U.S. patent application number 10/265404 was filed with the patent office on 2003-02-06 for apparatus for microinjection of sample into amphibian oocytes.
This patent application is currently assigned to Hitachi, Ltd.. Invention is credited to Matsunami, Shokichi, Moriya, Noboru, Nomura, Sayuri, Otomo, Jun, Saito, Sakae, Takeshita, Tomoko.
Application Number | 20030028908 10/265404 |
Document ID | / |
Family ID | 18744999 |
Filed Date | 2003-02-06 |
United States Patent
Application |
20030028908 |
Kind Code |
A1 |
Takeshita, Tomoko ; et
al. |
February 6, 2003 |
Apparatus for microinjection of sample into amphibian oocytes
Abstract
An apparatus for microinjection of samples into amphibian
oocytes incorporates a tray for holding a plurality of the
amphibian oocytes, an injection needle for injecting a sample into
the amphibian oocytes, a driving means for moving a relative
position between the tray and the injection needle and a
controlling device for controlling the movement by inputting a
depth of the injection needle for the tray or the amphibian oocytes
in the injection of the sample, and injecting the sample into the
amphibian oocytes at the given depth. According to the present
invention, the sample can be injected into the amphibian oocyte
with constant depth precisely , and quality of the oocytes and the
positional site of needle injection can be recorded as
information.
Inventors: |
Takeshita, Tomoko;
(Higashimatsuyama, JP) ; Otomo, Jun; (Tokyo,
JP) ; Nomura, Sayuri; (Higashimatsuyama, JP) ;
Matsunami, Shokichi; (Tokorozawa, JP) ; Moriya,
Noboru; (Tokorozawa, JP) ; Saito, Sakae;
(Tokorozawa, JP) |
Correspondence
Address: |
REED SMITH LLP
Suite 1400
3110 Fairview Park Drive
Falls Church
VA
22042
US
|
Assignee: |
Hitachi, Ltd.
|
Family ID: |
18744999 |
Appl. No.: |
10/265404 |
Filed: |
October 7, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10265404 |
Oct 7, 2002 |
|
|
|
09666411 |
Sep 20, 2000 |
|
|
|
Current U.S.
Class: |
800/8 ;
705/1.1 |
Current CPC
Class: |
C12M 35/00 20130101;
C12M 23/50 20130101 |
Class at
Publication: |
800/8 ;
705/1 |
International
Class: |
A01K 067/00; G06F
017/60 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 25, 2000 |
JP |
2000-256381 |
Claims
What is claimed is:
1. A method for selling or assigning a plurality of amphibian
oocytes, comprising selling or assigning a plurality of amphibian
oocytes as a set, into which a sample is injected under a
substantially same condition in injection depth.
2. The method for selling or assigning a plurality of amphibian
oocytes according to claim 1, wherein an information on the
injection of said sample into said plurality of said amphibian
oocytes is attached to the set.
3. The method for selling or assigning a plurality of amphibian
oocytes according to claim 1, wherein said information is at least
any one of a date and time of the injection, a term for guarantee
of quality, an area where said sample is injected, a depth at which
said sample is injected, and guaranteed injection efficiency or
expression efficiency.
4. A method for selling of assigning a plurality of amphibian
oocytes comprising, putting a plurality of the amphibian oocytes
into which a sample is injected under a substantially same
condition in the injection depth into a vessel, and attaching to
said vessel a label on which an information on the injection of the
sample into a plurality of said amphibian oocytes is described.
5. The method for selling or assigning a plurality of the amphibian
oocytes according to claim 4, wherein said vessel is maintained at
a temperature not lower than 18.degree. C. and not higher than
25.degree. C.
Description
[0001] This application is a Continuation of nonprovisional
application Ser. No. 09/666,411 filed Sep. 20, 2000.
BACKGROUND OF THE INVENTION
[0002] (1) Technical Field
[0003] The present invention relates to an automated apparatus for
injecting a sample of gene, pigment, protein, peptide or drug into
oocytes of Amphibia such as frog using a pipette-like needle. The
present invention further pertains a method for injecting the
sample of gene, pigment, protein, peptide or drug into the
specified position of the amphibian oocytes, the amphibian oocytes
with a guaranteed quality for injection of the sample, and a method
for selling or assigning the amphibian oocytes into which the
sample is injected at a specified position and depth.
[0004] (2) Background Art
[0005] Since oocytes of frogs such as Xenopus lavis, into which
gene, pigment, protein, peptide or drug is injected, have a
comparatively large size and can be obtained with a low cost and in
large quantities, they are widely used for the purpose of
confirming actions of pigment, protein, peptide and drug on viable
cells, analysis of gene functions, and production of proteins as a
gene product. To this purpose, the individual research scientists
breed frogs by themselves and collect the oocytes.
[0006] Heretofore, in the injection of a sample such as gene,
pigment, protein, peptide and drug into the oocytes of Amphibia
such as frog, technicians manually injected with a pipette-like
needle in which these samples are packed, into the oocytes under a
microscopic observation using a manipulator. The pipette is mounted
on the injector and a constant amount of sample is injected and
exhaled into cells by an action of an oil pressure or an air
pressure. Further, JP-A-5-192171 and JP-A-6-343478 disclose a
method for injection and exhalation of a constant amount of sample
into cells by applying a voltage. These prior arts disclose
techniques for injection of sample by approaching needle manually
to the cell under observing the cell microscopically.
SUMMARY OF THE INVENTION
[0007] (1) Problems to be Solved by the Invention
[0008] The above manual injection of samples such as genes had a
problem that a rate of oocytes, into which sample was successfully
injected, was varied depending on skills and experiences of
technicians. This was due to a difference in numbers of oocytes,
into which the sample could be injected within a fixed time,
depending on the individual technician, and as a result, a degree
of denaturation of the sample was varied in a time-dependent manner
among the technicians. It is an object of the present invention to
provide a method to give a constant numbers of cells per hour to be
treated independent of the skills and experiences of the
technicians.
[0009] Further, the above described prior art did not take into the
for unifying of the injection depth of samples into the oocytes at
a constant depth. Consequently, it was difficult to control the
depth even by a skilled technician. As a result, an injection of a
sample into a specified cell organelle such as a nucleus had to
depend on chance. It is another object of the present invention to
provide an easier method for injecting a sample into a cellular
organelle such as a nucleus, in which the injection can be
controlled in a depth direction by unifying the injection depth of
the sample.
[0010] Furthermore, in the above described prior art, since no
consideration was given for memorizing information of cells in the
injection of the sample, it was difficult to obtain a correlation
between the information on the sample injection and the subsequent
cell reaction. Consequently, it is further object of the present
invention to provide a method for obtaining the correlation between
them.
[0011] In addition, in the conventional techniques, the oocytes
were prepared individually and thus a mass production or production
on demand in good time was impossible.
[0012] Consequently, it is a further object of the present
invention to provide the oocytes, into which a specified sample is
injected, or the production, sale and transportation of the oocytes
with a guaranteed injection of the specified sample at a constant
position.
[0013] (2) Means for Solving the Problems
[0014] In order to solve the above problems, the present invention
provides an apparatus for automatically injecting the sample such
as gene, pigment, protein, peptide or drug into any constant
position and any constant depth of the oocytes of Amphibia such as
frogs by using a pipette-like needle.
[0015] Namely, the present invention provides an apparatus for
microinjection of samples into amphibian oocytes comprising a tray
for holding a plurality of the amphibian oocytes, an injection
needle for injecting a sample into the said amphibian oocytes, a
driving means for moving a relative position of the said tray to
the said injection needle and a controlling means for controlling
the said movement by inputting a depth of the said injection needle
for the said tray or the said amphibian oocytes in the injection of
the sample, and injecting the sample into the said amphibian
oocytes at the said depth.
[0016] The present invention further provides a system for
microinjection of samples into amphibian oocytes comprising a tray
for holding a plurality of the amphibian oocytes, an injection
needle for injecting sample into the said amphibian oocytes, a
driving means for moving a relative position of the said tray to
the said injection needle in a three dimensional direction, a
controlling means for controlling the said movement, an information
obtaining means for obtaining visual information on the said
amphibian oocytes in the microinjection, and a memorizing means for
accumulating the said information, and injecting the said sample
into the said amphibian oocytes.
[0017] As a result, the sample can be injected rapidly into the
plurality of amphibian oocytes at an almost constant depth.
[0018] Further, the tray has a plurality of wells having a
cylindrical structure with a flat base or with a conical base
having a maximum diameter of 105-150% of a diameter of the
amphibian oocytes. As a result, the sample can be injected into the
identical surface in about 80% of oocytes on the tray without
applying any special means.
[0019] We have found that when mRNA was injected into the oocytes,
in case that mRNA was injected into the animal hemisphere of the
oocyte or mRNA was injected into the vegetal hemisphere, expression
efficiency or the functional expression efficiency is different.
Namely, in order to suppress a variation of the expression
efficiency of the function of a protein in oocytes, it is important
to collect the oocytes, in which mRNA is injected into the same
hemispherical surface. In the present invention, it is possible to
memorize information of cellular area in the injection of the
sample and to induce easily a correlation between the information
and the subsequent cell reaction.
[0020] Further, the present invention provides a method for
automatic microinjection of samples into amphibian oocytes
comprising,
[0021] using an apparatus having a tray for holding a plurality of
the amphibian oocytes and an injection needle for injecting a
sample into a plurality of the said amphibian oocytes,
[0022] a step for setting a depth of the said injection needle for
the said tray or the said amphibian oocytes at the first depth,
[0023] a step for injecting the sample into the first oocyte in a
plurality of the said amphibian oocytes using the said injection
needle at the said first depth,
[0024] a step for automatically moving a relative position of the
said tray to the said injection needle, and
[0025] a step for subsequently injecting the sample into the second
oocyte in a plurality of the said amphibian oocytes by using the
said injection needle to the said first depth.
[0026] Furthermore, the present invention provides a method for
automatic microinjection of samples into amphibian oocytes
comprising,
[0027] using an apparatus having a tray for holding a plurality of
the amphibian oocytes and an injection needle for injecting a
sample into a plurality of the said amphibian oocytes,
[0028] a step for injecting the sample into the first oocyte in a
plurality of the said amphibian oocytes using the said injection
needle,
[0029] a step for moving a relative position of the said tray to
the said injection needle,
[0030] a step for subsequently injecting the sample into the second
oocyte in a plurality of the said amphibian oocytes using the said
injection needle,
[0031] a step for obtaining a condition of oocyte in the injection
of the said sample as a visual information, and
[0032] a step for accumulating the said visual information.
[0033] According to the invention of the above described apparatus
or method for injection of the sample into the amphibian oocytes,
the present invention further provides a plurality of the amphibian
oocytes wherein the sample is injected under a substantially equal
condition in the injection depth of the sample.
[0034] The present invention also provides the said amphibian
oocytes wherein the sample is further injected under a
substantially same condition in the injection position of the
sample.
[0035] In addition, the present invention provides the following
methods.
[0036] A method for preparation of a group of the amphibian oocytes
injected with a sample comprising,
[0037] using an apparatus having a tray for holding a plurality of
the amphibian oocytes and an injection needle for injecting sample
into a plurality of the said amphibian oocytes,
[0038] moving a relative position of the said injection needle to
the said tray,
[0039] injecting the sample into each of a plurality of the said
respective amphibian oocytes using the said injection needle,
[0040] obtaining each visual information of the said amphibian
oocytes in the injection, and
[0041] collecting a plurality of the oocytes in which the sample is
injected into the animal hemisphere of the oocyte, or a plurality
of the oocytes in which the sample is injected into the vegetal
hemisphere of the oocyte in a plurality of the said amphibian
oocytes based on the said visual information.
[0042] A method for selling or assigning a plurality of the
amphibian oocytes comprising selling or assigning as a set a
plurality of the amphibian oocytes, into which the sample is
injected under a substantially equal condition in the injection
depth of the sample, and attaching the information on the injection
of the sample into a plurality of the amphibian oocytes to the
set.
[0043] In addition, a method for selling or assigning a plurality
of the amphibian oocytes comprising putting a plurality of the
amphibian oocytes into which the sample is injected under a
substantially equal condition in the injection depth of the sample
into a vessel, and attaching a label on which the information on
the injection of the sample into a plurality of the said amphibian
oocytes is described to the said vessel.
[0044] In this connection, the information on the injection of the
sample into a plurality of the amphibian oocytes relates to at
least any one of a date and time of the injection, a term for
guarantee of a quality, a position where the sample is injected, a
depth at which the sample is injected and a probability of
expression.
[0045] As a result, according to the present invention, the oocytes
having substantially identical condition in relation to the
injections as well as their information can be obtained.
BRIEF DESCRIPTION OF THE DRAWINGS
[0046] FIG. 1: A construction of apparatus of the present
invention.
[0047] FIG. 2: An illustrative example of a tray used in the
present invention.
[0048] FIG. 3: An illustrative example of a vessel used in the
present invention.
[0049] FIG. 4: A correlation between fluorescence of GFP injected
into oocyte and a ligand response of histamine receptor expressed
by co-injection.
[0050] FIG. 5: A correlation between an area of gene injection
(animal hemisphere and vegetal hemisphere) and a ligand
response.
[0051] FIG. 6: Use of oocyte as a histamine sensor.
[0052] FIG. 7: A method of transferring oocytes after injection of
sample.
EXPLANATION OF SYMBOLS
[0053] 1: control unit, 2: auxiliary control unit, 3: monitor, 4:
moving table for injection needle, 5: injection device, 6:
injection needle, 7: CCD camera, 8: digital camera, 9: tray, 10:
light source, 11: orthogonal moving table, 12: horizontal moving
table, 13: oocytes, 14: physiological saline for Amphibia, 21:
vessel, 22: label, 23: cold insulator, 31: histamine receptor gene,
32: histamine receptor, 33: sample containing histamine, 34:
histamine response (positive), 35: sample without histamine, 36:
histamine response (negative), 41: syringe, 42: buffer solution for
Amphibia, 43: antibiotics, 44: outer vessel, 45: package.
DESCRIPTION OF THE PREFERRED EMBODIMENT
[0054] Referring to the drawings, the present invention will be
explained in detail in the following.
[0055] In FIG. 1, the principle of the present apparatus is
shown.
[0056] A tray, in which the oocytes are lined up and arranged,
having, for example, arrangements of 12 wells in a horizontal
direction and 8 wells in an orthogonal direction, in total 96 wells
with an uniform depth and form, can be used, however the number of
wells are not limited to 96 wells. The amphibian oocytes have
generally a heavier weight in the vegetal hemisphere than in the
animal hemisphere. Consequently, by designing a diameter of the
well in the tray to be slightly larger than that of the oocytes in
use, about 80% of the oocytes in average can be maintained to keep
their animal hemispheres upside, without changing the directions of
oocytes arranged on the tray by rotating the cells, and the
injection probability to the identical hemispherical surface can be
increased up without using any other special means. A form of a
well for arranging oocytes is preferably a cylindrical form, which
has a circular flat basal plane with a constant cross section
parallel to the base plane from the base plane to the open end
aperture of the above well, or a form with a conical base. A
diameter of the open end of the above well should be larger than
that of the amphibian oocytes in use. In addition, due to the
above-mentioned reason, it is preferable to keep a space for
rotating the oocytes in a well in which physiological saline is
filled, and specifically the fact that a maximum diameter of the
well set as 105-150% of the diameter of oocytes in use is
preferable, has been confirmed experimentally. For example, since
the diameter of Xenopus oocytes is approximately 1.3 mm, it becomes
possible to fix the oocytes in a specified direction without
damaging the oocytes by designing the diameter of a well as about
1.4-2 mm. Preferable form of a well in the present invention is,
for example, as shown in FIG. 2, a conical shaped well with an
angle of base at 90.degree., diameter 1.4 mm and depth in
cylindrical part 0.56 mm. Furthermore, by using a syringe in
addition to the use of the above tray, it is possible to operate
with an increased injection probability into the same surface sides
of different oocytes or it is possible to operate so that the
specified surface, for example, the vegetal hemispheres of the
oocytes are directed upward.
[0057] Examples of samples to be injected include, but are not
limited to gene, pigment, protein, peptide, and drugs. In the
example herein below, oocytes, into which histamine receptor cRNA
is injected, will be described, but an injectable gene is not
limited to cRNA and can be DNA, RNA and synthetic oligonucleotides.
A needle for injecting a sample is preferably a pipette-like
needle, but is not limited thereto. In order to obtain the cell
information such as the positional direction of oocytes, visual
information through the digital camera 8 is applied. Further, a
means for detecting the contact of the injection needle 6 with the
surface of oocyte is exemplified as the visual information through
CCD camera 7. However, a means required for obtaining the
information is not limited to the digital camera 8 and CCD camera
7. For example, a sensor, which can detect changes in pressure,
temperature, voltage, moisture or pH, may be mounted on the
injection apparatus, and the surface of oocytes can be detected
based on these types of information.
[0058] The oocytes before injection of the sample are arranged in
the wells on the tray 9, the physiological saline 14 is filled in
the tray 9, and then the tray is set on the orthogonal moving table
11 and the horizontal moving table 12. It is preferable to
determine the position of oocyte 13, to which the sample is
injected from the injection needle 6, by controlling movement of
the orthogonal moving table 11 and the horizontal moving table 12
to the direction for X-axis and the direction for Y-axis with the
control unit 1. Contrary to the constitution in FIG. 1, however,
the constitution can be applied by fixing the tray 9 with a movable
injection needle 6.
[0059] When the tray 9 is located at the position of the broken
line, the oocyte may be photographed by the digital camera 8, and
to transfer the image data to the control unit 1, and the
information of quality and positional direction of oocytes can be
accumulated.
[0060] The horizontal moving table 12 and the orthogonal moving
table 11 are operated by an indication of the control unit 1, and
the center of the first oocyte 13, which is located in the defined
position in the oocytes arranged on the tray 9, moves to the
downward position under the position of the gene injection needle
6. At this point, the injection needle moving table 4 is operated
by an indication of the control unit 1 for moving the injection
needle to the direction of Z-axis, a tip of the injection needle 6
mounted on the injection unit 5 moves downward to the position
slightly distant from the surface of the oocyte, for example, close
to the front by several hundred mm. At this point, by observing the
image taken by CCD camera 7 in the monitor 3, indication is given
from the auxiliary control unit 2, and the injection needle moving
table 4, operated to descending direction slowly. The contact of
the tip of the injection needle 6 with the surface of the oocyte 13
can be detected by visual information, pressure changes,
temperature changes, electric changes, moisture changes, or pH
changes, then the injection needle moving table 4 is stopped at
this position. This position is a reference point for the
subsequent gene injection operation. This point is made to be
memorized in the control unit 1 and the following operation is
performed. Namely, moving distance and depth of the injection
needle for the vertical direction against a plane of the tray from
the above reference point to the position of injecting sample are
set, and the injection needle 6 is stuck at the setting depth to
inject the previously fixed amount of the sample. For the injection
of the sample, a control for Z-axis direction can be performed, for
example, the injection needle moves downwardly to 0.2 mm from the
contact point of the injection needle 6 on the surface of oocyte
13. The optimum injection depth of the injection needle 6 into the
oocyte is different depending on the type of sample to be injected
and the object for injection and can be set freely. The sample can
not diffuse into the oocyte, if the injection depth is too shallow,
and if it is too deep, the probability for damaging a nucleus and a
oocyte is increased. Consequently, it is preferable to inject a
sample at the almost constant depth from the standpoint of
expression efficiency. For example, in the case that mRNA is
injected into the cytoplasm in order to express a protein, sticking
the needle to the depth of 0.02-0.1 mm from the cell surface is
preferable. On the other hand, in the case that DNA is injected
into a nucleus in order to express a protein, sticking the needle
to the depth of 0.05-0.2 mm from the cell surface is preferable on
the animal hemisphere. However, since the form of oocyte may be
deformed at the injection, actually the sample is injected at the
shallower position than the predetermined depth. Time for injecting
the sample is controlled by setting time for inserting the needle
in the oocyte depending on the amount of sample to be injected. In
order to improve injection efficiency, a plurality of the injection
needle 6 can be used. In this case, the movement of the relative
position between the injection needle and the tray by the
indication from the driving unit of the apparatus in
one-dimensional direction or two-dimensional direction may be
sufficient.
[0061] Subsequently, the sample is automatically injected at the
indicated time, rate and injection depth into the desired numbers
of other oocytes in the tray 9 by automatic control. Further, since
size of oocytes may have some deviation, a function for detecting a
position of the surface in each time of injection can be applied.
In addition, the information of oocyte condition is memorized in
the computer and is able to output on demand.
[0062] The system can be made so that the movement of the injection
needle 6 and the oocyte 13 in the injection or the visual
information of oocyte at the injection is memorized in the computer
and after termination of injection operation, position and depth of
sample injection, and characteristics of oocytes can be read out.
In this case, the visual information of each oocyte is preferably
related with its position on the tray by numbering the oocytes, for
example.
[0063] The amphibian oocytes are known to exist for the animal
hemisphere and the vegetal hemisphere, and each hemisphere has a
different function. We have found that in cases that mRNA was
injected into the oocyte, functional expression efficiency of
protein was different in each case of injecting mRNA into the
animal hemisphere and the vegetal hemisphere. In cases of injecting
histamine receptor mRNA, the expression efficiency is higher in the
injection into the animal hemisphere than the vegetal hemisphere;
as a result, the oocyte with a large ligand response can be
obtained. On the other hand, in cases that protein with fluorescent
protein, or gene there of and pigment are injected, information on
color and light with higher sensitivity can be obtained by the
injection into the vegetal hemisphere. As described above,
injection of sample into the animal hemisphere can be made for
about 80% of oocytes on the tray by using the above tray. In cases
that the oocytes, to which sample is injected into the specific
surface of oocytes such as only for the animal hemisphere or the
vegetal hemisphere, are expected to be obtained, as described
above, each oocyte is treated for arranging the upward direction of
the specific hemisphere by using a syringe before injection of
sample. Alternatively, the area information for sample injection on
the surface of oocytes is obtained by the detection means for
visual information or by the black and white discrimination sensor
in the injection of the sample, and as a result of the thus
obtained information, the oocyte, to which the sample is injected
into only the surface of interest directed for upward direction,
can be collected. As a result, the oocytes having substantially
identical conditions in the injected position can be obtained. In
the present invention, the "specific area" means the position
including the animal hemisphere, the vegetal hemisphere or the
equatorial area of the oocytes.
[0064] By using of the apparatus having the above constitution, the
sample can be injected into the specific area and depth of the
amphibian oocytes, and the oocytes which have almost the same
quality of the expression efficiency of the injected sample
(injection efficiency), can be produced rapidly in mass production.
Accordingly, the present invention provides a method for injecting
a sample to the specific position and depth of the amphibian
oocytes by using the apparatus of the present invention.
[0065] It is found that the sample injection efficiency can be
improved by using the apparatus of the present invention as
follows: Namely, in the case of beginners, who have no experience
for sample injection by manual operation, about 30 minutes are
required for injecting samples into 25 cells, and the injection
efficiency is about 30% in case of the expression rate using a
sample of gene. On the contrary, using the apparatus of the present
invention, time for injecting samples into 25 cells requires only 3
minutes, and the injection efficiency reaches about 80%. In case of
the sample injection performed by the experts with manual
operation, almost no shortage of time for injection is observed,
but the injection efficiency can be improved up to 90% by using the
apparatus of the present invention as compared with efficiency of
80% in the manual operation.
[0066] Consequently, as a result of using the apparatus and method
of the present invention, about 80-90% efficiencies can be achieved
without depending upon the skill of the operators, and to sell or
assign the plurality of oocytes with controlled condition of sample
injection can be possibly achieved by the present invention.
[0067] Therefore, in the other aspect, the present invention
provides the amphibian oocytes with guaranteed injection of the
sample into the specific position and depth.
[0068] Further, the oocytes, to which the sample is injected in the
specific area and depth, can be collected, sold or assigned. In the
occasion of sale or assignment, a plurality of oocytes are
packaged, and a label 22, in which the information on the the type
of sample, the injection date, the term for guarantee of quality,
setting condition such as the position and the depth of sample
injection, and guaranteed injection efficiency are described, can
be attached (FIG. 3).
[0069] According to the present invention, the oocytes can be sold
or assigned with the information on the specific depth and area of
the injection, and on the expression of protein encoded by the
injected gene. In the event of selling or assigning the guaranteed
oocytes regarding efficiency of sample injection or efficiency of
expression, the efficiency of sample injection can be guaranteed,
for example, by co-injecting a sample with protein containing
pigment or chromophore, fluorescent protein, or gene encoding these
proteins, counting numbers of oocytes emitting color or
fluorescence, and indicating the ratio as an indicator of
efficiency of sample injection. Although the coinjection can be
performed in the mixed form, in cases that both the sample to be
coinjected and protein for detection are injected in the form of
gene, a gene coding fused protein can be used.
[0070] Referring to FIG. 4, an example for calculating the
efficiency of sample injection or efficiency of expression by using
fluorescence from co-injected fluorescent protein as an indicator,
will be explained. In the present example, when that rate of
injection of histamine receptor gene is determined by using
expression of the green fluorescent protein derived from Aequorea
forbesiana (GFP) as an indicator, is explained. However, the
injected samples are not limited to genes. Further, substances used
for indicator of efficiency of sample injection are not limited to
GFP.
[0071] A mixture of histamine receptor gene and green fluorescent
protein gene is injected into the oocyte by using the apparatus
assembled with the above constitution or the above principles.
After 24 hours from gene injection manipulation, light of
wavelength at 488 nm is irradiated on the oocytes, and then the
expressed green fluorescent protein emits fluorescence of 507 nm.
Oocytes with emitted fluorescence of 507 nm are classified into
"light" and oocytes without emitted fluorescence is classified into
"dark". FIG. 4 shows that these oocytes are stimulated with
histamine and are classified by the presence or absence of
response. As shown in FIG. 4, in the "light" oocytes, 85% of
oocytes (34 out of 40 cells) respond to histamine. Namely, the
histamine receptor gene can be injected in more than 85% of the
oocytes. On the contrary, in the "dark" oocytes, 90% or more of
oocytes can not respond to histamine (27 out of 28 cells). Namely,
histamine receptor gene can not be injected in 90% or more of
oocytes. Accordingly, it is demonstrated that frequency of
injection of histamine receptor gene is high in the oocytes
expressing green fluorescent protein.
[0072] As clearly demonstrated by the above fact, when the
objective sample is co-injected with the fluorescent protein, the
efficiency of injection of the objective sample can be calculated
by existence of fluorescence as the indicator, as a result, sale or
assignment of oocytes with guaranteed efficiency of sample
injection is possible.
[0073] Next, a means for production, sale or assignment of oocytes
obtained by the present invention for the specific use, which is
exemplified by using oocytes injected with human histamine receptor
cRNA, will be described. However, gene for use of injection is not
limited to cRNA, and DNA, RNA and oligonucleotide can be used.
[0074] The histamine receptor cRNA is injected into oocyte by using
apparatus and method of the present invention. In this case, when a
means for obtaining visual information such as a sensor for
discriminating black and white color or CCD camera/digital camera
is used, the oocytes to which cRNA is injected in the animal
hemisphere, and the oocytes to which cRNA is injected in the
vegetal hemisphere can be differentiated.
[0075] After gene injection, histamine receptor is expressed in the
oocytes within 24 hours. After passing 24 hours from histamine
receptor gene injection, membrane potential of the oocytes, in
which histamine receptor may be expressed, is held at -60 mV by
clamping with two electrodes. Under such conditions, the addition
of a sample containing the histamine to the oocytes results in
interacts between the histamines in the sample and the histamine
receptors, and the signal transduction system in each oocyte is
activated to generate an ionic current, then the electric response
of the oocyte against histamine can be shown. The oocytes, after 24
hours, are stimulated with 1 .mu.M histamine and its electric
current response is measured. FIG. 5 shows comparison of electric
current response of the oocytes, to which a gene is injected in the
animal hemisphere and in the vegetal hemisphere. As shown in FIG.
5, differences between electric current responses against histamine
depending on the injected site of the gene are observed. Namely,
the oocytes with good ligand response can be obtained in the case
that gene is injected in the animal hemisphere rather than in the
case that gene is injected in the vegetal hemisphere.
[0076] Consequently, for example, the oocytes group, to which
histamine receptor cRNA is injected only in the animal hemisphere,
can be used as good sensors with high sensitivity for histamine.
Further, according to the present invention, sale or assignment of
oocytes, to which the sample is injected into the specific position
and depth, for the purpose of using them as sensors, can be made.
As easily recognized by a person skilled in the art, examples of
samples to be injected into oocytes according to the present
invention to be used as sensors, include, but are not limited to,
genes encoding receptors for any ligands other than for histamine,
antibodies having reactivity for specific antigens, and
glycoproteins having specific sugar chain limited.
[0077] A histamine receptor gene 31 is injected into the oocyte 13
by using the apparatus assembled with the above constitution or
using the above principles. In FIG. 6, the injection into the
vegetal hemisphere is shown. After injection of the histamine
receptor gene, histamine receptor 32 is expressed in the oocyte 13
within 24 hours. As discussed above, after passing 24 hours from
the histamine receptor gene injection, the membrane potential of
the oocytes, in which the histamine receptor 32 may be expressed,
is held at -60 mV by clamping with two electrodes. Under such
conditions, the addition of a sample 33 containing histamine to the
oocyte 13 results in interaction between the histamines in the
sample and the histamine receptors, and the signal transduction
system in the oocyte is activated to generate an ionic current,
then an electric response 34 of the oocyte 13 against histamine can
be shown. In case that a sample 35 without histamine is added,
since no substance, i.e., histamine, is available to anteract with
the receptor, the oocyte 13 cannot respond to histamine 36.
[0078] The oocyte expressing histamine receptor response (against
histamine containing samples) can be used as an indicator. Since
mass production of oocytes having identical condition for injection
of the sample is made possible by using the apparatus of the
present invention, the amphibian oocytes can be used for screening
ligand or antigen reacting with receptor and antibody respectively.
The screening can be performed by using plurality of oocytes, in
which a sample, such as gene, is injected under a substantially
equal condition such that protein or other substances is expressed,
and comparing the result of reactions of oocytes with defferent
ligands.
[0079] Further, in the other use of the present invention, for
example, the expressed protein can be extracted by crushing the
oocytes in which protein is expressed. The protein and other
products can be effectively extracted, for example, by controlling
the condition for injection using apparatus of the present
invention, and by using oocytes, to which a sample is injected into
the animal hemisphere.
[0080] A method for transporting the oocytes according to the
present invention, to each of which the sample is injected, will be
described referring to FIG. 6. In case of sale and transport of the
oocytes, the oocytes 13 are put into the vessel 21, to which the
solution of buffer conventionally used for the amphibian oocytes
dissolved with antibiotics, such as gentamicin sulfate, penicillin
and streptomycin, is filled. The vessel 21 is packaged using a
packaging material, such as styrene foam, and is preferably
transported by maintaining a temperature at 4-25.degree. C., more
preferably at 18-22.degree. C. by using a cold insulator 23 while
avoiding shock, as shown in FIG. 3.
[0081] The composition of the solution is not limited, and the
composition of the following can be used preferably. The solution
PH is adjusted to 7.5.
1 NaCl 96 mM KCl 2 mM CaCl.sub.2 1.8 mM MgCl.sub.2 1 mM HEPES 5 mM
Gentamicin sulfate 50 .mu.g/ml Sodium pyruvate 2.5 mM Penicillin 10
U/ml Streptomycin 10 .mu.g/ml
[0082] The preferable vessels for sale and transport are not
limited. Preferably, oocytes can move relatively freely inside the
vessels with cap being freely opened and closed, and the solution
is preferably filled to about 95% of the vessel volume. For
example, in case of a conical tube of 50 ml, preferably about
100-200 oocytes, more preferably about 130-180 oocytes, are
contained. This ratio corresponds to about 0.3-0.5 ml/oocyte, which
can be varied depending on types of oocytes and types of
vessels.
[0083] As shown in FIG. 7, the oocyte 13, to which the sample has
been injected, is recovered from the tray 9 by using a syringe 41.
In this time, based on the recorded information, any one of the
oocytes, to which the sample is injected into the animal hemisphere
or the vegetal hemisphere, can be recovered. The recovered oocytes
are transferred into the vessel 21. A buffer solution for Amphibia
42 is filled in the vessel 21, and the solution is exchanged for
several times. After several exchanges, new buffer solution for
Amphibia 42 is filled in the the vessel 21 again, and a preferable
amount of antibiotics 43 is added thereto. The cover of the tube is
closed and put into the outer case 44 with a cold insulator. The
outer case 44 is filled with a packaging material 45 to fix the
vessel 21 therein and transported to the customer by conventional
transporting means.
[0084] By this method, the vessel can be transported to the
customer without damaging the function of oocytes to which the
sample is injected.
[0085] In the occasion of sale or assignment, the information
including date of injection, place of injection of sample,
condition for injection such as depth, recovery rate and term for
guarantee of quality is provided. Ppaper describing such
information may be attached or a label 22 may be attached to the
vessel 21 containing the oocytes (FIG. 3).
[0086] If a gene is injected, about 24 hours is required for its
expression, and the life-span of the oocytes is about 7 days after
injection, preferably about 5 days for safety. Consequently, it is
preferable to describe a date and time of injection and, a
corresponding description indicating that "best use before X X
(day-month)" in order to clearly indicate an effective term for use
after injection.
[0087] As for the injection site of the oocyte, a sample can be
injected to the animal hemisphere in about 80% of oocytes by using
the above tray, and it is also possible to improve expression
efficiency based on the accumulated information in the apparatus as
well as using co-expression using the present apparatus.
EFFECT OF THE INVENTION
[0088] According to the present invention, the sample can be
injected into the oocytes of Amphibia, such as frog, with a
constant depth precisely such that mass production of such oocytes
having an identical quality and the injection can be rapidly and
efficiently performed. Quality of an oocyte or an area of needle
injection can be recorded as the information.
[0089] Further, the oocytes obtained by the method of the present
invention and into which the sample is injected in the specified
position and depth, can be recovered, and the efficiency of
injection of sample is guaranteed for the purpose of sale or
assignment. In addition, selling or assigning of the oocytes can be
made by specifying the use depending on the type of the injected
sample.
* * * * *