cells in the future.

Recently, from each of a plurality of vomeronasal nerve cells
By making a cDNA library and comparing them, subtle gene expression was investigated, and cloning of the pheromone receptor was performed. This method is very revolutionary, but it kills cells in the same way as previous methods, so it is not possible to examine changes over time with a single cell.

[0006]

As described above, the conventional method for collecting biomolecules is based on the premise of killing cells as a collection source, and attempts to collect biomolecules in a state where the cells are kept alive. The method was unknown.

The present invention has been made under such a technical background, and its purpose is to obtain RNA from living cells.
It is to provide a means for collecting biomolecules such as and continuously record changes over time in individual cells.

[0008]

Means for Solving the Problems The present inventor has the idea that it is necessary to extract biomolecules from cells in a living state in order to observe changes in gene expression in one cell. The present invention has been completed based on this idea.

That is, the present invention is at least the following (1)
And a method of collecting a biomolecule, which includes the steps (2) and (2). (1) A step of inserting a needle capable of binding to a biomolecule to be collected into a living cell by using a device capable of finely controlling the position (2) A step of inserting the needle from the living cell by using the device Further, the present invention is a method for identifying a gene expressed in a living cell, which comprises at least the following steps (1) and (2). (1) Step of collecting mRNA from living cells by the above method (2) The mRNA and an oligonucleotide containing a sequence specific to a gene whose expression is expected to coexist, the former as a template, and the latter as a primer The step of investigating whether or not the polymerase chain reaction occurs is further present, and the present invention is a method for identifying a gene expressed in a living cell, which comprises at least the following steps (1) to (3): . (1) Step of collecting mRNA from living cells by the above method (2) Step of synthesizing cDNA from said mRNA (3) Bringing said cDNA close to a DNA chip on which a plurality of gene fragments whose expression is expected are fixed Measuring the force generated between the cDNA and the fixed gene fragment

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below.

The method for collecting biomolecules of the present invention is characterized by including at least the following steps (1) and (2).

In step (1), a needle capable of binding to a biomolecule to be collected is inserted into a living cell using a device capable of finely controlling the position.

Biomolecules include mRNA, tRNA, rRNA, DN
A, protein, lipid, sugar, etc. can be mentioned.

Any needle may be used as long as it can bind to biomolecules, and for example, metal oxide whiskers (for example, ZnO whiskers) and carbon nanotubes can be used. Whiskers and the like can be bound to biomolecules by a physical adsorption force in a state as they are, but it is preferable to modify them with a substance capable of specifically binding to biomolecules in order to improve the binding property. Examples of the substance capable of specifically binding to a biomolecule include oligo dT in the case of collecting mRNA, and an antibody in the case of collecting proteins, lipids and sugars. it can. Further, when it is desired to collect a nucleic acid having a specific sequence, the needle may be modified with a nucleic acid having a sequence complementary to the nucleic acid.

The method of modifying the needle can be appropriately determined depending on the substance to be modified and the type of needle. For example, oligo dT
When the ZnO whiskers are modified with, the reaction can be performed by reacting the amino group-introduced whiskers with the oligo dT-containing 5'-thiolated oligo DNA with an appropriate cross-linking agent.

The device capable of finely controlling the position may be any device capable of piercing the needle and pulling out the needle as will be described later without killing living cells. A commercially available micromanipulator or atom A force microscope can be used.

The insertion of a needle into a living cell is carried out, for example, by fixing the needle to a cantilever of an atomic force microscope, moving the cantilever above the living cell to be collected, and then moving it downward. be able to. The needle can be fixed to the cantilever using a general method, for example, epoxy. It is preferable to perform such operations such as inserting the needle under a microscope.

Any living cells may be used as a collection source, and examples thereof include, but are not limited to, fibroblasts, nerve cells, and glial cells.

In step (2), the needle is pulled out from the living cells by using the device.

The time from the insertion of the needle to the withdrawal is not particularly limited, but it is preferably short considering the damage to cells, and long is preferable for taking out more biomolecules. It usually takes about 5 to 10 seconds from insertion to removal.

By utilizing the above method of collecting biomolecules, the gene expressed in living cells can be identified. That is, it is possible to know what kind of gene was expressed in living cells by collecting mRAN from living cells and examining which gene the mRNA originated from. Specific methods include the following first method and second method.

The first method is characterized by including at least the following steps (1) and (2). (1) Step of collecting mRNA from living cells by the above method (2) The mRNA and an oligonucleotide containing a sequence specific to a gene whose expression is expected to coexist, the former as a template, and the latter as a primer The step of examining whether or not the polymerase chain reaction occurs If the mRNA is derived from the expected gene, an amplified fragment by the polymerase chain reaction is produced, but if not from the expected gene, the amplified fragment is not produced. Therefore, by examining the presence or absence of the amplified fragment by electrophoresis or the like, it is possible to know what kind of gene was expressed in living cells.

The second method is at least the following (1) to
It is characterized by including the step (3). (1) Step of collecting mRNA from living cells by the above method (2) Step of synthesizing cDNA from said mRNA (3) Bringing said cDNA close to a DNA chip on which a plurality of gene fragments whose expression is expected are fixed The step of measuring the force generated between the cDNA and the fixed gene fragment, if the cDNA is derived from the expected gene fragment, cD
When NA is brought closer to a gene fragment, the attractive force and repulsive force between them change. Therefore, by examining this change, it is possible to know what kind of gene was expressed in living cells. The change in attractive force or the like can be measured by an atomic force microscope or the like.

[0023]

[Example] 1. Modification of ZnO Whiskers After washing the ZnO whiskers with anhydrous toluene, the mixture was stirred overnight in a toluene solution containing 50% aminopropylsilane with a rotator to introduce amino groups on the surface of the ZnO whiskers. After completion of the reaction, ZnO was washed with methanol three times and then stored in methanol. Next, cross-linking agent (Sulfo-LC
-SPDP) 5'-thiolated oligo DNA (AAACGACG
GCCAGTGAATTGTAATACGACTCACTATAGGCGC (T) ₂₄ ) was chemically bound to the ZnO whisker surface.

Whether the modification was successful was examined by reaction with poly A having a red fluorescent dye attached thereto.
FIG. 1B shows ZnO whiskers dyed red by the reaction with poly A. 2. Fixed stereo microscope (SZX12, Olympus) on the AFM cantilever of ZnO whiskers and heater (MP200DM)
SH, Kitazato) and a micromanipulator (Narishige Kagaku) were set. The temperature of the heater was set to 70 ° C.
Three pieces of slide glass (about 1.5 cm square) cut into small pieces were prepared, and epoxy (grade 1001, oiled shell, melting point 64 ° C), ZnO whiskers, and AFM chip were placed on each.

When the epoxy melted and the ZnO whiskers dried, the following work was started. While observing with a stereomicroscope, the micropipette of the manipulator was slightly dipped in epoxy. This caused a very small amount of epoxy to become spherical and adhere to the pipette tip. The tip of the pipette was lightly touched with the tip of the AFM cantilever this time.
This left a very small amount of epoxy on the tip of the cantilever. Next, using another manipulator pipette, I picked up only one ZnO whisker. Touching the ZnO whiskers with the needle tip causes ZnO
Whiskers attached to the tip of the pipette. Then, the ZnO whiskers picked up were placed on the place where the epoxy was attached, and when the temperature was returned to room temperature, the resin solidified and the ZnO whiskers were fixed to the tip of the AFM cantilever. 3. Preparation of Live Cells as Samples In this example, BALB 3T3 cells, which are BALB / c mouse-derived fibroblasts, were used as the RNA collection source. The culture environment was kept at 37 ° C. in the presence of 5% CO ₂ .
The medium is 100 U / mL penicillin-100 mg / mL streptomycin (Gibco BRL) in D-MEM / F-12 medium (Gibco BRL).
FBS (fetal (fetal)
Bovine serum, JRH Biosciences) A mixed medium was used so that the ratio of serum was 9: 1. The medium was exchanged about 3 days after the passage. Passaging was performed about every week. As a subculture method, confluent cells were treated with 0.25% trypsin-1 mM EDTA (Gibco BRL) and spun down (70
(0 rpm, 10 minutes), and then passaged 1/10 of the whole. Usually, a square dish (T-25) is used for culturing, but a 35 mm petri dish was used in this example. 4. Collection of mRNA from living cells The above-mentioned BALB 3T3 cells were placed on the sample stand of an atomic force microscope, and the ZnO whiskers fixed to the cantilever were moved to the site where the cells were to be stabbed. This work was performed while observing with an optical microscope attached to the atomic force microscope. After completing the ZnO whisker position adjustment, move the piezo,
Cells were stabbed with ZnO whiskers. A photograph immediately after the cells were stabbed with ZnO whiskers is shown in FIG. The cells indicated by circles in the figure were stabbed with ZnO whiskers. The ZnO whiskers, which are not visible in this figure, are attached to the tip of the cantilever (triangle part).

After puncturing the cells with ZnO whiskers, the cantilevers were quickly removed from the atomic force microscope. 5. mRNA analysis QIAGEN PCR using mRNA attached to ZnO whiskers as a template
RT-PCR and nested PCR were performed using the kit. As the primers, two sets of primers specific to the β-actin gene were used. With these primers, ZnO
If β-actin gene-derived mRNA is attached to the whiskers, a fragment of about 400 bp is amplified (Fig. 3).

The PCR conditions and primer sequences are as follows. <Primer of ^{1 st RT-PCR> Foward Primer} : 5'-GTG GGC CGC TCT AGG CAC CAA-3 '
(SEQ ID NO: 1) Reverse Primer: 5'-CTC TTT GAT GTC ACG CAC GAT TTC-
3 '(SEQ ID NO: 2) <2 ^nd nested PCR primers> Foward Primer: 5'-GAC TCC TAT GTG GGT GAC GAG G-3'
(SEQ ID NO: 3) Reverse Primer: 5'-GGA TCT TCA TGA GGT AGT CCG TCA-
3 '(SEQ ID NO: 4) After performing the above two-step PCR, the amplification product was electrophoresed. A photograph of the gel is shown in FIG. As shown in this figure,
A band of about 400 bp was detected in the center lane. This band is considered to be derived from β-actin mRNA. The leftmost lane in the figure is a marker and the rightmost lane is a control lane.

[0028]

INDUSTRIAL APPLICABILITY The present invention provides a method for collecting biomolecules from living cells. In the case of examining the time course of gene expression in a certain cell, conventionally, a plurality of cloned cells were used, but the method of the present invention makes it possible to examine the time course of one cell. This makes it possible to obtain more accurate expression information than when using clonal cells. In addition, it becomes possible to investigate the time-dependent changes in the expressed gene even in cells that cannot be cloned.

Further, in the method of the present invention, by changing the place where the whisker is stabbed, the difference in expression depending on the place of the cell can be examined.

[0030]

[Sequence list]                                SEQUENCE LISTING        <110> President of Tokyo Institute of Technology <120> METHOD FOR PICKING BIOMOLECULES FROM A VIABLE CELL <130> P01-054 <160> 4 <170> PatentIn Ver. 2.1 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: SYNTHETIC DNA <400> 1 gtgggccgct ctaggcacca a 21 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: SYNTHETIC DNA <400> 2 ctctttgatg tcacgcacga tttc 24 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: SYNTHETIC DNA <400> 3 gactcctatg tgggtgacga gg 22 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: SYNTHETIC DNA <400> 4 ggatcttcat gaggtagtcc gtca 24

[Sequence list free text]

[SEQ ID NO: 1] SEQ ID NO: 1 is of RT-PCR performed in the examples.
The nucleotide sequence of the Foward Primer is shown.

[0031]

[SEQ ID NO: 2] SEQ ID NO: 2 is the RT-PCR sequence of the example.
The base sequence of Reverse Primer is shown.

[0032]

[SEQ ID NO: 3] SEQ ID NO: 3 is the nested P sequence in the example.
The forward sequence of the CR forward primer is shown.

[0033]

[SEQ ID NO: 4] SEQ ID NO: 4 is the nested P sequence in the example.
The nucleotide sequence of the Reverse Primer of CR is shown.

[Brief description of drawings]

FIG. 1 is a micrograph showing the shape of ZnO whiskers (FIG. 1
A), ZnO whiskers modified with oligo dT and fluorescent poly A
Fluorescence micrograph showing the annealing process (Fig. 1B)

[Figure 2] Photo taken immediately after ZnO whiskers were stabbed into cells

[FIG. 3] PCR primers used in Examples and m used as a template
Diagram showing the position of RNA

FIG. 4 is a photograph showing a fragment amplified by PCR using mRNA recovered from cells as a template.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Kim, Toru             2-12-1 Ookayama, Meguro-ku, Tokyo Tokyo             Inside the university F-term (reference) 4B024 AA20 CA04 CA09 CA12 DA02                       GA11 HA14                 4B063 QA01 QA13 QQ53 QR32 QR55                       QR62 QS12 QS25 QS34

Claims (7)

[Claims] 1. A method for collecting biomolecules, which comprises at least the following steps (1) and (2): (1) A step of inserting a needle capable of binding to a biomolecule to be collected into a living cell by using a device capable of finely controlling the position (2) A step of inserting the needle from the living cell by using the device Extraction process 2. The method for collecting biomolecules according to claim 1, wherein the device capable of fine position control is an atomic force microscope. 3. The method for collecting a biomolecule according to claim 1, wherein the needle is a whisker modified with a substance capable of specifically binding to the biomolecule. 4. The method for collecting biomolecules according to claim 3, wherein the whiskers are ZnO whiskers. 5. The biological molecule is mRNA, according to any one of claims 1 to 4.
The method for collecting a biomolecule according to any one of 1. 6. A method for identifying a gene expressed in a living cell, which comprises at least the following steps (1) and (2): (1) A step of collecting mRNA from a living cell by the method according to claim 5, (2) the mRNA and an oligonucleotide containing a sequence specific to a gene whose expression is expected to coexist, and the former is used as a template, Step of investigating whether the polymerase chain reaction using the latter as a primer occurs 7. A method for identifying a gene expressed in a living cell, which comprises at least the following steps (1) to (3): (1) A step of collecting mRNA from living cells by the method according to claim 5 (2) A step of synthesizing cDNA from the mRNA (3) A plurality of gene fragments whose expression is expected to be fixed in the cDNA Step of measuring the force generated between the cDNA and the fixed gene fragment by bringing it close to the DNA chip