CN111763692B - Method for measuring enucleated volume of nuclear transfer blind suction method - Google Patents
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Abstract
The invention relates to the technical field of biology, in particular to a metering method for accurate removal of chromosome and cytoplasm of somatic cell nuclear transfer receptor cells. The method for measuring the enucleation volume of the nuclear transfer blind suction method mainly comprises the following steps of oocyte collection and maturation; preparing a coring tool; firstly, dyeing mature oocytes, measuring the amounts of enucleated chromosomes, first polar bodies and surrounding cytoplasm by using an enucleated needle, and enucleating by a blind aspiration method; only one UV observation was performed to see if the chromosome and the first polar body were removed or if the oocyte still contained a chromosome, and it was observed that the non-enucleated clean oocyte was directly discarded. In the invention, in the operation of the nuclear transfer blind suction method for enucleating, operators can quantify the enucleated volume, the enucleation can be accurately measured, the number of times of ultraviolet nuclear display for oocyte dyeing is reduced, the damage to the oocyte is reduced, the later development quality of the reconstructed embryo is improved, and the invention is economical, practical and convenient to operate.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a metering method for accurate removal of chromosome and cytoplasm of somatic cell nuclear transfer receptor cells.
Background
At present, the methods mainly applied to the aspects of mammal somatic cell nuclear transfer and enucleation comprise a half-egg enucleation method, a blind enucleation method, a DNA dyeing enucleation method, a phase difference imaging enucleation method, a chemical induction enucleation method and the like, and the methods applied to sheep are the most widely applied to the blind enucleation method and the dyeing enucleation method.
The DNA staining and enucleation method is to treat oocyte with 10 mug/mL Hoechst33342 for 10-15min, then to put the oocyte under a microscope, to excite ultraviolet light by means of an exciter to observe the positions of the first polar body and the nucleus of the oocyte, to suck the first polar body and the cytoplasm 1/6-1/5 below the first polar body by means of an injection needle, and to again observe whether enucleation is complete one by one. Since the method uses ultraviolet light to irradiate the metakaryotic chromosome and the first polar body, damage to each oocyte, especially damage to mitochondria, can be caused, and the development quality of the later cloned embryo is reduced.
The blind aspiration method is free from other chemical substances, can reduce the damage to oocytes possibly caused by certain chemical substances, and has simple and rapid operation and is widely adopted. The biggest problem of the blind suction method is that the positions of cell nuclei are invisible, whether the cell nuclei are removed or not cannot be guaranteed during the nuclear removal operation, operators can only carry out the nuclear removal operation by experience, the nuclear removal rates of operators with different technical proficiency are greatly different, and the consistency and the repeatability of the nuclear removal operation are poor.
Blind aspiration enucleation is usually performed by using a chromosome located near the polar body of the oocyte immediately after the first polar body of the oocyte is discharged, and sucking the first polar body and a part of cytoplasm in the vicinity with a micro enucleation needle to achieve the purpose of enucleation. With the extension of the enucleation operation time, the first polar body and the position of the oocyte chromosome of the same batch of non-enucleated oocytes are greatly separated, so that the enucleation quality of the oocytes is increased (1/6-1/4 of the cytoplasmic volume of the ovum is unequal), the enucleation efficiency is reduced from 95% to 71%, and the abnormal probability of the chromosome of the late-stage development embryo is increased by the reconstructed embryo containing multiple nuclei, so that the acquisition of cloned animals is reduced.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide the method for measuring the enucleated volume of the blind suction method for nuclear transfer, which is economical and practical, is convenient to operate, and can enable an operator to quantify the enucleated volume in the enucleated operation of the blind suction method for nuclear transfer, accurately measure enucleation, reduce the number of times of ultraviolet nuclear indication for oocyte dyeing, reduce the damage to the oocyte and promote the improvement of the development quality of the reconstructed embryo, and provides the method for measuring the enucleated volume of the blind suction method accurately, quickly and practically.
The invention discloses a method for measuring the enucleation volume of a nuclear transfer blind suction method, which mainly comprises the following steps:
(1) Oocyte collection and maturation: collecting sheep ovaries, cleaning, and then collecting oocytes by a thorn-dissection method, picking out mature oocytes discharged with a first polar body under a microscope for standby, and recording the duration of maturation;
(2) Preparing a coring tool: the stoning needle is a stoning needle subjected to bending treatment, the structure of the stoning needle comprises a tip, a stoning section and an extension section, the tip is provided with an oblique angle incision of 30-45 degrees, the length S of the stoning section is 360-400 mu m, and the stoning section and the extension section are bent and arc-shaped in transition; taking the diameter of fibroblasts of herbivores as a reference, wherein the inner diameter of a denucleation needle is the diameter of one fibroblast, namely 20 mu m, and the length S of the denucleation section is a denucleation critical point;
(3) Mature oocytes were first stained and then enucleated by the blind aspiration method using an enucleation needle to measure the amount of enucleated chromosomes and first polar bodies and surrounding cytoplasm, the enucleation procedure being as follows: determining an enucleation amount based on the maturation duration of the mature oocyte, said enucleation amount being measured in terms of length L of chromosome and first polar body and surrounding cytoplasm inhaled by the enucleation needle:
the maturation duration of the mature oocyte is 16 h-18 h: the length L of the suction of the enucleation needle is 0.25 fibroblast;
mature oocyte maturation duration is 18-20 h: the length L of the suction of the enucleation needle is 3.77-4.05 fibroblasts;
maturation duration of mature oocytes exceeds 20 h: the length L of the suction of the enucleation needle is 3.77-4.05 fibroblasts; when loose and discontinuous cytoplasm of the enucleated content is present, the length L of the enucleated needle is 17.11-18.33 fibroblasts, or the length L of the enucleated needle is determined according to the length of the enucleated critical point S, namely S=L;
(4) And (3) only performing one ultraviolet observation to observe whether the chromosome and the first polar body are removed or whether the oocyte also contains the chromosome, and observing that the non-enucleated clean oocyte is directly discarded.
The invention provides a method for measuring the enucleated volume, which comprises the following deduction process and principle:
1. in the research, the relative position change of the chromosome and the first polar body of each maturation stage of the oocyte is found to show a certain rule:
transferring the mature oocyte discharged from the first polar body into 10 mug/mL Hoechst33342 staining solution for 10-15min, placing the mature oocyte into a microscope for observing the relative positions of the chromosome and the first polar body, and setting the mature oocyte as a sphere approximately because of the good morphological quality of the mature oocyte, setting the oocyte as a sphere, taking the center of the sphere as a vertex, taking the radius of the first polar body as one side, taking the radius of the chromosome as the other side, and setting an included angle as an included angle a; the position of the first polar body is plotted as the position of the 12 o 'clock, with the position of the oocyte chromosome occurring from 12:00 to 14:00 o' clock, with reference to fig. 3: mature oocyte chromosome is mapped relative to the first polar body. When the chromosome is located at point 12:30, then the angle a is 15 ° when the chromosome is located at the position of the table pointer at the position of the point 13:00, the included angle a is 30 degrees, and so on; the positions of oocyte chromosomes and the first polar phase relative to the in vitro maturation time (16-26 h) are subjected to staining observation statistics, and the statistics are as follows:
TABLE 1 relative positions of mature oocyte chromosomes to first polar bodies at different time periods
As shown in table 1:
1. the maturation time is within 18h, and the first polar body of the mature oocyte and the position where the chromosome appears are all included angle a is between 0 and 15 degrees (namely, expressed by the position of the table pointer and between 12:00 and 12:30);
2. within 20h of maturation time, 98.30% (58/59=98.30%) of the mature oocytes have a first polar body and the position of the chromosome present, between 0 ° and 30 ° (i.e. expressed as the position of the table pointer, between 12:00-13:00);
3. within 22h of maturation time, 94% (47/50=94%) of the mature oocytes have a first polar body and chromosome appearance position between 0 ° -30 ° (i.e. expressed as the position of the table pointer, between 12:00-13:00); 98% (49/50=98%) of the mature oocytes were placed at an angle a between 0 ° -45 ° (i.e. between 12:00-13:30 in terms of position of the table pointer) with the first polar body of the mature oocyte and the position of the chromosome. The first polar body of all mature oocytes is located at an angle a of between 0 and 60 (i.e. expressed as the position of the table pointer, between 12:00 and 14:00) to the position where the chromosome appears;
4. within 24h of maturation time, 95.03% (41/43= 95.03%) of the first polar body of the mature oocyte is at an angle a of 0 ° -30 ° (i.e. expressed as the position of the table pointer, between 12:00-13:00) with the position of the chromosome; 97.67% (42/43= 97.67%) of the mature oocyte has a first polar body at an angle a of between 0 ° -45 ° (i.e. expressed as the position of the table pointer, between 12:00-13:30) with the position of the chromosome; the first polar body of all mature oocytes is located at an angle a of between 0 and 60 (i.e. expressed as the position of the table pointer, between 12:00 and 14:00) to the position where the chromosome appears;
5. within 26h, 96.07% (49/51=96.07%) of the mature oocyte has a first polar body and a chromosome appearance position within an included angle a of 0 ° -30 ° (i.e. expressed as the position of the table pointer, between 12:00-13:00); 98.03% (50/51=98.03%) of the position where the first polar body of the mature oocyte appears with the chromosome, appears at an angle a of between 0 ° -45 ° (i.e. expressed as the position of the table pointer, between 12:00-13:30); the first polar body of all mature oocytes is located at an angle a of between 0 and 60 (i.e. expressed as the position of the table pointer, between 12:00 and 14:00) to the position where the chromosome appears;
6. from the above, the position change of the oocyte chromosome and the first polar body basically varies between 0 ° and 60 ° at an included angle a, and the position change of 96.925% (252/260=96.92%) of the mature oocyte chromosome and the first polar body varies between 0 ° and 30 ° at an included angle a, and the position change of 98.84% (257/260=98.84%) of the mature oocyte chromosome and the first polar body varies between 0 ° and 45 ° at an included angle a.
2. Distance relationship of mature oocyte chromosome to first polar body at different time periods:
1. oocyte diameter R measurement:
the diameter and zona pellucida thickness of the oocyte were measured through the microscope eyepiece with the mature oocyte containing the first polar body, and non-uniform and abnormal oocytes were not measured. The measurement results are shown in table 2 below.
TABLE 2 difference in diameter of oocytes and zona pellucida in follicles of different diameters.
| Type of follicle | Number of oocytes | Oocyte diameter (μm) | Transparent belt thickness (mum) |
| 1.0-2.0 | 98 | 152.19±2.44 | 12.5±0.2 |
| 2.1-4.0mm | 75 | 154.29±2.49 | 12.2±0.4 |
| ≥4.0mm | 40 | 155.86±2.54 | 12.5±0.6 |
As can be seen from the measurement results, the diameter R of the oocyte varies between 152.19 and 155.86 μm.
2. The oocyte chromosome is at a height h from the sphere of the first polar body in the sphere model.
The mature oocyte has better morphological quality, can be approximately identified as a sphere, and is set as the sphere; since the oocyte is enucleated by nuclear transfer, the first polar body, the chromosome and a small amount of cytoplasm around the first polar body are removed, so that the relative volume of enucleated flat enucleated sphere can be regarded as the sphere segment of one sphere, and the internal volume of the sphere where the first polar body and the chromosome are located is set as the sphere segment.
Setting the radius of the sphere as R, the diameter as R, the height of the sphere as h, and the included angle between the chromosome and the first polar body in the sphere as a, and calculating the height of the sphere at which the chromosome and the first polar body are positioned as h by utilizing the positive and residual relationship, wherein the calculation result is as follows:
because a varies between 0-60 °, r= 152.19-155.86 μm, i.e. r= 76.09-77.93, h=r (1-Cosa);
when a=15°,
h=2.59 to 2.65 μm;
when a=30°,
h=10.19 to 10.44 μm;
when a=45°,
h= 22.29 to 22.83 μm;
when a=60°,
h= 38.04 to 38.96 μm.
Thus, it was found that the sphere height h of the entire mature oocyte chromosome and the first polar body in the oocyte was in the range of 2.59 to 38.96 μm.
3. And calculating the proportion of the enucleated mass of the oocyte in different time periods.
1. Oocyte cytoplasmic V:
the mature oocyte has better morphological quality, can be approximately identified as a sphere, can be known according to a sphere volume formula,
V＝18844383.11～1981446.79μm 3 。
2. oocyte nuclear transfer enucleated pellet volume V1:
in the case of enucleation of oocyte nuclear transfer, the relative volume of enucleation of flat enucleation can be regarded as a sphere segment because the first polar body and chromosome and a small amount of cytoplasm surrounding the first polar body are removed. Thus the segment v1=pi h 2 (r-h/3), r= 76.09 to 77.93 the results of calculating the segment volume from the segment height are as follows:
when a=15°, h=2.59 to 2.65 μm, v1= 510.52 to 547.26 μm 3 ；
When a=30°, h=10.19 to 10.44 μm, v1= 7548.19 to 8114.57 μm 3 ；
When a=45°, h= 22.29 to 22.83 μm, v1= 34113.32 to 36651.41 μm 3 ；
When a=60°, h= 38.04 to 38.96 μm, v1= 91756.91 to 98576.29 μm 3 。
3. Ratio of enucleation of oocyte nuclear transfer:
the degree of enucleation of the oocyte directly influences the development potential of the subsequent embryo, the more the enucleation volume is less beneficial to the development of the reconstructed embryo, the less enucleation is likely to not achieve the aim of clean enucleation, therefore, the determination of the enucleation amount and the visual measurement are necessary, and the enucleation amount can be calculated through the volume ratio of the spherical segment:
when a=15°, h=2.59 to 2.65 μm, V1/v= 0.000847 to 0.000859 ≡8/10000;
when a=30°, h=10.19 to 10.44 μm, then V1/v= 0.01472 to 0.01474 ≡1/68;
when a=45°, h= 22.29 to 22.83 μm, then V1/v= 0.05807 to 0.05808 ≡1/17;
when a=60°, h= 38.04 to 38.96 μm, V1/v= 0.1562 to 0.1778≡1/6.
4. Measurement of the enucleation amount of oocytes in enucleated needles for different time periods.
1. Mathematical model relationship of segment and enucleated needle:
the volume to be removed due to enucleation of mature oocytesThe above-mentioned segment is assumed, as shown in fig. 2; the enucleation tool is an enucleation needle with a grinding tip by a needle grinding instrument, and has a shape like a cylinder, as shown in fig. 1; enucleation procedure referring to fig. 4, the volume of oocyte removal can thus be scaled to the length of the enucleated needle cylinder, so that the following mathematical functional relationship exists: pi h 2 (r-h/3)＝πr Column 2 L。
2. Donor cell diameter measurement of different breeds of sheep:
ear skin cells of different sheep breeds were established by tissue mass method, and the following is briefly described: removing villi at the edge of ear with blade, scrubbing ear tissue with 75% alcohol cotton ball for 2-3 times, sterilizing for 30s, taking ear tissue with sampling forceps, adding physiological saline (containing double antibody), taking back to laboratory, removing cartilage tissue with ophthalmic scissors and forceps in ultra clean bench, washing with physiological saline for 4-5 times, soaking in 75% alcohol for 15-20s, cutting ear skin tissue in 30mm dish, culturing with tissue block method, and culturing liquid of DMEM plus 15% FCS. After primary culture for 3-5d, observing tissue block adherence and fibroblast migration, and changing liquid once every 2 d. After primary fibroblasts grow to 80% -90% and are connected into slices, the primary fibroblasts are digested by pancreatin for 3min, subcultured by DMEM culture solution containing 15% FBS (containing EGF10ng/mL and IGF10 ng/mL), changed for 1 time every 2d, transferred for 3-5 generations and frozen. Donor cells were thawed to 24-well plates using 3d before use, washed 1-2 times with PBS, digested with pancreatin, collected by centrifugation at 2.500 r/mm to 1.5mL EP tubes, and resuspended in 100 μl of culture medium for later use.
Donor cells were transferred into droplets and diameters were recorded by inverting the microscope eyepiece scale, with the following results:
table 3. Diameter of fibroblasts of the common sheep variety.
From the above measurement of the fibroblast diameter of several common sheep species, the fibroblast diameter varied around 20 μm.
3. Theoretical measurement of somatic cell nuclear transfer enucleation:
from the above-mentioned measurement results of the diameter of the fiber, the diameter of the prepared enucleated needle was 20 μm, and thus the enucleated needle diameter 2r was set Column The quantity of enucleated oocytes is 20 μm, and the volume height in the cylinder of the enucleated needle can be converted into the suction length L of the enucleated needle (also called theoretical enucleated volume quantity) or the quantity of the fibroblast number, so that the quantitative measurement of the enucleated quantity can be realized.
From pi h 2 (r-h/3)＝πr 2 Column L, r= 76.09 to 77.93 μm, known as 2r Column As can be seen from =20μm, l=h 2 (r-h/3)/r 2 Column ；
When a=15°, h=2.59 to 2.65 μm, then l=5.10 to 5.47 μm, i.e. 0.5 lattice of the eyepiece scale, also 0.25 fibroblast length, wherein eyepiece scale 1 lattice=10 μm;
when a=30°, h=10.19 to 10.44 μm, l= 75.48 to 81.14 μm, i.e. the length of 8 cells of the eyepiece scale is 3.77 to 4.05 fibroblasts;
when a=45°, h= 22.29 to 22.83 μm, l= 341.13 to 366.51 μm, i.e. the length of 35 lattices of the eyepiece scale, the length of 17.11 to 18.33 fibroblasts;
when a=60°, h= 38.04 to 38.96 μm, l= 917.57 to 985.76 μm, i.e. the length of 95 lattices of the eyepiece scale, 45.88 to 49.29 fibroblasts.
From the above, the position of the clock pointer pointing to the 12 points is taken as the position of the first polar body, and when the oocyte chromosome is at the position with the included angle of a=15°, namely, the clock pointer points to the position of 12:30, the theoretical enucleation volume L is 0.25 length of fibroblasts; when the chromosome is at the position with an included angle of a=30°, namely, when the table pointer points to the position of 13:00, the theoretical enucleation volume quantity L is 3.77-4.05 times of the length of the fibroblast; when the chromosome is at the position with an included angle of a=45°, namely, the position of the table pointer pointing to 13:30, the theoretical enucleation volume quantity L is 17.11-18.33 fibroblasts in length; when the chromosome is at the position with an included angle of a=60°, namely, the position with the table pointer pointing to 14:00, the theoretical enucleation volume quantity L is 45.88-49.29 fibroblasts in length;
4. physical measurement of somatic cell nuclear transfer enucleation:
since the first polar body of the oocyte is a sphere in which the meiotic chromosome is highly concentrated, the chromosome is also an approximate sphere, and when the calculation of the theoretical enucleated volume amount is performed in the above fourth step (3), the first polar body and the chromosome are reduced to two fluorescent spots, and the calculated theoretical enucleated volume amount L is different from the actual enucleated volume amount by two hemispheres of which the volume amounts are equivalent in size to the volume amount of 1 fibroblast by measurement, so that the actual enucleated volume amount=the theoretical enucleated volume amount l+1 fibroblast volume amount.
5. Manufacturing and measuring a enucleated needle:
referring to fig. 1, the tip of the enucleated needle was beveled at an angle of 30-45, and the volumetric volume of the tip, as measured, was equivalent to the volumetric volume of 1 fibroblast. And the remaining metric of the denucleated amount is based on the calculation result of the theoretical metric value of denucleation.
Because the enucleation needle is embedded on the operating arm sleeve of the micromanipulator, as shown in figure 1, the front end of the glass enucleation needle needs to be bent by a calciner to be in a V shape, the front end of the glass enucleation needle is also beneficial to be kept in a parallel state with the horizontal plane, the distance between the bent position and the top end is limited by the length L which is 341.13-366.51 mu m through the theoretical calculation, 98.03-98.84% (which is calculated by the number ratio of mature oocytes in theoretical data of 50/51-257/260 and the process of 16-26h maturation is a dynamic process) is limited in the range of table pointers 12:00-13:30 (the included angle a is 0-45 degrees), and the bent length of the enucleation needle is 341.13-366.51 mu m, namely the position of an eyepiece scale 35 lattice. Because the glass needle is bent to form a sharp angle, but has a certain radian, the range needs to be enlarged, and therefore, bending is required to be carried out at the positions of 36-40 grids of the eyepiece scale, namely, the positions of the length S of the denucleated sections in the figure 1 are 360-400 mu m.
The chromosomes of most mature oocytes change within the range of 12:00-13:00 in terms of the included angle a of 0-30 degrees, and the included angle a is expressed by the position of a table pointer, so that the enucleation of most mature oocytes can be realized only by completing the enucleation of the included angle a within the range of 0-30 degrees, and the enucleation ratio is 96.07-96.92% by calculating the number ratio 49/51-252/260 of the mature oocytes obtained by the theory; when the operator fails to know the microoperation instrument to perform the enucleation or the enucleation content is loose and discontinuous cytoplasm, the length L inhaled by the enucleation needle can be used for enucleating 17.11-18.33 fibroblasts, or the length S of the enucleation needle bending position can be used for performing the maximum enucleation, namely, the included angle a is 0-45 degrees, the enucleation is expressed by the position of a pointer in a range of 12:00-13:30, and the enucleation of mature oocyte chromosome with the maximum enucleation number proportion is formed, which is calculated according to the number proportion 257/260 of mature oocyte in theoretical data, namely, 98.84%. Then, an ultraviolet observation is performed to see whether the chromosome and the first polar body are removed or whether the oocyte also contains the chromosome, and the non-enucleated clean oocyte is directly discarded.
5. Mature oocyte enucleation effect.
1. Blind inhalation method enucleation effect in each time period of different classification methods:
the model results constructed according to the invention described above reclassify the cloning steps according to different literature as follows:
TABLE 4 relative positional distribution of mature oocyte chromosomes to first polar region by different taxonomies
| Time period | Total number of eggs | 12:00 | 12:30 | 1:00 | 1:30 | 2:00 |
| 16-18h segmentation method | 57 | 36 | 21 | |||
| 18-20h segmentation method | 59 | 27 | 12 | 19 | 1 | |
| 20-22h segmentation method | 50 | 7 | 31 | 9 | 2 | 1 |
| 22-24h segmentation method | 43 | 29 | 12 | 1 | ||
| 24-26h segmentation method | 51 | 1 | 39 | 9 | 1 | 1 |
| Early 16-20h method | 116 | 63 | 33 | 19 | 1 | 0 |
| Advanced 18-22h method | 109 | 34 | 43 | 28 | 3 | 1 |
| 16-26h | 260 | 71 | 132 | 49 | 4 | 2 |
The sectional cloning is a one-step method, the enucleation and injection are completed simultaneously, the enucleation reconstructed embryo completed in 2-2.5h is mainly used as a batch, the cloning, the fusion and the activation are carried out in batches, and the method is adopted by researchers in the laboratory for nearly three years. Thus, more than 95% of mature oocytes in each time period are enucleated with 3.7-4.0 fibroblast diameters.
Both the early and late stage methods described above are two-step cloning, i.e., concentrated enucleation followed by concentrated nuclear injection, which is employed by most laboratories. Early method 99.13% of oocytes completed 3.7-4 fibroblasts to achieve enucleation. Late stage 96.33% oocyte enucleation was achieved by 3.7-4 fibroblasts.
2. Sorting treatment and use after enucleation:
according to the mathematical model relationship of the invention, most mature oocytes can realize the measurement and the enucleation of the blind suction method. Firstly, the mature oocyte is subjected to Hoechesst33342 staining, then, the enucleated chromosome and the first polar body and the quantity of surrounding cytoplasm are measured by an enucleation needle, and then, the blind aspiration enucleation method is carried out, and then, the ultraviolet observation is carried out only once, whether the chromosome and the first polar body are removed or whether the oocyte also contains the chromosome or not is observed, the non-enucleated clean oocyte is directly discarded, the reconstructed embryo can be distinguished through the injected donor cell, and the 100% enucleated embryo reconstruction can be realized.
Compared with the prior art, the invention has the advantages that: in the operation of the nuclear transfer blind suction method, operators can quantify the enucleation volume, the enucleation can be accurately measured, the number of times of ultraviolet nuclear indication for oocyte dyeing is reduced, the damage to the oocyte is reduced, the later development quality of the reconstructed embryo is improved, and the nuclear transfer blind suction method is an economical, practical and convenient-to-operate measurement method of the enucleation volume.
Drawings
Fig. 1 is a schematic structural view of a enucleated needle in an embodiment of the present invention. L in FIG. 1 is the aspiration length of the enucleated needle, also the theoretical enucleated volume, and S is the length of the enucleated segment.
FIG. 2 is a schematic structural diagram of the relationship between the position model of the mature oocyte, the first polar body and the chromosome in the embodiment of the present invention.
FIG. 3 is a map of the relative positions of the chromosome of a mature oocyte and a first polar body in an embodiment of the present invention.
FIG. 4 is a schematic diagram showing a process of removing nuclei in somatic cell nuclear transfer according to an embodiment of the present invention. Wherein the fifth picture is a graphical representation of the volumetric size comparison of the first polar body and chromosome of the fibroblast cell with the oocyte. The first row of 5 spots in the picture represents 5 stained fibroblasts, and the following row of 1 spot is the enucleated length 1+3.77-1+4.05 fibroblasts measured according to the enucleated model, this spot representing the first polar body of the oocyte and the complete measured removal of the chromosome.
The figure shows: 1 is a tip, 2 is a denucleated substance, 3 is a denucleated segment, 4 is an extension segment, 5 is an operating arm sleeve, 6 is a sphere segment, 7 is a sphere radius, 8 is a first pole body, 9 is a sphere segment height, 10 is a chromosome, 11 is an included angle a, and 12 is a sphere diameter.
Detailed Description
The test methods used in this example are conventional methods unless otherwise specified. Materials, reagents and the like used are commercially available unless otherwise specified.
Part of the reagents are as follows: the reagents were obtained from Nippon and light pharmaceutical Co., ltd unless otherwise specified. M199 (GIBCO), DMEM (GIBCO), FCS (Hyclone), pancreatin (Gbico), four-well plate (NUNC), 60mm Petri dish (NUNC), micromanipulation needle, paraffin oil (sigma M8410), split microscope (Nikon SMZ 800), needle drawing instrument (NARISHIGE PN-30), needle grinding instrument (NARISHIGE EG-400), calciner (NARISHIGE MF-900), micromanipulator (Eppendorf Transferman NK), CO 2 Incubator (New Branswick Galaxy S).
Referring to fig. 1-4, which are schematic structural diagrams of an embodiment of the present invention, a method for measuring a enucleated volume of a blind suction method for nuclear transfer according to the present embodiment mainly includes the following steps:
first, oocyte collection and maturation: collecting sheep ovaries, cleaning, and then collecting oocytes by a thorn-dissection method, picking out mature oocytes discharged with a first polar body under a microscope for standby, and recording the duration of maturation;
the specific method for in vitro maturation and treatment of oocytes in the examples is as follows:
collecting sheep ovaries, cleaning once, shearing off oviduct connecting parts and redundant connective tissues, cleaning 3-4 times by using normal saline, and collecting oocytes by a thorn-dissecting method: briefly, the ovaries were drained and placed in a 100mm petri dish with an egg collection solution (M199+0.1% BSA+0.1mg/mL heparin+0.1 mg/mL gentamicin) and left hand-held pointed ophthalmic forceps were used to hold the ovaries in the egg collection solution, and the right hand-held surgical blade was used to puncture the bright follicles protruding from the cortex of the ovaries and multiple knives into the egg collection solution, and the collected ovaries were gently shaken in the egg collection solution to prevent adhesion. After sedimentation for several minutes, the supernatant was discarded, and Cumulus Oocytes Complexes (COCs) with normal morphology and uniform cytoplasm were selected under a split microscope by using a Lagrangian tube for maturation. The collected COCs are washed 2-3 times by egg-picking liquid, then washed 1-2 times by maturation liquid, put into maturation liquid microdrops (M199 +15% FBS +0.1I mu/mL FSH +0.2I mu/mL LH +1.0 mu g/mL estradiol) pre-equilibrated for 2 hours, and cultured at a density of 10 pieces/50 mu L under the conditions of 38.5 ℃ and 5% CO 2 Saturated humidity.
The early method, the late method and the segmentation method used in the example are respectively to mature oocytes in vitro, the maturation time of the early method is 16h, the maturation time of the late method is 18h, the maturation time of the segmentation method is 16h, 18h, 20h, 22h and 24h respectively, in operation, the diffused granulosa cells are respectively blown off by a pipettor, the granulosa cells are continuously blown off after 0.1% hyaluronidase digestion for 1min to remove the granulosa cells, the oocytes are washed 2 to 3 times by maturation liquid and transferred into maturation culture drops which are pre-balanced in a 60mm dish, and the mature oocytes with the first polar body are picked out under a split microscope for standby.
Step two, preparing a required reagent, an operating instrument and other enucleation tools, wherein the enucleation needle is a enucleation needle subjected to bending treatment, the structure of the enucleation needle comprises a tip 1, an enucleation section 3 and an extension section 4, the tip 1 is provided with an oblique angle incision of 30-45 degrees, the length S of the enucleation section 3 is 360-400 mu m, and the enucleation section 3 and the extension section 4 are bent and arc-shaped in transition; taking the diameter of fibroblasts of herbivores as a reference, wherein the inner diameter of a denucleation needle is the diameter of one fibroblast, namely 20 mu m, and the length S of the denucleation section 3 is a denucleation critical point;
thirdly, firstly, staining mature oocytes, and then measuring the amounts of enucleated chromosomes, first polar bodies and surrounding cytoplasm by using an enucleation needle to carry out enucleation by a blind aspiration method, wherein the enucleation operation is as follows: determining an enucleation amount based on the maturation duration of the mature oocyte, said enucleation amount being measured in terms of length L of chromosome and first polar body and surrounding cytoplasm inhaled by the enucleation needle:
the maturation duration of the mature oocyte is 16 h-18 h: the length L of the suction of the enucleation needle is 0.25 fibroblast;
mature oocyte maturation duration is 18-20 h: the length L of the suction of the enucleation needle is 3.77-4.05 fibroblasts;
maturation duration of mature oocytes exceeds 20 h: the length L of the suction of the enucleation needle is 3.77-4.05 fibroblasts; when loose and discontinuous cytoplasm of the enucleated content is present, the length L of the enucleated needle is 17.11-18.33 fibroblasts, or the length L of the enucleated needle is determined according to the length of the enucleated critical point S, namely S=L;
(4) And (3) only performing one ultraviolet observation to observe whether the chromosome and the first polar body are removed or whether the oocyte also contains the chromosome, and observing that the non-enucleated clean oocyte is directly discarded.
The somatic cell nuclear transfer in the experiment is specifically performed as follows:
the operating fluid (M199+25 mmol/L hepes+20% FBS) was prepared in a 60mm dish and covered with paraffin oil, and the injection needle and the fixed needle were moved into the operating field of view by moving into the micromanipulation station. Recipient oocytes were incubated with maturation solution containing 7.5. Mu.g/mL CB+1.5. Mu.g/mL host 33342 for 10min, and donor cells and oocytes were transferred into the working fluid using a Pasteur tube for oocyte reconstitution.
The early and late methods are two-step cloning, i.e., concentrated batch enucleation followed by concentrated batch nuclear injection. Briefly, the oocytes stained by Hochest3342 are washed 3-4 times in maturation liquid, then concentrated enucleation and nuclear injection are carried out, after nuclear injection is finished, the enucleation condition is marked one by one through liquid drops, the enucleation is clean, concentrated reconstruction is carried out, and the oocytes with fluorescence display residual nuclei are discarded. After removing granular cells in the early method 16h, according to the method for removing the core, when the core is removed in the reconstruction time of 16-20h, the core removing amount, namely the length L of the sucking-in of a core removing needle is 3.77-4.05 fibroblasts; after the late stage method 18h passes through the enucleated granulosa cells, when the enucleation is carried out in the reconstruction time of 18-22h according to the enucleation method of the invention, the enucleation amount, namely the length L of the enucleation needle suction is 3.77-4.05 fibroblasts, when the enucleation content is loose, the enucleation amount, namely the length L of the enucleation needle suction can be increased to 17.11-18.33 fibroblasts, or the length L of the enucleation needle suction is determined according to the length of the enucleation critical point S, namely S=L, and the enucleation amount of the oocyte does not exceed the enucleation needle bending position.
The segmentation method is one-step cloning, namely, the core removing and the core injecting are completed simultaneously. The fixed needle attracts the oocyte, the first polar body points to the 12-point direction, the enucleation needle pierces the enucleated polar body and a small amount of cytoplasm around, the enucleation method according to the invention carries out the blind aspiration method for measuring enucleation, then the enucleation condition is observed through ultraviolet light, the oocyte with the cell nucleus is discarded, and the enucleated oocyte is used for reconstruction. The enucleation amount within 18h, namely the length L of the enucleation needle suction is 0.25 fibroblast; the enucleation amount is that the length L of the enucleation needle is 3.77-4.05 fibroblasts when the enucleation time is within 20 hours; when the reconstruction time exceeds 20h of in-vitro maturation, the enucleation amount is 3.77-4.05 microscopic cells when enucleation content is more compact, and the enucleation amount can be increased to 17.11-18.33 fibroblasts when enucleation content is loose, or the enucleation needle suction length is determined according to the length of the enucleation critical point S, namely S=L, and the enucleation amount of the oocyte is not more than the enucleation needle bending position.
The results of the somatic cell reconstitution measurements verification in this example are shown in the following table:
early method denucleation results
Advanced enucleation results
Segmentation method denucleation result
Claims (1)
1. The method for measuring the enucleated volume of the nuclear transfer blind suction method is characterized by mainly comprising the following steps of:
(1) Oocyte collection and maturation: collecting sheep ovaries, cleaning, and then collecting oocytes by a thorn-dissection method, picking out mature oocytes discharged with a first polar body under a microscope for standby, and recording the duration of maturation;
(2) Preparing a coring tool: the stoning needle is a stoning needle subjected to bending treatment, the structure of the stoning needle comprises a tip, a stoning section and an extension section, the tip is provided with an oblique angle incision of 30-45 degrees, the length S of the stoning section is 360-400 mu m, and the stoning section and the extension section are bent and arc-shaped in transition; taking the diameter of fibroblasts of herbivores as a reference, wherein the inner diameter of a denucleation needle is the diameter of one fibroblast, namely 20 mu m, and the length S of the denucleation section is a denucleation critical point;
(3) Mature oocytes were first stained and then enucleated by the blind aspiration method using an enucleation needle to measure the amount of enucleated chromosomes and first polar bodies and surrounding cytoplasm, the enucleation procedure being as follows: determining an enucleation amount based on the maturation duration of the mature oocyte, said enucleation amount being measured in terms of length L of chromosome and first polar body and surrounding cytoplasm inhaled by the enucleation needle:
the maturation duration of the mature oocyte is 16 h-18 h: the length L of the suction of the enucleation needle is 0.25 fibroblast;
mature oocyte maturation duration is 18-20 h: the length L of the suction of the enucleation needle is 3.77-4.05 fibroblasts;
maturation duration of mature oocytes exceeds 20 h: the length L of the suction of the enucleation needle is 3.77-4.05 fibroblasts; when the enucleated content is loose and discontinuous cytoplasm, the length L of the enucleated needle is 17.11-18.33 fibroblasts, or the length L of the enucleated needle is determined according to the length of the enucleated critical point S, namely S=L, and the enucleated amount of the oocyte is not more than the bending position of the enucleated needle;
(4) And (3) only performing one ultraviolet observation to observe whether the chromosome and the first polar body are removed or whether the oocyte also contains the chromosome, and observing that the non-enucleated clean oocyte is directly discarded.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE102012200461A1 (en) * | 2012-01-13 | 2013-07-18 | Voith Patent Gmbh | Roller cover for roller in web manufacturing or processing machine, has circumferential grooves and multiple holes formed as blind holes or suction holes, where cross section of circumferential grooves and blind holes has continuous profile |
| CN104789523A (en) * | 2015-05-18 | 2015-07-22 | 河南省格林金斯生物科技有限公司 | Simple, effective and low-consumption porcine oocyte in vitro mature cloning and culturing method |
| CN105087620A (en) * | 2015-08-31 | 2015-11-25 | 中国农业大学 | Overexpression porcine co-stimulatory 4-1BB vector and application thereof |
| CN106086080A (en) * | 2016-07-08 | 2016-11-09 | 西北农林科技大学 | A kind of method utilizing miRNA to improve cattle cloning efficiency |
| WO2018015509A1 (en) * | 2016-07-21 | 2018-01-25 | Vernet | Mixing unit and mixer tap comprising such a mixing unit |
| CN107858376A (en) * | 2017-11-10 | 2018-03-30 | 南开大学 | A kind of measuring method of stoning amount |
| CN108611319A (en) * | 2018-05-02 | 2018-10-02 | 南开大学 | A kind of Enucleating Cells method being quantitatively enucleated |
| CN109321518A (en) * | 2018-10-23 | 2019-02-12 | 温氏食品集团股份有限公司 | A kind of method of mammal ovocyte stoning |
-
2020
- 2020-07-07 CN CN202010647703.9A patent/CN111763692B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE102012200461A1 (en) * | 2012-01-13 | 2013-07-18 | Voith Patent Gmbh | Roller cover for roller in web manufacturing or processing machine, has circumferential grooves and multiple holes formed as blind holes or suction holes, where cross section of circumferential grooves and blind holes has continuous profile |
| CN104789523A (en) * | 2015-05-18 | 2015-07-22 | 河南省格林金斯生物科技有限公司 | Simple, effective and low-consumption porcine oocyte in vitro mature cloning and culturing method |
| CN105087620A (en) * | 2015-08-31 | 2015-11-25 | 中国农业大学 | Overexpression porcine co-stimulatory 4-1BB vector and application thereof |
| CN106086080A (en) * | 2016-07-08 | 2016-11-09 | 西北农林科技大学 | A kind of method utilizing miRNA to improve cattle cloning efficiency |
| WO2018015509A1 (en) * | 2016-07-21 | 2018-01-25 | Vernet | Mixing unit and mixer tap comprising such a mixing unit |
| CN107858376A (en) * | 2017-11-10 | 2018-03-30 | 南开大学 | A kind of measuring method of stoning amount |
| CN108611319A (en) * | 2018-05-02 | 2018-10-02 | 南开大学 | A kind of Enucleating Cells method being quantitatively enucleated |
| CN109321518A (en) * | 2018-10-23 | 2019-02-12 | 温氏食品集团股份有限公司 | A kind of method of mammal ovocyte stoning |
Non-Patent Citations (2)
| Title |
|---|
| Atsushi Kawaguchi .Comparison of two methods to clear the airways of critically ill children and adults with COVID-19 infection: a structured summary of a study protocol for a pilot randomized controlled trial.Trials.2020,第21卷(第21期),全文. * |
| 王文亭.哺乳动物胚胎细胞核移植显微操作用针的制备与应用.黑龙江动物繁殖.2006,全文. * |
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