JP6870178B2 - Egg cytoplasmic injection method - Google Patents

Description

The present invention relates to an injection pipette that punctures an egg, a cell, or the like, and more particularly to an egg cytoplasmic injection method using an injection pipette that is used in an egg cytoplasmic injection method and is microdriven by a piezoelectric element.

Conventionally, in biotechnology, a new genetic information body, a fertilized egg, has been created by artificially manipulating genes, cells, and the like. Micromanipulators that apply physical and mechanical manipulations to cells, nuclei, fertilized embryos, etc. that can be observed with an optical microscope are used. Micromanipulators have become indispensable for artificial manipulation of cells and the like.

The micromanipulator is equipped with a microinstrument (micropipette) and a micromoving device for moving the micropipette in a linear direction. The micropipette is also called an injection pipette.

[Microinjection operation into the egg cytoplasm by the conventional method]
FIG. 13 is a diagram illustrating a conventional method of a micropipette in an intracytoplasmic egg injection method. As shown in FIG. 13 (a), in the conventional method, a micropipette (hereinafter referred to as “conventional pipette CV”) having a sharp-pointed spike-processed tip on the ruptured membrane of the egg 60 fixed to the holding pipette 68 is used. It is used. The sperm 70 is sucked into the conventional pipette CV.

Therefore, as shown in FIG. 13B, while the conventional pipette CV penetrates the zona pellucida 61 of the egg 60, the entire egg is deformed inward and the zona pellucida 61 is pushed to the egg cytoplasm 65. An increase in internal pressure may occur.

Further, since the tip portion 75 (FIG. 13 (a)) of the conventional pipette is sharply spiked, it is indicated by an arrow while the egg cytoplasmic membrane 64 is not sufficiently stretched as shown in FIG. 13 (c). The rate of film rupture due to stress on the site increases.

Further, as shown in FIG. 13D, in the puncture of the egg cytoplasmic membrane 64, a part of the egg cytoplasm 65 is sucked into the conventional pipette CV, so that the egg cytoplasm 65 may be agitated.

For this reason, instead of the conventional method using a micropipette with a sharp tip, a piezoelectric microdrive device (piezomicromanipulator (also referred to as "PMM") that punctures an egg with a micropipette with a flat tip using a piezoelectric element. )It has been known.

Patent Document 1 discloses a piezoelectric micro drive device. According to Patent Document 1, the piezoelectric microdrive device includes a micropipette microdevice that is supported by an appropriate frictional force on a holder via an abutting member, a flange portion formed on the microdevice, and the flange portion. The piezoelectric element is provided in the above and is arranged concentrically with the micro device, and an inertial body attached to the piezoelectric element and applying an impact force to the micro device by driving the piezoelectric element. It is disclosed that the impact force generated by the inertial body is generated by the drive of the inertial body, and the impact force is used to pierce the egg.

Further, Patent Documents 2 to 4 also disclose that an impact force is generated by an inertial body by driving a piezoelectric element, and an egg is punctured by using this force.

As described above, in the piezoelectric micro drive device in the so-called piezo ICSI method, an inertial body fixed to the rear end portion of the piezoelectric element is provided as described in the prior art document.

[Microinjection operation into the egg cytoplasm by the piezo ICSI method]
Here, a microinjection operation of sperm into the egg cytoplasm using a piezoelectric microdrive device will be described. FIG. 14 is a diagram illustrating the piezo ICSI method of a micropipette in the intracytoplasmic egg injection method.

As shown in FIG. 14A, the first polar body 66 of the egg 60 to be injected is held by the holding pipette 68 at the 6 o'clock or 12 o'clock position, and is directly below the polar body when the egg 60 is punctured. Avoid damaging the egg nucleus of the egg.

As shown in FIG. 14B, the tip of the injection pipette PI is lightly pressed against the zona pellucida 61. The sperm 70 at this time is located inside the injection pipette PI slightly away from the tip of the injection pipette PI.

With the tip of the injection pipette PI lightly in contact with the zona pellucida 61, the piezoelectric element (piezo) is driven, and the injection pipette PI pierces the zona pellucida 61 and pushes the injection pipette PI to the last layer. Leave the last layer, stop the piezoelectric element and push it open manually.

The drive setting of the piezoelectric element is performed with respect to the number of vibrations of the piezoelectric element and the magnitude of the vibration intensity. Further, the drive setting of the piezoelectric element is changed according to the puncture condition of the transparent band 61 or the like. If a fragment of the zona pellucida 61 is contained in the injection pipette PI, it is discharged to the outside of the egg 60 or into the ovum 67.

As shown in FIG. 14 (c), the sperm 70 is moved to the tip of the injection pipette PI, and the tip of the injection pipette PI is pressed against the egg cytoplasmic membrane 64 to extend the egg cytoplasmic membrane 64. Advance the needle tip from half to two-thirds of the egg cytoplasm 65.

At this time, the egg cytoplasmic membrane 64 is stretched and deformed as shown in FIG. 14 (c). The piezoelectric element 48 is driven at a position of half to two-thirds of the egg cytoplasm 65. That is, when the egg cytoplasmic membrane 64 in the microinsemination operation is punctured by using the piezoelectric microdrive device, the tip of the injection pipette PI is brought into contact with the egg cytoplasmic membrane 64 and moved so as to push it, and the egg cytoplasmic membrane 64 is sufficiently extended. After that, the piezoelectric element of the piezoelectric microdrive device is operated to break the film, and the sperm 70 is injected into the egg 60 to complete the operation.

After penetrating (breaking) the egg cytoplasmic membrane 64, sperm 70 is injected so as not to inject the culture medium as much as possible. At this time, as shown in FIG. 14 (d), the egg cytoplasm 65 moves in the direction indicated by the arrow so as to restore the original state.

If the ovum cytoplasmic membrane 64 cannot be sufficiently stretched and the ovum cytoplasmic membrane 64 is ruptured in the middle of stretching from the extension start point toward the center, the sperm 70 is injected without operating the piezoelectric microdrive device. To do. After injecting the sperm 70, slowly pull out the injection pipette or the like. This completes the microinjection operation into the ovum cytoplasm of the pig.

In this case, in the intracytoplasmic injection method, the setting of the injection pipette for sperm injection is a micropipette, which is a filler in which the operation liquid (fluorinert) is sucked in advance before being attached to the pipette holder 35 (shown in FIG. 3). Use to fill the vicinity of the center of the injection pipette with operation liquid (fluorinert) with a width of about 1 to 1.5 cm. The filling device is not limited to the micro pipette, and may be any device that can fill the operation liquid in the injection pipette.

The filling of the operation liquid (fluorinert) into the injection pipette is performed in order to efficiently transmit the impact force from the piezoelectric element to the tip of the injection pipette, and acts as an impact transmission liquid.

In these so-called piezo ICSI micromanipulators, the piezoelectric microdrive device is used to puncture an egg, and normally, an injection pipette punctures an egg due to an impact force from an inertial body driven by a piezoelectric element. In addition to the minute drive of the egg, other positioning operations are possible with other manipulators.

Special fair 6-98582 Special fair 6-488975 Special fair 6-98583 Special fair 7-73830

As described above, the setting of the injection pipette for sperm injection is such that the operation liquid (florinate) is sucked in advance before attaching to the pipette holder in order to efficiently transmit the impact force from the piezoelectric element to the tip of the injection pipette. Using a pipette, fill the vicinity of the center of the injection pipette with Operation Liquid (Florinate) with a width of about 1 to 1.5 cm. As a result, it was necessary to fill the injection pipette with the operation liquid before attaching it to the pipette holder, which required time for operation.

Therefore, it is required to simplify the operation related to the injection pipette and realize efficient driving of the PMM without using an operation liquid such as Fluorinert.

As described above, there is a demand for an injection pipette having excellent puncture property, operability and high membrane extensibility without using an operation liquid.

Therefore, the present invention is an injection pipette in an egg cell in mammals, and uses an injection pipette that is minutely driven by an impact force of an inertial body connected to the piezoelectric element by driving the piezoelectric element. It is not necessary to fill the operation liquid (florinate) near the center of the PMM, and by using the existing components, it is possible to realize efficient driving of the PMM, further improve the operability, and drive the piezoelectric element. An injection pipette was used, which has excellent extensibility and puncture properties of the egg membrane by the previous injection pipette, and can improve the blastocyst development rate, which can lead to the improvement of the egg survival rate . It is an object of the present invention to provide an intraplasmic egg injection method.

In order to achieve the above object, the intra-cytoplasmic injection method according to the present invention is performed in the cytoplasm of mammals in mammals by using an injection pipette that is micro-driven by the impact force of an inertial body connected to the piezoelectric element by driving the piezoelectric element. In the injection method, the injection pipette has a main tapered portion in which the outer peripheral surface and the inner peripheral surface on the distal end side are tapered, and the inner peripheral surface of the injection pipette is tapered on the distal end side of the main tapered portion. It has a tip taper whose angle is larger than the taper angle of the main tapette, and the tip of the injection pipette is pressed against the egg cytoplasmic membrane to extend the egg cytoplasmic membrane, and then the injection pipette is microdriven. It is characterized by breaking the egg cytoplasmic membrane and injecting sperm into the egg cytoplasm.

Further, the tip tapered portion is characterized by having a heat shrinking portion formed by applying heat to the tip of the tip tapered portion to shrink the glass tube.

Further, the tip tapered portion further has an outer peripheral surface having a taper angle larger than the taper angle of the outer peripheral surface of the main tapered portion between the heat shrinking portion and the main tapered portion, and the main taper portion. It is characterized by having a sub-tapered portion having an inner peripheral surface larger than the taper angle of the inner peripheral surface of the portion.

Further, the tip tapered portion is composed of an outer peripheral surface having a taper angle larger than the taper angle of the outer peripheral surface of the main tapered portion and an inner peripheral surface having a taper angle larger than the taper angle of the inner peripheral surface of the main tapered portion. It is characterized by that.

Further, the annular portion in contact with the transparent body of the egg cell or the egg cytoplasmic membrane located at the tip of the injection pipette is formed in a direction orthogonal to the axial direction on the tip side of the injection pipette, and the said. The transition portion of the injection pipette from the annular portion to the outer peripheral surface and the inner peripheral surface is chamfered.

Further, it is characterized in that a culture solution can be used as a shock transmitting solution to be injected into the injection pipette.

Further, the mineral oil is sucked adjacent to the tip side of the injection pipette of the impact transmission liquid injected into the injection pipette, and further injected into the egg cell substance adjacent to the tip side of the injection pipette of the mineral oil. It is characterized by sucking the liquid to be used.

Further, the inner diameter of the injection pipette is larger than the maximum diameter of the sperm head to be injected into the egg cytoplasm.

Further, the inner diameter of the tip of the injection pipette is larger than the maximum diameter of the sperm head to be injected into the egg cytoplasm, and the minimum required predetermined amount for the sperm to be taken in and out from the tip of the injection pipette. It is characterized by having a clearance other than the value of the diameter of the sperm head.

Further, the heat-shrinkable portion is characterized in that the ratio of the inner diameter to the outer diameter is smaller than that of the main taper portion.

Further, the ratio of the inner diameter to the outer diameter of the main taper portion of the injection pipette is 80% or more and less than 100%.

Further, the injection pipette is micro-driven by the impact force of the inertial body connected to the electric strain element by replacing the piezoelectric element with the electric strain element.

According to the intracytoplasmic infusion method of the present invention, although the principle of the intracytoplasmic instillation method for work in a very small area is unknown in many parts, the inventor has the following effects as a result of trial and error. I found that I could get it.

That is, according to the present invention, the injection pipette having the main taper portion on the tip side is provided with the tip taper portion whose inner peripheral surface taper angle is larger than the taper angle of the main taper portion on the tip side further than the main taper portion. As a result, the inner peripheral surface is reduced in diameter toward the tip, so that the flow of the culture solution or the like in the injection pipette can be adjusted, and the operability of the injector is improved.

Further, the tip tapered portion of the injection pipette has a heat-shrinkable portion formed by contracting the glass tube by applying heat to the tip, and the heat-shrinkable portion has a curved tip with a rounded tip and a narrow inner diameter of the tip. Therefore, the suction and discharge of the culture solution or the like by the injector can be finely adjusted, and the operability of the injector of the micromanipulator in the piezo ICSI method is improved.

In the injection pipette used in the intracytoplasmic injection method of the present invention, the ring portion located at the most advanced end is formed in a direction orthogonal to the axial direction on the tip side of the injection pipette, and the ring of the injection pipette is formed. As a result of trial and error, it was found that the internal pressure to the egg cytoplasm did not increase because the transitional portion from the portion to the outer peripheral surface and the inner peripheral surface was chamfered. Thereby, it is possible to provide an intracytoplasmic injection method using an injection pet having excellent puncture property, operability and high membrane extensibility.

Further, the injection pipette used in the intracytoplasmic injection method of the present invention is the same as the conventional one by using the culture solution, which is a constituent element in the first place, as a shock transmission solution, without preparing an operation liquid such as fluorinert. Injection into the egg cytoplasm by the Piezo ICSI method becomes possible.

Further, according to the injection pipette used in the injection method into the egg cytoplasm of the present invention, it is not necessary to inject the operation liquid into the injection pipette, and the injection step in the egg cytoplasm can be simplified.

Further, the injection pipette used in the intracytoplasmic injection method of the present invention has a reliable puncture performance when manipulating an egg / embryo by PMM. That is, in the conventional method, the puncture may vary depending on the shape of the pipette, the quality of the egg, and the skill level of the operator, but the injection pipette according to the present invention sufficiently extends the membrane with stable puncture performance. It is possible to perform the same puncture every time.

In addition, the injection pipette used in the intracytoplasmic injection method of the present invention can sufficiently extend the fragile egg cytoplasmic membrane of an aged egg by rounding the tip shape, and the egg survival rate after sperm injection can be improved. You can expect it.

It is a figure which shows the appearance and structure of the injection pipette used in this invention, (a) is the figure which shows the whole of the injection pipette, (b) is the main taper part of the injection pipette, and the tip which is located at the tip side of the main taper part. FIG. 3C is an enlarged cross-sectional view showing the configuration of the tip tapered portion of the region A in the broken line circle shown in FIG. 1B. It is an enlarged cross-sectional view which shows the shape of the side surface of the tip in the heat shrink part of the injection pipette used for ICSI operation, and the size of each part. The shape of the heat-shrinkable portion, (c) shows the shape of the heat-shrinkable portion with the tip greatly rounded. It is a block diagram which shows the structure of the piezoelectric microdrive device to which the injection pipette used in this invention is connected. It is a figure which shows the measurement evaluation flow of the puncture property and the operability to the transparent pig egg by an injection pipette. It is a graph which shows the measurement result of the puncture property of PMM by the difference in the amount of culture liquid sucked into an injection pipette, (a) is the measurement result of puncture at speed 2 of a controller, and (b) is the measurement result of puncture at speed 5 of a controller This is the measurement result. It is a figure which shows the evaluation result of the operability of an injector by the difference in the amount of culture liquid sucked into the injection pipette shown in FIG. It is a figure which shows the measurement result of the puncture property of the porcine zona pellucida without operation liquid. It is a figure which shows the measurement evaluation result of the puncture property and operability of various injection pipettes. It is a figure which shows the measurement result of the extensibility of the egg cytoplasmic membrane of various injection pipettes. It is a figure which shows the result of the pig microinsemination without the operation liquid in an injection pipette. Operation with injection pipettes of 7 types The number and proportion of surviving eggs in intracytoplasmic sperm injection of pigs with and without liquid (egg survival rate (%)) and the number of blastocysts and their proportion (blastocyst incidence (%)) It is a figure which shows the result of). It is a figure which shows the result of the blastocyst development rate in the pig microinsemination with and without the operation liquid by the injection pipette of 7 kinds of shapes shown in FIG. It is a figure explaining the conventional method of the micropipette in the intracytoplasmic egg injection method. It is a figure explaining the piezo ICSI method of a micropipette in the intracytoplasmic egg injection method.

Hereinafter, a mode for carrying out the intracytoplasmic injection method according to the present invention will be described with reference to the drawings. The present invention is an intracytoplasmic sperm injection method (ICSI) in mammals , which is an intracytoplasmic sperm injection using an injection pipette which is micro-driven by an impact force of an inertial body connected to the piezoelectric element by driving the piezoelectric element. The injection pipette used in the present invention has a tip taper portion in which the taper angle of the inner peripheral surface of the injection pipette is larger than the taper angle of the main taper portion on the tip side of the main taper portion. As a result of trial and error, the inventor has found that the culture solution to be injected inside without an operation liquid can be used as a shock transmission solution.

[Composition of injection pipette]
1A and 1B are views showing the appearance and configuration of the injection pipette used in the present invention, FIG. 1A is a view showing the entire injection pipette, and FIG. 1B is a main taper portion and a main taper portion of the injection pipette. FIG. 1 (c) is a view showing the outer shape of the tip tapered portion located on the tip side of the above, and is an enlarged cross-sectional view showing the configuration of the tip tapered portion of the region A in the broken line circle shown in FIG. 1 (b).

The present invention is an improvement on the shape of the injection pipette PI of the piezo ICSI method of the micropipette in the intracytoplasmic egg injection method described in FIG. 14 and the operation method thereof, and the same contents as those in FIG. 14 have the same agreement. Will be explained using.

As shown in FIG. 1A, the injection pipette 1 is made of a glass tube and is opened with a main taper portion 3 formed in a tapered shape and a tip taper portion 7 located on the tip side of the main taper portion 3. It has a bent portion 25 provided at a predetermined distance from the tip of the glass, and a straight tubular base 27 attached to a pipette holder 35 (shown in FIG. 3) and having a predetermined length and a constant outer diameter. ing. In FIG. 1A , the boundary between the main taper portion 3 and the base portion 27 is shown by a broken line.

As shown in FIG. 1 (b), the main tapered portion 3 of the injection pipette 1 has a length of, for example, about 15 mm from the tip of the injection pipette 1 and is formed in a tapered shape that reduces in diameter toward the tip. And has a taper angle θ1.

A tip taper portion 7 is provided on the tip side of the main taper portion 3. The tip tapered portion 7 located on the tip side of the main taper portion 3 has a taper angle θ2. Further, the tip tapered portion 7 has a heat shrinking portion 12 whose tip is heat-shrinked. In FIG. 1B, the boundary between the main taper portion 3 and the tip taper portion 7 is indicated by a broken line.

As shown in FIG. 1 (b), the taper angle (θ2 shown in FIG. 1 (b)) of the tip tapered portion 7 located on the tip side of the main tapered portion 3 is the taper angle of the main tapered portion 3 (FIG. 1 (B). It is larger than θ1) shown in b).

The tip tapered portion 7 shown in FIG. 1B has an outer peripheral surface 10 in which the taper angle θ2 between the heat shrinking portion 12 and the main tapered portion 3 is larger than the taper angle θ1 of the outer peripheral surface 4 of the main tapered portion 3. And the taper angle (not shown) is larger than the taper angle (not shown) of the inner peripheral surface (not shown) of the main tapered portion 3 (shown in FIG. 1 (c)). ) Is provided. The sub-tapered portion 9 has a length of, for example, about 0.25 mm from the tip of the injection pipette 1.

As described above, the tip tapered portion 7 is composed of the heat shrinking portion 12 and the sub-tapered portion 9, and the sub-tapered portion 9 is composed of the outer peripheral surface 10 having a taper angle θ2 larger than the taper angle θ1 of the outer peripheral surface of the main tapered portion 3. Moreover, it is composed of an inner peripheral surface 11 (shown in FIG. 1 (c)) larger than the taper angle (not shown) of the inner peripheral surface of the main tapered portion 3. As a result, the taper state is sharply tapered from the main taper portion 3 toward the tip taper portion 7. The injection pipette 1 having the tip tapered portion 7 is referred to as a radish type (denoted as RD).

Further, when the taper angles of the outer peripheral surface and the inner peripheral surface of the tip tapered portion 7 are formed at the same angle as the taper angles of the outer peripheral surface and the inner peripheral surface of the main tapered portion 3, the injection pipette 1 Is composed of only the main taper portion 3. Hereinafter, the injection pipette 1 composed of only the main taper portion 3 will be referred to as a parallel type (referred to as P). The heat shrinkage portion 12 may be formed even in the parallel type.

The heat-shrinkable portion 12 of the injection pipette 1 shown in FIG. 1 (c) is formed by applying heat to the tip of the injection pipette 1 by fire polishing to shrink the glass tube. By applying heat to the tip, the surface is melted, and a chamfered annular portion 14 is formed at the tip of the heat-shrinkable portion 12.

That is, the annular portion 14 is formed in a direction orthogonal to the axial direction on the distal end side of the injection pipette 1, and the end surface of the transition portion 19 from the annular portion 14 to the outer peripheral surface 9 of the injection pipette 11 is It is chamfered. Further, in the injection pipette 1, the end surface of the transition portion 21 from the annular portion 14 at the tip to the inner peripheral surface 11 is chamfered. In other words, in FIG. 1C, the apex portion sandwiched between the transition portion 19 and the transition portion 21 is the annular portion 14.

As a result, it was invented that the annular portion 14 comes into contact with the zona pellucida of the egg cell or the egg cytoplasmic membrane during intracytoplasmic injection to prevent the occurrence of an increase in internal pressure into the egg cytoplasm, and the egg cytoplasmic membrane can be sufficiently extended. It turned out as a result of trial and error of the person.

The size of the inner diameter of the injection pipette 1 shown in FIG. 1 (c) (di shown in FIG. 1 (c)) is larger than the maximum diameter of the sperm head injected into the egg cytoplasm 65 (shown in FIG. 12). It has become. For example, the diameter of the sperm head of a pig is about 3.5 micronmeter (μm), and the inner diameter of the injection pipette 1 used for a pig (also referred to as a pig) is due to the stickiness peculiar to pig sperm. In order to prevent adhesion with other sperm, a clearance other than the maximum diameter of the sperm head is required, and as a result, the minimum required inner diameter of the injection pipette 1 used for pigs is about 6 μm, and the injection Since the required clearance differs depending on the experience and operating characteristics of the person who operates the pipette 1, the maximum value is about 9 μm.

In addition, the diameter of the head of a human sperm is also about 3.5 micrometers (μm), and since it is not sticky like a pig, it is not necessary to set a large clearance, and it is also referred to as a human (also referred to as a human). ), The minimum required inner diameter of the injection pipette 1 is a size exceeding about 3.5 μm.

Therefore, normally, if the injection pipette 1 maintains a clearance of more than 0% and 65% or less with respect to the inner diameter of the injection pipette 1 with respect to the diameter of the sperm head, the inside of the injection pipette 1 can be compared with that of the mammalian sperm. It can be taken in and out, and the inner diameter of the injection pipette 1 is set in consideration of the diameter of the sperm head of the mammal and the clearance other than this diameter (inner diameter-maximum diameter of the sperm head).

That is, the inner diameter of the injection pipette 1 depends on the characteristics of sperm depending on the animal species (adhesiveness due to sperm acrosome reaction, for example, shape of sperm head such as hook-shaped sperm head like mouse sperm head). A size that is larger than the maximum diameter of the head is required. In addition, the required clearance differs depending on the experience and operating characteristics of the person who operates the injection pipette 1. Therefore, the clearance of the inner diameter of the injection pipette 1 is 65% at the maximum with respect to the inner diameter. On the other hand, since the inner diameter of the injection pipette 1 may be substantially the same as the maximum diameter of the sperm head, the clearance of the inner diameter of the injection pipette 1 is 0 at the minimum with respect to the inner diameter. % (Slightly above the maximum diameter of the sperm head).

As shown in FIG. 1A, the bending portion 25 provided at a predetermined distance from the opened tip is provided with a bending position at a distance of, for example, 1 to 5 mm from the tip of the injection pipette 1, and is bent. The bending angle of the portion 25 is, for example, 20 degrees.

As for the bending angle, when the bending angle from the base 27 forming the base of the injection pipette 1 is 0 degrees, the entire injection pipette 1 is formed straight. The injection pipette 1 is not limited to the one having a bending angle, and a straight one may be used.

The base portion 27 attached to the pipette holder 35 has a length of, for example, about 50 mm, and has an internal space communicating with the heat shrinking portion 12 located at the tip end. One end of the straight tubular base 27 is connected to the main taper 3, and the other end is fixed to the pipette holder 35.

[Shape of the tip of the injection pipette]
Next, the shape of the tip of the heat-shrinkable portion of the injection pipette used for ICSI operation for pigs will be described.

FIG. 2 is an enlarged cross-sectional view showing the shape of the side surface of the tip of the heat-shrinkable portion of the injection pipette used for ICSI operation for pigs and the size of each portion, and FIG. 2A is a shape with a flat tip. FIG. 2B shows the shape of the heat-shrinkable portion with a small rounded tip, and FIG. 2C shows the shape of the heat-shrinkable portion with a large rounded tip.

The outer diameter do1 of the injection pipette 1 having a flat tip shown in FIG. 2A is 10 μm (micron meter), and the inner diameter di1 is 9 μm. An injection pipette with a flat tip is referred to as flat F. The outer diameter do1 of the injection pipette having a flat tip is also referred to as the tip outer diameter.

In FIG. 2B, the tip of a glass tube having an outer diameter of 11 μm is rounded to a small size by fire polishing so that the tip outer diameter df1 is 10 μm and the inner diameter di2 is 8 μm. Further, from the annular portion 14 of the heat shrinking portion 12 located at the most advanced end of the injection pipette 1 to the outer peripheral surface 10 of the sub-tapered portion 9 corresponding to 25 to 30 μm in the axial direction (t1 shown in FIG. 2B). , Transition unit 19. The injection pipette 1 having the heat-shrinkable portion 12 having the tip rounded to a small size is referred to as a small rounding type R.

In FIG. 2C, the tip of a glass tube having an outer diameter of 12 μm is rounded by fire polishing so that the tip outer diameter df2 is 10 μm and the inner diameter di3 is 6.8 μm. .. Further, from the annular portion 14 of the heat shrinking portion 12 located at the most advanced end of the injection pipette 1 to the outer peripheral surface of the sub-tapered portion 9 corresponding to 35 to 40 μm in the axial direction (t2 shown in FIG. 2C). The transition unit 19. An injection pipette 1 having a heat-shrinkable portion 12 having a large rounded tip is referred to as a large rounded type HR.

As shown in FIGS. 2 (b) and 2 (c), the thickness of the glass tube of the heat-shrinkable portion 12 at the tip is increased by rounding the tip of the injection pipette 1. The dimensions of the injection pipette described above are merely examples, and are not limited thereto.

[Preparation of pipette for microinsemination]
Next, the preparation of the injection pipette 1 will be described. To prepare the injection pipette 1 shown in FIG. 1, the material is a glass tube, the outer diameter is 1 mm, the wall thickness of the glass tube is about 50 μm, that is, the ratio of the inner diameter to the outer diameter of the glass tube is 90% (hereinafter referred to as glass tube). “90 tubes”), a glass tube wall thickness of about 75 μm (hereinafter referred to as “85 tubes”), and a glass tube wall thickness of about 125 μm (hereinafter referred to as “75 tubes”) are used. Set the glass tube in the glass tube enlarger and stretch the glass tube thinly by the program according to the desired shape.

The stretched glass tube is attached to the pipette holder of the glass tube processing machine. The glass tube processing machine heats a glass ball on a platinum wire attached to a heater to perform various processing. The glass tube is broken by quickly adhering a heated glass ball to a portion of the glass tube having a predetermined thickness by a glass tube processing machine. For the folded glass tube, the position of the heated glass ball of the glass tube processing machine is adjusted to a portion having a length of 1 mm or more from the tip, and if necessary, the folded glass tube is bent at a predetermined angle.

Further, in the injection pipette 1 made of a glass tube, a heat-shrinkable portion 12 is formed by applying heat to the tip of the injection pipette 1 by a fire polish to melt the surface. Further, the fire polish forms a transition portion 19 which is a transition portion from the outer peripheral surface 10 of the injection pipette 1 to the annular portion 14 and a transition portion 21 which is a transition portion from the annular portion 14 to the inner peripheral surface 11. To. By adjusting the heating time of the fire polish and the like, the tip shapes shown in FIGS. 2 (b) and 2 (c) can be obtained.

[Piezoelectric microdrive device configuration]
Next, the configuration of the piezoelectric microdrive device to which the injection pipette 1 is connected will be described.

FIG. 3 is a block diagram showing a configuration of a piezoelectric microdrive device to which the injection pipette 1 is connected. As shown in FIG. 3, the piezoelectric microdrive device 30 includes a pipette holder 35 for attaching the injection pipette 1, a slide base 40 for attaching the piezoelectric microdrive device 30 to the XYZθ manipulator 55 of the microscope, and a piezoelectric element 48. It has a drive unit 45 and the like.

The pipette holder 35 includes a holder grip 36 for attaching and fixing the injection pipette 1 to the tip thereof. An O-ring is provided inside the holder grip 36. The end of the straight tubular base 27 of the injection pipette 1 is inserted into the pipette holder 35 and tightened with the O-ring to tighten the injection pipette 1. To fix.

The injection pipette 1 used in the present invention is used without an operation liquid, but it is also possible to fill the injection pipette 1 with an operation liquid and use it as in the conventional case.

As shown in FIG. 3, a drive unit 45 is attached between the holder grip 36 of the pipette holder 35 and the slide base 40. The pipette holder 35 is fixed to the slide base 40 by the clamp screw 41 provided on the slide base 40. The slide base 40 has a connection portion (not shown) for connecting to the XYZθ manipulator 55 provided in the microscope. By connecting the connecting portion of the slide base 40 and the XYZθ manipulator 55 of the microscope, the tip of the injection pipette 1 moves in conjunction with the movement of the XYZθ manipulator 55.

In the so-called piezo ICSI method micromanipulator of the present invention, the piezoelectric microdrive device 30 is used when puncturing an egg, and the injection pipette 1 uses an egg due to an impact force from an inertial body driven by a piezoelectric element. In addition to the minute drive at the time of puncturing, other positioning operations are possible by the movement of the XYZθ manipulator 55.

In this way, the piezoelectric microdrive device 30 to which the injection pipette 1 is connected is connected to the XYZθ manipulator 55 of the microscope via the slide base 40. The connection between the piezoelectric microdrive device and the XYZθ manipulator 55 of the microscope is not limited to the use of the slide base 40, and may be other than that.

The drive unit 45 located between the holder grip 36 of the pipette holder 35 and the slide base 40 incorporates an inertial body 49 connected to one main surface (vibrating surface) of the piezoelectric element 48, and the other of the piezoelectric elements 48. It is fixed to the pipe of the pipette holder 35 via a flange portion 47 to which the main surfaces of the pipette holder 35 are connected. By locating the drive unit 45 between the holder grip 36 of the pipette holder 35 and the slide base 40, the force can be efficiently transmitted to the tip of the injection pipette 1. The slide base 40 may be attached between the holder grip 36 of the pipette holder 35 and the drive unit 45.

The piezoelectric micro drive device 30 includes a controller 54 that controls the piezoelectric element 48, a display panel (not shown) that is connected to the controller 54 and sets driving conditions for the piezoelectric element 48, and a switch that drives the piezoelectric element 48 (not shown). (Not shown) and are provided.

The controller 54 is piezoelectric by, for example, a speed (denoted as Speed in the drawing) which is a frequency applied to the piezoelectric element 48 and an intensity (denoted as Intensity in the drawing) which is the magnitude of vibration applied to the piezoelectric element 48. The element 48 is controlled. As an example, the controller 54 can set the speed from 1 to 16 and the intensity from 1 to 16, and the minimum value of the output is 1 and the maximum value is 16. The output to the piezoelectric element 48 with respect to the set value of the controller 54 changes linearly.

The injection pipette 1 is minutely driven by the impact force of the inertial body 49 connected to the piezoelectric element 48 by driving the piezoelectric element 48. Details of the impact force generated in the injection pipette 1 by the inertial body 49 driven by the piezoelectric element 48 are known and disclosed in Japanese Patent Application Laid-Open No. 6-98582 and the like, and thus the description thereof will be omitted. Even if the piezoelectric element 48 is an electrostraining element, the injection pipette 1 can be minutely driven by the impact force of the inertial body 49.

Further, as shown in FIG. 3, a pneumatic injector 52 is connected to the end of the pipette holder 35 opposite to the holder grip 36 via a tube 53. The air supplied from the pneumatic injector 52 flows through the drive unit 45 and the pipette holder 35 and is supplied to the other end of the injection pipette 1.

The pneumatic injector 52 only connects the tube 53 to the drive unit 45, and can finely adjust suction and discharge. Therefore, a pneumatic injector is suitable as the injector. A hydraulic injector may be used as the injector instead of the pneumatic injector.

In the piezoelectric microdrive device 30 having the above configuration, the injection pipette 1 has a main taper portion on the tip side, and the taper angle of the inner peripheral surface is larger than the taper angle of the main taper portion on the tip side of the main taper portion. By providing the large tip taper portion, the inner peripheral surface is reduced in diameter toward the tip, so that the flow of the culture solution or the like in the injection pipette can be adjusted and the operability of the injector is improved.

Further, the tip tapered portion of the injection pipette 1 has a heat-shrinkable portion formed by contracting the glass tube by applying heat to the tip, and the heat-shrinkable portion has a curved tip with a rounded tip and an inner diameter of the tip. Since the space is narrowed, the suction and discharge of the culture solution and the like by the injector can be finely adjusted, and the operability of the injector of the micromanipulator in the piezo ICSI method is improved.

[Measurement of puncture property of PMM by different amount of culture solution]
Next, the experimental evaluation of the puncture property and operability of PMM into the egg transparent body by the difference in the amount of the culture solution for the injection pipette used in the present invention will be described. The puncture property shown below is a measurement of whether or not the ovum zona pellucida can be punctured by the amount of the culture solution or the like without using an operation liquid for the injection pipette.

FIG. 4 is a diagram showing a flow for measuring and evaluating the punctureability and operability of a transparent pig egg by the injection pipette used in the present invention.

First, the preparation and operation of puncture of the egg zona pellucida by the intracytoplasmic injection method in mammals will be described. The egg zona pellucida in the intracytoplasmic injection method described below uses a pig as a mammal. The intracytoplasmic egg injection method uses a piezoelectric microdrive device 30 using a piezoelectric element 48 (piezo), and the piezoelectric microdrive device 30 is attached to an XYZθ manipulator 55 provided in a microscope.

A holding pipette 68 for fixing the egg 60 (shown in FIG. 12) to be used is attached to a pipette holder (not shown). At that time, if it is connected to a hydraulic type injector, the holding pipette 68 is attached after draining the oil in the pipette holder and firmly discharging the air bubbles, but in the case of the pneumatic injector 52, it is attached as it is.

As shown in FIG. 4, the injection pipette 1 is attached to the PMM (step S1). Two types of injection pipettes 1 were used. One of the injection pipettes 1 had 85 tubes, a tip outer diameter of 6 μm, a parallel type P with only the main taper portion 3, and a tip shape of flat F. It is called 85-6-PF.

The other injection pipette 1 is a 90-tube, a radish type RD having a tip outer diameter of 5 μm, a tip taper portion 7 on the tip side of the main taper portion 3, and a rounded type R having a small tip shape. It is called -5-RD-R.

As shown in FIG. 3, a holder grip 36 for connecting the injection pipette 1 and the pipette holder 35 is provided at the tip of the piezoelectric microdrive device 30. Loosen the holder grip 36 of the pipette holder 35 attached to the piezoelectric microdrive device 30 to attach the injection pipette 1.

When the pneumatic injector 52 is connected to the end side of the pipette holder 35 opposite to the holder grip 36, loosen the holder grip 36 of the pipette holder 35 to which the piezoelectric microdrive device 30 is attached. Attach the injection pipette 1 as it is.

Insert the injection pipette 1 so as to penetrate the four O-rings in the holder grip 36, and firmly tighten the holder grip 36. First, the injection pipette 1 of 85-6-PF attached to the PMM is rinsed with a 7% PVP (polyvinylpyrrolidone) solution, and the PVP solution is discharged after the rinse (step S2).

After spitting out the PVP solution, the mixture is moved to the PZM drop which is the culture solution, and the pneumatic injector 52 is operated to suck the PZM (Porcine Zygote Medium) which is the culture solution by 5 mm from the tip of the injection pipette 1 (step S3). ..

Next, mineral oil is sucked into the tip of the injection pipette 1 (step S4). The aspirated mineral oil has the shape of an oil ball as an index indicating the boundary between different liquids.

After sucking the mineral oil, the PVP liquid is sucked from the tip of the injection pipette 1 to the position of 1 mm of the bent portion 25 of the injection pipette 1 (step S5).

Next, the puncture of the transparent porcine egg is measured (step S6). For the measurement of puncture, the first polar body 66 (see FIG. 12 (a)) of the egg 60 to be injected first is held at the 6 o'clock or 12 o'clock position by the holding pipette 68.

The controller 54 sets the drive setting of the number of vibrations of the piezoelectric element 48 and the magnitude of the vibration intensity. First, the speed, which is the number of vibrations of the piezoelectric element 48, is set to 2. With the tip of the injection pipette 1 lightly in contact with the zona pellucida 61, the piezoelectric element (piezo) 48 is driven, and it is confirmed that the injection pipette 1 pierces the zona pellucida 61 and a hole is opened in the zona pellucida 61. Measure the value of Intensity when a hole is made in. The state in which the PVP liquid is sucked into the tip of the injection pipette 1 is referred to as PVP +.

Further, the operability of the injector with the tip of the injection pipette 1 lightly pressed against the zona pellucida 61 is confirmed, and the operability of the injector is evaluated in five stages from 1 to 5 (step S7). The evaluation on a 5-point scale indicates that the injection pipette 1 can be sucked into the injection pipette 1 and discharged out of the pipette without any problem. 4 indicates that some caution is required for the operation. 3 indicates that the operation requires more attention than 4 and 5, but the puncture operation and the like can be controlled. 2 and 1 indicate a level at which the puncture operation and the like are difficult to control and cannot be operated.

Next, using the oil ball as an index, the PVP solution is discharged from the injection pipette 1 and the tip is replaced with the culture solution (PZM) (step S8). The state in which the PVP solution is discharged and the tip is replaced with the culture solution (PZM) is referred to as PVP-.

Next, the puncture is measured by the same operation as in step S6 (step S9). The operability of the injector is confirmed by the same operation as in step S7, and a five-step evaluation is performed (step S10).

Next, the process proceeds to step S3, and PZM is sucked by 7 mm from the tip. Similarly, the measurement of puncture property is repeated from step S3 by sucking PZM by 10, 12, 13, and 15 mm from the tip (step S11).

Further, the speed, which is the number of vibrations of the piezoelectric element 48, is set to 5, and the same experimental evaluation is performed from step S3.

Further, the injection pipette 1 of 90-5-RD-R is also experimentally evaluated for the same punctureability to the egg transparent body and the operability of the injector.

[Results of measurement of puncture property due to difference in culture solution volume]
The measurement results of the puncture property of PMM due to the difference in the amount of the culture solution from the tip of each injection pipette will be described below with reference to FIG. FIG. 5 is a graph showing the measurement result of the puncture property of PMM due to the difference in the amount of the culture solution sucked into the injection pipette 1, and (a) is the measurement result of the intensity value of the punctureable controller at the controller speed 2. , (B) are the measurement results of the intensity value of the punctureable controller at the controller speed 5.

The horizontal axis of the graph showing the measurement result of the puncture property of PMM shown in FIG. 5 shows each distance (width) of the aspirated culture solution amount from the tip of the injection pipette 1, and the vertical axis of the graph is the intensity of the controller. The numerical value of is shown.

As shown by 85-6-PF / PVP + in FIG. 5 (a), in the injection pipe of 85-6-PF, the amount of the culture solution from the tip of the injection pipette is 5 mm under the setting condition of Speed value 2. When aspirated with a width of 16.0, the Intensity value was 16.0, when the amount of the culture solution was aspirated with a width of 7 mm, the Intensity value was 15.0, and when the amount of the culture solution was aspirated with a width of 10 mm. The Intensity value is 11.0, the Intensity value is 6.5 when the amount of the culture solution is aspirated with a width of 12 mm, and the Intensity value is the value when the amount of the culture solution is aspirated with a width of 13 mm. 5.5. When the amount of the culture medium was aspirated with a width of 15 mm, the porcine egg could be punctured with the Intensity value set to 5.0.

As shown by 85-6-PF / PVP-in FIG. 5 (a), the PVP solution in front of the oil ball in the injection pipette of 85-6-PF is discharged into the culture solution (PZM). When replaced, under the setting condition of Speed value 2, when the amount of the culture solution from the tip of the injection pipette was aspirated with a width of 5 mm, the Intensity value was 14.0 and the amount of the culture solution was aspirated with a width of 7 mm. In this case, the Intensity value is 7.0, the Intensity value is 4.0 when the amount of the culture solution is aspirated with a width of 10 mm, and the Intensity value is the value when the amount of the culture solution is aspirated with a width of 12 mm. Is 2.5, the Intensity value is set to 2.0 when the amount of the culture solution is aspirated with a width of 13 mm, and the Intensity value is set to 1.5 when the amount of the culture solution is aspirated with a width of 15 mm. I was able to pierce the pig egg.

Further, as shown by 85-6-PF / PVP + in FIG. 5 (b), the injection pipette of 85-6-PF is a culture solution from the tip of the injection pipette under the setting condition of Speed value 5. When the amount is aspirated with a width of 5 mm, the Intensity value is 14.0, and when the amount of the culture solution is aspirated with a width of 7 mm, the Intensity value is 12.0 and the amount of the culture solution is 10 mm. When aspirated, the Intensity value was 9.0, when the amount of the culture solution was aspirated with a width of 12 mm, the Intensity value was 6.0, and when the amount of the culture solution was aspirated with a width of 13 mm, When the Intensity value was 5.0 and the amount of the culture medium was aspirated with a width of 15 mm, the porcine egg could be punctured with the Intensity value set to 4.5.

As shown by 85-6-PF / PVP-in FIG. 5 (b), the PVP solution in front of the oil ball in the injection pipette of 85-6-PF is discharged into the culture solution (PZM). When replaced, when the Speed value is set to 5, the amount of culture medium from the tip of the injection pipette is sucked with a width of 5 mm, the Intensity value is 12.0, and the amount of culture medium is sucked with a width of 7 mm. The Intensity value is 6.0, the Intensity value is 3.0 when the amount of the culture solution is aspirated with a width of 10 mm, and the Intensity value is the value when the amount of the culture solution is aspirated with a width of 12 mm. 2.0. When the amount of culture solution is aspirated with a width of 13 mm, the Intensity value is set to 1.5, and when the amount of culture solution is aspirated with a width of 15 mm, the Intensity value is set to 1.5. The pig egg could be pierced.

Similarly, in FIGS. 5 (a) and 5 (b), the measurement result of PVP + of the injection pipette 90-5-RD-R is 90-5-RD-R / PVP +, and the measurement result of PVP- is 90-5. It is indicated by -RD-R / PVP-.

From the puncture property measurement result shown in FIG. 5, the 85-6-PF attached without the operation liquid has the Intensity value of the controller 54 when the suction amount of the culture solution from the tip of the injection pipette is less than the width of 12 mm. Was high and the puncture property was extremely reduced. Further, it was confirmed that the puncture property was slightly improved when the suction amount of the culture solution was 12 mm or more and the PVP solution was contained.

The 90-5-RD-R attached without the operation liquid improved the puncture property when the suction amount of the culture solution was 7 mm or more, and the puncture property was stable even when the PVP solution was contained.

As described above, as a result of investigating the influence of the suction amount of the culture solution as a setting condition of the injection pipette attached without the operation liquid by the test, if the culture solution is sucked with a width of 12 mm or more, the puncture property of both pipettes is stable. However, 90-5-RD-R is more stable than 85-6-PF because the puncture property is improved and stable when the suction amount of the culture solution is 7 mm or more. It turned out to be superior.

The difference between the two types of injection pipettes tested is that the thickness of the glass tube, the shape of the tapered part and the tip shape are different, but the 90-5-RD-R, which is an injection pipette with a radish type RD taper, has no operation liquid. The puncture property is extremely stable.

The 85-6-PF has a shape in which the tapered portion gradually narrows, so that the width of the culture solution sucked from the tip is the same as that of 90-5-RD-R, but the actual volume of the culture solution is small. Therefore, it was found from the experimental results that the puncture property was low.

Further, both 90-5-RD-R and 85-6-PF injection pipettes can obtain puncture property by aspirating a large amount of the culture solution, but the intensity under the condition that the PVP solution is contained. Considering the stability such as a low value of and the time and effort required for setting the time for sucking the culture solution, 90-5-RD-R is much more practical.

[Operability due to differences in the amount of culture medium]
Next, the operability of the injector due to the difference in the amount of the culture solution sucked into the injection pipette shown in FIG. 5 will be described. The operability of this injector means the operability of a delicate operation such as suction and discharge of a culture solution or the like into an injection pipette by the injector.

FIG. 6 is a diagram showing the evaluation results of the operability of the pneumatic injector due to the difference in the amount of the culture solution sucked into the injection pipette.

As shown in FIG. 6, when the PVP solution is sucked at the tip with respect to the suction amount of the culture solution in the evaluated injection pipettes 85-6-PF and 90-5-RD-R (denoted as PVP +), the PVP solution is used. The evaluation results in the case of discharge (denoted as PVP-) are shown in 5 stages from 1 to 5.

As shown in FIG. 6, there was no difference in the operation with the pneumatic injector depending on the suction amount of the culture solution, but when the PVP solution was not contained, 85-6-PF was the injector. The operability was slightly lowered, and 90-5-RD-R was more stable.

As a result, it was found that 90-5-RD-R was superior to 85-6-PF in terms of punctureability to the transparent porcine egg and operability of the injector. That is, an injection pipette having 90 tubes, a radish type RD having a tapered tip, and a rounded type R having a small tip shape is suitable.

As described above, considering the measurement results of the puncture property of the PMM and the operability of the injector due to the difference in the amount of the culture solution sucked into the injection pipette 1 shown in FIGS. 5 and 6, the culture solution is different from the conventional operation liquid. It was confirmed that when the same amount (amount corresponding to 10 to 15 mm in the injection pipette) was used, the puncture effect was exhibited without the operation liquid by combining with the shape of the injection pipette 1 according to the present invention.

As for the culture solution, an example using PZM for pig puncture was described. Although the type of culture solution differs depending on the mammal species, the injection pipette 1 according to the present invention has almost no effect on the puncture performance due to the difference in the culture solution. Confirmed not to give.

[Evaluation of injection pipette puncture performance]
Next, the puncture performance when the 90-5-RD-R injection pipette 1 prepared using 90 ultra-thin glass tubes was used in a piezo micromanipulator (PMM) under the condition of no operation liquid was evaluated. Similarly, an 85-6-P-HR injection pipette made of 85 tubes with an outer diameter of 6 μm, a parallel type P composed of only the main taper, and a rounded type HR with a large tip shape is also operated. An evaluation test of the puncture performance of PMM was conducted under the condition of no liquid. As shown in FIG. 7, the evaluation of the puncture performance is divided into five test groups of C1, T1, T2, T3, and T4, and the speed of the controller 54 for puncturing the porcine zona pellucida in each test group. , Intensity figures were investigated.

Using the injection pipette 90-5-RD-R for which the setting was completed, the porcine egg was punctured under the setting conditions of the test plots C1, T1, T2, and T3.

The outline of each test plot will be described first. In the test group C1, a small amount of oil ball (also referred to as a small oil ball) is sucked into the tip of the injection pipette 1 of 90-5-RD-R, and after sucking the small oil ball, a larger amount of culture solution is sucked. , A small oil ball is held at the bending position of the injection pipette. The bent position portion is about 1 mm from the tip of the injection pipette and is shown below and as “rear” in FIG. In the test group T1, the puncture operation was performed not only in the rear position of the small oil ball in the injection pipette but also in the vicinity of the tip of the injection pipette. The vicinity of the tip of the injection pipette is about 500 μm from the tip of the injection pipette, and is shown below and in FIG. 7 as “forward”. The test group T2 is a state in which the amount of oil balls is increased 10 times as much as that of the test group C1 and the oil balls in the injection pipette are located in the front and the rear. Test group T3 is a state in which a large amount of PVP solution is sucked into the injection pipette instead of the culture solution. As described above, the test group T3 is in a state in which a large amount of PVP solution is sucked in place of the large amount of the culture solution in the test group C1.

Next, using the injection pipette 85-6-P-HR for which the setting was completed, the porcine zona pellucida was punctured under the conditions of the test group T4, and the values of Speed and Intensity were investigated. In addition, it was confirmed whether the injection pipette of 85-6-PR made of rounded type R having a small tip shape also had puncture property into the zona pellucida of the porcine egg. Test group T4 is a state in which each amount of the culture solution to be sucked, each amount of the oil ball, and the oil ball in the injection pipette are located rearward.

Next, the detailed results of each test plot will be described. FIG. 7 is a diagram showing the measurement results of the puncture property of the porcine zona pellucida without the operation liquid. As shown in FIG. 7, in the test group C1, when the 90-5-RD-R injection pipette prepared using 90 tubes was attached to the PMM without the operation liquid, a large amount of culture solution was sucked into the injection pipette. Then, under the condition that a small amount of oil ball (small oil ball) is sucked and the position of the small oil ball is set to the rear, the pig egg can be pierced with the setting of Intensity value 3.5-4.5 and Speed value 5. The operability of the injector was also stabilized.

In test group T1, when the position of the small oil ball in the injection pipette was held behind (A), puncture was possible with an Intensity value of 1.5 and a Speed value of 8, but the position of the oil ball was set to (B). ), The puncture was not possible with an Intensity value of 1.5 and a Speed value of 8, and the same puncture property was not obtained.

In the test group T2, even if the amount of oil balls in the injection pipette is about 10 times that of the test group C1, if the position of the oil balls is held behind (A), the puncture performance similar to that of a small amount of oil balls can be obtained. Obtained. Even when the oil ball is large, if the position of the oil ball is held in front of (B), puncture cannot be performed and the puncture performance deteriorates. However, even if the position of the oil ball is set to the front as in (C), the controller 54 It was possible to puncture by raising the setting value of Intensity of.

In the test group T3, when the culture solution in the injection pipette is replaced with the PVP solution and the position of the small oil ball is rearward as in (A), the Intensity value is 5.0 and the Speed value is 3, and the culture solution is used. The puncture performance was lower than that of. Similarly, in (B), the position of the small oil ball was rearward, the Intensity value was 3.5, and the Speed value was 8, and the puncture performance was lower than that in the case of the culture solution.

In the test group T4, the puncture operation was performed using the 85-6-P-HR produced by using the 85 tube, and the position of the oil ball was set to the rear in all of (A) to (E). Operation of environment in pipette (A) No liquid As a result of puncture test under the condition of sucking a large amount of culture solution and a small amount of oil balls (small oil balls), puncture was possible with an Intensity value of 4.5 and a Speed value of 5. It was. By aspirating a large amount of culture solution in the in-pipette environment (B), puncture can be performed with an Intensity value of 3.0 and a Speed value of 5, and when the Speed value is increased to 8 as in the in-pipette environment (C). Puncture was performed with an Intensity value of 2.0 to 2.5. Further, as in the pipette environment (D), when the amount of oil balls was double that of the test group C1, when the Speed value was increased to 8, puncture was performed with an Intensity value of 2.5 to 3.0. As shown in the pipette environment (E) for these (A) to (D), when the culture solution is made small with a small oil ball, the Intensity value is 8.0 and the Speed value is increased to 8. I couldn't puncture.

Further, as a result of performing a puncture test using an 85-6-PR prepared using an 85 tube under the same conditions as the in-pipette environment (A) in the test group T4 shown in FIG. 7 without an operation liquid, Intensity The puncture was performed with a value of 4.5 to 5.5 and a Speed value of 5.

As described above, from the results of the test group C1 and the test group T3 using the 90-5-RD-R injection pipette, when a large amount of the culture solution was sucked into the injection pipette, the PVP solution was contained in the injection pipette. It was found that the puncture performance was higher than that in the case of a large amount of suction.

In addition, from the results of the test group T1 and the test group T2, it was found that the position of the oil ball made of mineral oil in the injection pipette affects the puncture property to the egg. That is, the puncture property is reduced in the front where the position of the oil ball is near the tip of the injection pipette. Therefore, keep this in mind in actual operation.

In addition, from the results of the test group T4, it was found that puncture was not possible when a small amount of the culture solution was sucked into the injection pipette.

Next, the measurement and evaluation results of the puncture property of the injection pipette 1 and the operability of the injector in various glass tube materials will be described.

FIG. 8 is a diagram showing the measurement and evaluation results of the puncture property and the operability of the injector of various injection pipettes 1. The measurement and evaluation were performed on 75 pipes, 85 pipes, and 90 pipes.

A 75-7-PF injection pipette made of 75 tubes is attached to the PMM without an operation liquid, a large amount of culture solution is sucked from the tip of the injection pipette, a small amount of oil is sucked, and then the PVP solution is sucked up to the bent portion 25. A suction test was performed on the zona pellucida of the pig egg. As shown in FIG. 8, in the state where the PVP solution is sucked into the tip of 75-7-PF marked with + in the column of presence / absence of PVP, the speed value is set to 2 and the intensity value is set to 16.0 to the pig egg. It was possible to puncture, and when the Speed value was increased to 5, it was possible to puncture with an Intensity value of 15.0. In the state where the PVP solution marked with-in the presence / absence of PVP column is discharged and the tip is replaced with the culture solution (PZM), the pig egg can be punctured with the setting of Speed value 2 and Intensity value 12.0, and the Speed value can be changed. When raised to 5, the puncture was possible with an Intensity value of 9.0.

Further, as shown in FIG. 8, an injection pipette made of 85 tubes has an 85-6-PF having a flat tip shape and an 85-6-PR having a rounded type R having a small tip shape. In the case of 85-6-P-R with a rounded tip, the Intensity value was lower than that of 85-6-PF with a flat tip, and the puncture property was improved.

Further, as shown in FIG. 8, when the injection pipette 90-5-RD-R made of 90 tubes is attached to the PMM without the operation liquid, the PVP is + and the speed value is 2 and the intensity value is 4.5. It was possible to puncture, and when the Speed value was increased to 5, it was possible to puncture with an Intensity value of 3.0. When the PVP was −, the pig egg could be punctured with the Speed value of 2 and the Intensity value of 2.5, and when the Speed value was increased to 5, the Intensity value of 2.0 could be punctured.

Thus, the puncture property of the 75-7-PF attached to the PMM without the operation liquid was extremely reduced compared to other pipettes. On the other hand, it was confirmed that the injection pipette 90-5-RD-R has higher puncture performance than other injection pipettes.

Further, as shown in FIG. 8, regarding the operability of the injector, it was possible to control the injection, extraction, etc. of the culture solution in all of the 75 tubes, 85 tubes, and 90 tubes.

As a result, it was found that the injection pipette 90-5-RD-R can be used with a small intensity value of the controller 54, so that the injection pipette 90-5-RD-R has good puncture property and the injector operability is also good.

[Measurement of extensibility of egg cytoplasmic membrane with various injection pipettes]
Next, the measurement results regarding the extensibility of the egg cytoplasmic membrane by various injection pipettes will be described.

FIG. 9 is a diagram showing the measurement results of the extensibility of the egg cytoplasmic membrane of various injection pipettes. The extensibility of the egg cytoplasmic membrane was measured with a culture medium or the like without using an operation liquid for the injection pipette 1.

As shown in FIG. 9, the injection pipette 1 used has a material of 90 tubes and a tip outer diameter of 10 μm, and the shape of the main taper portion 3 is RD (radish type) and P (parallel type), respectively. The tip shape of is F, R, HR, and consists of a total of 6 types. The test was performed four times for each injection pipette 1, the puncture operation was performed without the operation liquid, the number of operated eggs and the number of surviving eggs were totaled, and the number of eggs with membrane fragility was counted.

As shown in FIG. 9, in the PMM in which the operation liquid is not used for the injection pipette 1, the "puncture% of the membrane fragility" is 40% or more when the taper shape is RD and the tip shape is F, but the tip shape is R. In HR, the tip shape was about 20% of half of F. Further, when the taper shape is P and the tip shape is R and HR, the ratio of "puncture% of membrane fragility" is lower than that of the tip shape F, and in particular, when the tip shape is HR, superiority is observed. ..

The "survival rate" of the number of eggs is 99.0% when the taper shape is RD and P, the tip shape of the injection pipette is R and HR, and the taper shape is RD.

From this, it was confirmed that in the PMM in which the operation liquid is not used for the injection pipette 1, the injection pipette 1 having a tapered shape of RD or a tip shape of R and HR is effective in improving the egg survival rate.

In addition, by rounding the tip shape of the injection pipette, it is possible to sufficiently extend the fragile egg cytoplasmic membrane of an aged egg (an egg found in elderly women) during microinsemination, and the egg after sperm injection. It can be expected to improve the problem of reduced survival rate.

[Results of microinsemination of pigs without operation liquid]
Next, the results of microinsemination of pigs in an injection pipette without an operation liquid will be described.

FIG. 10 is a diagram showing the results of microinsemination of pigs in an injection pipette without an operation liquid. Two types of injection pipettes 1 were used: 90-10-RD-R, whose tip was rounded less by fire polishing, and 90-10-RD-HR, whose tip was rounded more by fire polishing.

As shown in FIG. 10, microinsemination was performed using 90-10-RD-R and 90-10-RD-HR prepared using 90 tubes under the condition that the culture solution was aspirated without filling with the operation liquid. As a result, the conditions for PMM that could puncture the porcine zona pellucida (zona) were an Intensity value of 3.5-5.0 and a Speed value of 5-8 for both pipettes. The egg cytoplasmic membrane was punctured with an Intensity value of 4.0 and a Speed value of 1 for both pipettes.

The survival rate after microfertilization was 100% for both pipettes. As a result of in vitro culture of microfertilized ova for 6 days, embryos that had developed to blastocysts were observed in both groups. The blastocyst development rate was higher in 90-10-RD-R.

As a result of microinsemination of pigs without operation liquid, it was found that 90-10-RD-R was superior as an injection pipette.

[Differences in blastocyst development rate due to differences in the shape of injection pipettes with and without operation liquid]
Furthermore, in order to clarify the superiority of blastocyst development due to the difference in the shape of the injection pipette with and without the operation liquid in the Piezo ICSI method, blastocyst development was performed using PMM, and blastocysts after porcine intracytoplasmic sperm injection were performed. The injection pipette was evaluated based on the measurement of the incidence.

The injection pipette 1 used for the evaluation has 90 tubes, a tip outer diameter of 10 μm, and taper shapes of RD and P, and the tip shapes of 90-10-RD-HR and 90-10 are composed of HR, R, and F, respectively. 6 types of -RD-R, 90-10-RD-F, 90-10-P-HR, 90-10-P-R, 90-10-PF, 75 pipes, tip outer diameter 12 μm, There are a total of seven types, one type of 75-12-PF having a taper shape of P and a tip shape of F. Pig microinsemination with or without operation liquid was performed with 7 types of injection pipettes 1 to evaluate the blastocyst development rate.

[Operation procedure for pig microinsemination]
The outline of the operation procedure of porcine intracytoplasmic sperm injection with an injection pipette with and without operation liquid is described below.

The petri dish for ICSI used in the injection pipette with operation liquid has one drop of 10-20 μl high osmotic PZM (hPZM) in the center, and a drop of 7% PVP solution for rinsing and sperm suspension. Two were prepared and covered with mineral oil.

The petri dish for ICSI used in the injection pipette without operation liquid has one 10-20 μl hPZM drop in the center, one PZM drop for setting the injection pipette, 7 for rinsing and 7 for sperm suspension. Two drops of% PVP solution were prepared and covered with mineral oil.

The high osmotic pressure PZM (hPZM) is obtained by adding sorbitol to PZM to make the osmotic pressure slightly higher than that of PZM. The reason for using high osmotic pressure PZM (hPZM) in the culture medium containing the egg in the microinsemination operation is that the egg cytoplasm is slightly contracted, making it easy to observe the first polar body and grasp the position of the egg nucleus. This is to increase the size of the egg-circulating cavity located in the gap between the zona pellucida and the egg cytoplasm. By enlarging the egg-circumferential cavity, it is possible to prevent the egg-circumferential cavity from softening the impact and damaging the egg cytoplasmic membrane when the Zona pellucida is punctured by driving the PMM. Ordinary PZM is used as a culture medium by sucking a large amount into an injection pipette for setting without an operation liquid.

The prepared petri dish was kept warm at 37-38.5 ° C. in an incubator 1-2 hours before use, and the pig in vitro matured egg to be operated was transferred to a drop of hPZM of the warmed petri dish. In addition, an appropriate amount of sperm solution was placed in a drop of 7% PVP solution for sperm suspension and suspended to prepare a petri dish for ICSI operation.

When setting an injection pipette with an operation liquid, in order to efficiently transmit the impact force from the piezoelectric element to the tip of the injection pipette, injection is performed using a micropipettor that has previously sucked the operation liquid (florinate) before attaching it to the pipette holder. Operation liquid (florinate) was filled near the center of the pipette with a width of about 1 to 1.5 cm.

After attaching the injection pipette with operation liquid to the pipette holder, it was first dropped into a drop of 7% PVP solution for rinsing, and suction and discharge were repeated by the injector to check the operability and at the same time rinse the inside and outside of the injection pipette. ..

The injection pipette without operation liquid was attached to the pipette holder, and then PZM was sucked into the injection pipette, and then mineral oil was sucked to form an oil ball for a liquid boundary. After moving to a drop of 7% PVP solution for rinsing, suction and discharge were repeated with an injector, and the operability was confirmed using the movement of the oil ball as an index, and at the same time, the inside and outside of the injection pipette were rinsed.

The following operations are the same for the injection pipette with the operation liquid and the injection pipette without the operation liquid.

The injection pipette was moved to a drop of 7% PVP solution in which sperm was suspended to initiate the operation of sperm immobilization. Sperm were selected for active movement and no deposits on the head and tail. For sperm immobilization, a method was used in which the PMM was driven to immobilize the sperm while the tip of the injection pipette was touched to the tail when the sperm was sucked and discharged from the tail into the injection pipette.

The sperm confirmed to have completely stopped moving was sucked into the injection pipette and used for the injection operation into the egg.

After sucking the sperm into the injection pipette, the sperm was transferred from the 7% PVP solution containing the sperm to the hPZM drop for injecting the egg.

For puncture of the egg, a microinjection operation into the egg cytoplasm was performed by the piezo ICSI method of the micropipette in the intracytoplasmic egg injection method shown in FIG.

After injecting sperm into the eggs, the injection pipette was pulled out to check the number of surviving eggs. Surviving eggs were transferred to PZM drops for culture and cultured in vitro for 6 days in an incubator under the conditions of _{5% O 2} , 5% CO ₂ , and 20% N _2. The in vitro cultured ICSI embryos were observed under a microscope on the 6th day, and the number of blastocysts developed was counted.

[Evaluation results of blastocyst incidence in porcine intracytoplasmic sperm injection]
Next, the evaluation results of the blastocyst development rate in porcine intracytoplasmic sperm injection with and without operation liquid due to the difference in the shape of the injection pipette will be described.

FIG. 11 shows the number of surviving eggs and their ratio (egg survival rate (%)) and the number of blastocysts and their ratio (blastocyst development) in porcine intracytoplasmic sperm injection with and without operation liquid using injection pipettes of 7 types. It is a figure which shows the result of rate (%)). FIG. 12 is a graph showing the results of blastocyst development rate in porcine intracytoplasmic sperm injection with and without operation liquid using the injection pipettes of the seven types shown in FIG.

In addition, + in the presence or absence of OL in FIG. 11 and OL + shown in FIG. 12 indicate that the operation liquid was filled in the injection pipette and the puncture operation was performed (indicated as having OL) in FIG. The presence / absence of OL-and the OL-shown in FIG. 12 indicate that the puncture operation was performed without using the operation liquid inside the injection pipette (indicated as no OL).

[Effect of blastocyst development rate depending on the presence or absence of OL for each injection pipette]
FIG. 11 shows the number of eggs used for ICSI operation (the number of operated eggs shown in FIG. 11) and the number of surviving eggs (the number of surviving eggs shown in FIG. 11) in various injection pipettes having different tapered shapes and tip shapes with and without OL. ) And its ratio (egg survival rate (%)). In addition, the number of cultured embryos used, the number of blastocysts developed, and the ratio thereof (blastocyst development rate) are shown in the column of embryonic development.

As shown in FIGS. 11 and 12, with OL + in 90-10-RD-HR, 90-10-RD-R, 90-10-P-HR and 90-10-P-R, respectively. The blastocyst development rate after microinsemination of pigs without OL (OL-) was almost the same value with little difference between with and without OL.

In addition, the blastocyst development rates after microinsemination of pigs with and without OL in 90-10-RD-F and 90-10-PF, respectively, were not significantly different, but 90-10. For both -RD-F and 90-10-PF, the blastocyst development rate tended to be higher with OL.

Further, in 75-10-PF, there is a remarkable difference in the blastocyst development rate after microinsemination of pigs with and without OL, and the blastocyst development rate may be higher with OL. It became clear. On the other hand, in the case of 75-12-PF without OL, the puncture ability to the egg was low, and the micro-fertilized embryo that could be produced did not develop up to the blastocyst.

[Effect of blastocyst development rate by combination of injection pipette shape and presence / absence of OL]
90-10-RD-HR and 90-10-RD-R had a high blastocyst development rate with and without OL, respectively. In particular, the blastocyst development rates with and without OL of 90-10-RD-HR and without OL of 90-10-RD-R are blastocysts with and without OL of 90-10-PF. It was confirmed that the blastocyst development rate and the blastocyst development rate of 75-12-PF without OL were significantly higher. That is, it was confirmed that 90-10-RD-HR and 90-10-RD-R are effective against the blastocyst development rate even without OL.

In addition, the blastocyst development rate of 75-12-PF with OL is as high as the blastocyst development rate of 90-10-RD-HR and 90-10-RD-R with or without OL. It was significantly higher than the blastocyst development rate of 90-10-PF with or without OL and the blastocyst development rate of 75-12-PF without OL.

In addition, the incidence of blastocysts without OL of 75-12-PF was low in the ability to puncture eggs, and the microfertilized embryos that could be produced did not develop into blastocysts.

Thus, a piezo using 90-10-RD-HR, 90-10-RD-R, 90-10-P-HR and 90-10-P-R without OL, which is not filled with operation liquid (OL). The ICSI method has the same result as the case of the injection pipette filled with the conventional operation liquid OL, and it has been confirmed that the shapes of HR and R for polishing the tip can be effectively used with the piezo micromanipulator (PMM) without the OL. did it.

In particular, in 90-10-RD-HR and 90-10-RD-R, the blastocyst development rate is high in both with and without OL, and since without OL is equal to or higher than that with OL, the taper shape is radish. The superiority of RD, which is a type (radish type), was recognized. Further, it was found that HR, which is a heat-shrinkable portion with a large rounded tip, and R, which is a heat-shrinkable portion with a small rounded tip, are dominant in the tip shape.

That is, in the 90-10-RD-HR and 90-10-RD-R without OL, the puncture performance and the operation of the injector can be ICSI operation comparable to the conventional condition with OL. Minimize the handling time of eggs and sperms outside the body (outside the injector) because it is not necessary to fill the injection pipette with the operation liquid as with OL and the time required to confirm the setting can be shortened. It is presumed that this has made it possible and has brought about good results in embryogenesis.

On the other hand, pipettes 90-10-RD-F and 90-10-PF having a flat tip using 90 tubes are 90-10-RD-HR, 90-10-RD-R, 90-10. The reason why the blastocyst development rate was lower than that of -P-HR and 90-10-P-R is considered to be the material of the glass tube and the shape of the tip. That is, since the 90-tube glass tube is ultra-thin, in the case of 90-10-RD-F and 90-10-PF with flat tip processing, the shape becomes sharp and the puncture performance of PMM becomes high. Is thought to affect the egg cytoplasmic membrane.

The blastocyst development rate was slightly higher in the 90-10-RD-F and 90-10-PF with and without the OL because the injector was filled with the OL. It is considered that the operability is stable. Furthermore, 90-10-RD-F tended to have a higher blastocyst development rate than 90-10-PF, but this was because RD, which is a radish type (radish type), became ICSI. It is presumed that the operability of the injector is suitable. Thus, from the results of the blastocyst development rate in 90-10-RD-HR and 90-10-RD-R, it was confirmed that the radish type RD is effective.

Further, in the case of 75-12-PF having a flat tip shape consisting of 75 tubes used for manipulation operations in the past, the thickness of the glass tube is thin, but it is not too thin, so the tip shape is 90. Not as sharp as a tube. Therefore, if the conventional method of using PMM for filling the operation liquid, sufficient puncture performance was obtained and the operation could be performed without affecting the egg cytoplasmic membrane, so that good embryogenesis was obtained after ICSI. It was thought to be. However, since the injection pipette consisting of 75 tubes has a slightly thicker glass tube, the puncture performance by PMM is greatly reduced under the condition without operation liquid, and accurate microinsemination operation cannot be performed. It is presumed that it did not occur. From this result, it became clear that the wall thickness of the glass tube in the injection pipette is important for the puncture performance.

As described above, according to the present invention, the injection pipette having the main taper portion on the tip side has a tip in which the taper angle of the inner peripheral surface is larger than the taper angle of the main taper portion on the tip side of the main taper portion. By providing the tapered portion, the inner peripheral surface is reduced in diameter toward the tip, so that the flow of the culture solution or the like in the injection pipette can be adjusted and the operability of the injector is improved.

Further, the tip tapered portion of the injection pipette has a heat-shrinkable portion formed by contracting the glass tube by applying heat to the tip, and the heat-shrinkable portion has a curved tip with a rounded tip and a narrow inner diameter of the tip. Therefore, the suction and discharge of the culture solution or the like by the injector can be finely adjusted, and the operability of the injector of the micromanipulator in the piezo ICSI method is improved.

In the injection pipette used in the intracytoplasmic injection method of the present invention, the ring portion located at the most advanced end is formed in a direction orthogonal to the axial direction on the tip side of the injection pipette, and the ring of the injection pipette is formed. As a result of trial and error, it was found that the internal pressure to the egg cytoplasm did not increase because the transitional portion from the portion to the outer peripheral surface and the inner peripheral surface was chamfered. Thereby, it is possible to provide an intracytoplasmic injection method using an injection pet having excellent puncture property, operability and high membrane extensibility.

Further, the injection pipette used in the intracytoplasmic injection method of the present invention is the same as the conventional one by using the culture solution, which is a component in the first place, as a shock transmission solution, without preparing an operation liquid such as fluorinert. Injection into the egg cytoplasm by the piezoixie method is possible.

Further, according to the injection pipette used in the injection method into the egg cytoplasm of the present invention, it is not necessary to inject the operation liquid into the injection pipette, and the injection step in the egg cytoplasm can be simplified.

Further, the injection pipette used in the intracytoplasmic injection method of the present invention has a reliable puncture performance when manipulating an egg / embryo by PMM. That is, in the conventional method, puncture may vary depending on the shape of the pipette, the quality of the egg, and the skill level of the operator, but the injection pipette used in the intracytoplasmic injection method of the present invention has stable puncture performance. It is possible to fully extend the membrane and perform a similar puncture each time.

In addition, the injection pipette used in the intracytoplasmic injection method of the present invention can sufficiently extend the fragile egg cytoplasmic membrane of an aged egg by rounding the tip shape, and the egg survival rate after sperm injection can be improved. You can expect it.

The injection pipette according to the present invention is particularly effective for DNA and RNA injections and ES cell injections into blastocysts, where punctureability and injector operability are important. In addition to these, it can be effectively used for denuclearization operation in nuclear transplantation and nuclear cell collection by biopsy instead of the intracytoplasmic injection method.

According to the injection pipette of the present invention, it is possible to pierce the egg transparent band and the egg cytoplasmic membrane, so that not only sperm injection but also nuclear replacement technology and somatic cell nuclear transfer for the purpose of producing a somatic cell clone can be performed. It can also be applied to RNA injection for controlling gene expression, ES cell injection for producing recombinant animals, DNA injection, and genome editing technology.

The present invention can be embodied in many forms without departing from its essential properties. Therefore, it goes without saying that the above-described embodiments are merely explanatory and do not limit the present invention.

1 Injection pipette 3 Main taper part 4 Outer surface of main taper part 7 Tip taper part 9 Sub-taper part 10 Outer surface of sub-taper part 11 Inner peripheral surface of sub-taper part 12 Heat shrinkage part (tip part)
14 Ring part 19, 21 Transition part 25 Bending part 27 Base part (straight pipe part)
30 Piezoelectric Micro Drive (PMM)
35 Pipette holder 36 Holder grip 40 Slide base 41 Clamp screw 45 Drive unit 47 Collar 48 Piezoelectric element (piezo)
49 Inertial body 52 Pneumatic injector 53 Tube 54 Controller 55 XYZθ Manipulator 60 Egg 61 Zona pellucida 64 Egg cytoplasmic membrane 65 Egg cytoplasm 66 First polar body 67 Surrounding egg cavity 68 Holding pipette 70 Sperm 75 Conventional pipette tip CV conventional pipette PI Injection pipette F Flat type R Small round type HR Large round type do1, do2, do3 Outer diameter of injection pipette di, di1, di2, di3 Inner diameter of injection pipette df1, df2 Outer diameter of tip of injection pipette t1, t2 Length of transition Sa

Claims (12)

It is an intracytoplasmic injection method in mammals performed using an injection pipette that is micro-driven by the impact force of an inertial body connected to the piezoelectric element by driving the piezoelectric element.
The injection pipette has a main tapered portion having a tapered outer peripheral surface and inner peripheral surface on the distal end side.
Further on the tip side of the main taper portion, the injection pipette has a tip taper portion in which the taper angle of the inner peripheral surface is larger than the taper angle of the main taper portion.
The tip of the injection pipette is pressed against the egg cytoplasmic membrane to extend the egg cytoplasmic membrane.
A method for intracytoplasmic injection, which comprises finely driving the injection pipette to break the egg cytoplasmic membrane and injecting sperm into the egg cytoplasm. The intracytoplasmic injection method according to claim 1, wherein the tip taper portion has a heat shrinkage portion formed by applying heat to the tip of the tip taper portion to contract the glass tube. The tip tapered portion further has an outer peripheral surface having a taper angle larger than the taper angle of the outer peripheral surface of the main tapered portion between the heat shrinking portion and the main tapered portion, and the main tapered portion has an outer peripheral surface. The method for intraplasmic injection according to claim 2 , further comprising a sub-tapered portion having an inner peripheral surface larger than the taper angle of the inner peripheral surface. The tip tapered portion is composed of an outer peripheral surface having a taper angle larger than the taper angle of the outer peripheral surface of the main tapered portion and an inner peripheral surface having a taper angle larger than the taper angle of the inner peripheral surface of the main tapered portion. The intraocular injection method according to claim 1, wherein the method is characterized. The ring portion in contact with the transparent body of the egg cell or the egg cytoplasmic membrane located at the tip of the injection pipette is formed in a direction orthogonal to the axial direction on the tip side of the injection pipette, and the injection pipette is formed. The intracytoplasmic injection method according to any one of claims 1 to 4 , wherein the transition portion from the annular portion to the outer peripheral surface and the inner peripheral surface is chamfered. .. Egg intracytoplasmic injection method according to claim 1 or of claim 5, or 1, wherein the availability of the culture solution as a shock transfer liquid to be injected into the interior of the injection pipette. A liquid that sucks mineral oil adjacent to the tip side of the injection pipette of the impact transmission liquid injected into the inside of the injection pipette, and further injects the mineral oil into the egg cell substance adjacent to the tip side of the injection pipette. The method for intraplasmic injection of an egg according to claim 6 , wherein the injection method is characterized by sucking the pipette. The intra-egg cytoplasmic injection method according to any one of claims 1 to 7 , wherein the inner diameter of the injection pipette is larger than the maximum diameter of the sperm head to be injected into the egg cytoplasm. .. The inner diameter of the tip of the injection pipette is larger than the maximum diameter of the sperm head to be injected into the egg cytoplasm, and the minimum predetermined clearance required for the sperm to be taken in and out of the tip of the injection pipette is provided. The intracytoplasmic injection method according to any one of claims 1 to 7, wherein the sperm has a value other than the diameter of the head of the sperm. The intracytoplasmic injection method according to claim 2 , wherein the heat-shrinkable portion has a smaller ratio of the inner diameter to the outer diameter than the main taper portion. The intracytoplasmic injection method according to any one of claims 1 to 10 , wherein the ratio of the inner diameter to the outer diameter of the main taper portion of the injection pipette is 80% or more and less than 100%. .. The substituted on the piezoelectric element, of claims 1 to 11, characterized in that finely drives the injection pipette by impact force due linked inertial body to the electrostrictive element by driving the electrostrictive elements, The intracytoplasmic injection method according to any one.

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