CN111793652A - Method for efficiently constructing chimera gonad chick embryo through embryo transfer technology - Google Patents
Method for efficiently constructing chimera gonad chick embryo through embryo transfer technology Download PDFInfo
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Abstract
In order to solve the technical problems of low operation efficiency of embryo transplantation and low success rate of chimera gonad chick embryos obtained after transplantation in the prior art, the invention provides an efficient method for constructing chimera gonad chick embryos by an embryo transplantation technology, which comprises the following four steps: material preparation, donor tissue isolation, recipient embryo preparation, and donor tissue transplantation. Through the genotype determination of donor and acceptor tissues, the sex of the donor and the acceptor can be judged, and the cross-sex chimera gonadal chick embryo is obtained. The operation efficiency and the success rate of obtaining the chimera gonad chick embryo after transplantation are obviously improved. The chimera gonad chicken embryo is used as model for researching the expression rule of relevant gene in gonad of different sex, so as to further research the sex determination and gonad development mechanism of chicken.
Description
Technical Field
The invention belongs to the technical field of poultry embryo engineering. In particular to a method for constructing chimeric gonad chick embryos of cross sex by embryo transfer technology and researching the sex determination mechanism of the chick by taking the chimera gonad chick embryos as a model. Can be used for determining sex and embryo development of chicken.
Background
Chickens are important economic and model animals. The animal model can be used as an easily-obtained model animal for scientific research work and is a main food source for human beings. The poultry industry worldwide is now looking for ways to regulate the sex differentiation and development of chickens. Particularly in the layer chicken industry, cocks are usually directly eliminated due to the fact that only hens are needed, and therefore the problems of animal welfare and economic loss are serious. If a single sex chicken (such as a hen) can be produced by regulating the sex, the method brings great value to the poultry industry. The regulation of sex depends on the deep understanding of the normal sex determination and development mechanism of the chicken.
Unlike mammals such as humans, chickens and other birds have a ZZ/ZW sexual chromosome system, where males are homogametic sex. The mechanisms of sex determination and development of chickens are not yet fully understood. The gonad differentiation of the chicken occurs about day 6 of the development of the chicken embryo, and various embryo engineering and molecular biology techniques are utilized to research the chicken embryo at this stage, which is beneficial to deeply exploring the sex determination and development mechanism of the chicken and lays a foundation for further production and application. The chicken embryo with chimera gonad constructed by chicken embryo transplantation technology is one of the most necessary and effective technologies.
At present, few research teams mastering the construction technology of chimera gonad chick embryos in the world are available. A recent report on this technology is from the article published in Nature in 2010 by doctor Debiao Zhao (one of the patent applicants). The core idea of the technology is to transplant tissues of specific parts of mesoderm of a donor embryo with a fluorescent label, which develops to a specific stage, into corresponding positions of a wild-type donor embryo. However, the method of embryo transfer is only briefly described in this paper, and the efficiency of embryo transfer operation and the success rate of obtaining chimeric gonadal chick embryos after transfer are both low. In view of this, the team has been working on the improvement and perfection of the technology in recent years, and the operation details and the used reagents in the embryo transplantation process are optimized in multiple ways, so that the operation efficiency of the embryo transplantation and the acquisition rate of the chimera gonadal chicken embryos are greatly improved. The invention provides a system and a detailed explanation of the improved chick embryo transplantation method.
Disclosure of Invention
The invention aims to solve the problems of low operation efficiency and low success rate when the chimera gonad chick embryo is constructed by embryo transplantation. The embryo transplantation technology of the chicken has higher operation difficulty and has higher requirements on the control of the embryo development stage and reagent materials used in the operation process, so the problems of time-consuming operation, low efficiency, low acquisition rate of chimera embryo after transplantation and the like are easily caused. On the basis of the embryo transplantation technology mastered in the previous stage, the research team optimizes various details such as the preparation of embryo transplantation materials, the separation of donor tissues, the operation key points of the transplantation technology and the like through years of research, forms a set of complete and efficient chick embryo transplantation technology, and greatly improves the operation efficiency and success rate of the construction of chimera gonadal chick embryos.
In order to achieve the purpose of the invention, the technical scheme is as follows:
the invention provides a method for constructing a chimaera gonad chick embryo, which comprises the following steps:
(1) preparing chicken embryos: taking a fertilized egg with Green Fluorescent Protein (GFP) in the whole embryo as a donor (the GFP part does not comprise an eggshell), taking a normal fertilized egg as a receptor, and incubating the donor and the receptor until a chicken embryo develops into 13-15 somites;
(2) donor tissue isolation: taking out a donor chick embryo from a donor, predicting to develop into the 16 th to 23 th body sections of the spine of the donor chick embryo by micromanipulation, and separating mesoderm from left tissues predicted to develop into the 16 th to 23 th body sections of the spine of the donor chick embryo and cutting the mesoderm into small sections with the length and the width of each 1 to 3 body sections so as to obtain donor tissues; preferably, cutting into small sections of 2 body sections with length and width; the method for predicting (developing into) the 16 th to 23 rd body sections of the donor chick embryo spine comprises the following steps: the approximate length of each body segment is observed under a microscope, then the calculation is carried out on the basis of the approximate length, if 13 body segments exist, 10 body segments are calculated downwards (the position is at the tail part of the spine), and if 14 and 15 body segments exist, 9 or 8 body segments are calculated downwards.
(3) Preparing a receptor embryo: removing mesoderm of left tissues of 21 st to 23 rd body sections of the spinal column predicted to develop from the receptor chick embryo, and reserving the residual receptor embryo to be transplanted; the prediction method is the same as the step (2).
(4) Embryo transplantation: taking donor tissue and transplanting the donor tissue into a receptor according to the epiblast orientation; continuously incubating until the embryo age or the day age to be tested is reached after the sealing (incubating for 21 days, if the time to be tested is before hatching, the embryo age is reached, and if the time to be tested is after hatching, the embryo age is reached); determining the sex of the donor and the acceptor by performing genotype measurement on donor and acceptor tissues, and selecting a sex combination of the donor and the acceptor according to the required target sex to obtain the target cross sex chimeric gonadal chick embryo. (Cross sex means sex difference between donor and recipient. in practice, the donor used for each recipient is recorded, sex between donor and recipient is determined by genotyping, and individuals with different sex between donor and recipient are selected as required.)
Further, in the step (1), the fertilized egg as a donor is incubated with the blunt end facing upward; the fertilized eggs as the receptors are horizontally placed when being hatched; after incubation, the donor and recipient were sterilized.
Further, the step (2) is specifically operated as:
(2-a) removal of donor chick embryos: opening a small window at the blunt end of the donor, cutting off the embryo along the periphery of the blood island, and taking out the donor chick embryo;
(2-b) isolation of donor tissue bands: the donor chick embryo is right-side-up, under a stereo microscope, a donor tissue band with a length of 8 body segments and a width of 2 body segments is cut from a mesoderm predicted to be developed to the 16 th to 23 rd body segments from the left side of the spine of the donor chick embryo, and the sex determination is carried out on the rest donor chick embryos;
(2-c) isolation of donor tissue: dividing the tissue strip into small sections with length and width of each 2 body sections, namely donor tissue; the donor tissue was placed in carbon dioxide independent medium/10% chicken serum medium for use.
Further, the step (3) is specifically operated as:
(3-a) windowing the embryonated egg: horizontally placing a receptor, sticking an adhesive tape on the topmost part, and forming a small window in the middle of the adhesive tape; the adhesive plaster has the function that the edge is more neat when the opening (the eggshell is cut), so that fragments are not easy to generate, the fragments enter the egg to cause pollution, and the operation success rate is improved.
(3-b) shows embryo: carefully adding penicillin-streptomycin (1 ×) -PBS buffer over the recipient chick embryos through the small window opened in step (3-a) to keep the embryos moist and to allow the recipient chick embryos to slightly rise close to the window; connecting a micropipette with a diameter of about 50 μm (pulled at 90 ° right angle in advance) with a mouth pipette, sucking penicillin-streptomycin (1 ×) -PBS buffer solution containing India ink, and injecting into the lower part of recipient chick embryo to make the recipient chick embryo clearly visible
(3-c) removing recipient tissue: using a micromanipulation needle to carefully open the yolk membrane of the left tissue of the donor tissue predicted to develop into 16 th-23 rd body segment, exposing the ectoderm tissue; cutting the ectoderm tissue corresponding to the 21 st to 23 rd body segments with a micromanipulator and turning to one side to expose mesoderm; removing mesoderm at the corresponding part of the recipient chick embryo, and reserving the residual recipient chick embryo to be transplanted.
Further, the diameter of the small window in the step (3-a) is 0.5-1 cm.
Further, in the penicillin-streptomycin (1 ×) -PBS buffer solution in the step (3-b), the concentration of penicillin is 100U/ml streptomycin and the concentration of penicillin is 100. mu.g/ml.
Further, the step (4) is specifically operated as:
(4-a) transfer of donor tissue: transferring the donor tissue to the receptor embryo processed in the step (3) in the direction of the ectoderm, and re-covering the turned-up ectoderm and yolk membrane on the donor tissue;
(4-b) resealing: carefully sucking 0.5-1.0ml of protein from a needle hole at the blunt end of a receptor by using an injector, so that the position of an embryo sinks to be lower than an opening on an eggshell; observing the embryo under a GFP microscope, and tightly sealing the opening and the blunt-end pinhole on the eggshell of the receptor by using an adhesive tape after ensuring that the donor tissue is still at the corresponding position;
(4-c) Re-hatching: horizontally placing the receptor in an incubator, incubating again at 37.5 ℃ and 60% humidity until the receptor is incubated to the age of the embryo to be tested or the age of the embryo in days; determining the sex of the donor and the acceptor by performing genotype measurement on the donor and the acceptor, and selecting a sex combination of the donor and the acceptor according to the required target sex to obtain the cross-sex chimeric gonadal chick embryo. Egg turning was not performed on the first day of re-hatching. Care is taken during the sealing and incubation of the eggs so as not to cause separation of donor tissue from the recipient.
Compared with the prior art, the technical scheme of the invention has the following beneficial effects:
1) the method effectively improves the success rate of cross sex chimera gonad chick embryos, the success rate is about 12.5% from the method of the 2010 paper, and the success rate of the method reaches 38%.
According to the method, the blunt end of the donor fertilized egg is placed upwards for incubation, so that the success rate of donor taking is effectively improved, the donor fertilized egg is transversely placed for incubation, and the PBS buffer solution is added to enable the chick embryo to be closer to the top end, so that the donor implantation is facilitated, and the success rate of donor transplantation is improved. The adhesive tape is pasted on the surface of the receptor fertilized egg before the opening, so that the opening smoothness is improved, the pollution of eggshell fragments to the chick embryo is reduced, and the success rate of transplantation is improved. The direction of the donor tissue to be placed into the recipient is defined to ensure success of the transplantation (if the donor tissue is placed in the wrong place, the chimeric gonad cannot be obtained)
2) The method of the invention can be used for obtaining cross sex chimera gonad chick embryos. The chimera gonad chicken embryo is used as model for researching the expression rule of relevant gene in gonad of different sex, so as to further research the sex determination and gonad development mechanism of chicken.
3) The sex determination mechanism of the chicken is researched through the chimera gonad chicken embryo model, so that the sex control of the chicken is realized, and the method has great market benefits in the industries of laying hens and broilers. In the broiler industry, people want to obtain more male offspring due to the fact that males grow faster than females; in the laying hens, only the hens are needed, and the cocks are usually killed directly after the chicks are hatched, so that the economic loss is caused and the serious welfare problem is also caused. If more (or all) male offspring are generated in the broiler breeds and more (or all) female offspring are generated in the laying hen breeds through the sex regulation of the chickens, great economic benefit can be brought to the industry, and meanwhile, the welfare problem caused by the fact that cocks in laying hen production are directly killed can be effectively avoided.
Drawings
FIG. 1: chick embryo transplantation technical route diagram
FIG. 2: donor tissue preparation flow chart
FIG. 3: recipient embryo preparation flow chart
FIG. 4: tissue transplantation flow chart
FIG. 5: detection map of chimera chicken (embryo) and chimera gonad
FIG. 6: immunostaining of chimera gonad tissue section
Detailed Description
The invention is specifically illustrated below by taking a batch of chicken embryo transfer tests as an example:
EXAMPLE 1 Material preparation
(1) Chick embryo preparation
(1-1) preparation of donor embryos: 8 fertilized eggs with Green Fluorescent Protein (GFP) are taken, placed with blunt ends facing upwards, and begin to hatch at 11 am, the temperature is 37.5 ℃, the humidity is 60%, the eggs are turned once every 30 minutes, and the eggs are hatched to an H & H (Hamburger Hamilton cultures) stage 11/12, the development state of the chick embryos is 13-15 somites, the time required by the development is about 42-45 hours, namely the chick embryos are hatched to about 8 am on the third day and taken out. Because the air chamber is at the blunt end, the chick embryo always floats at the topmost end, and the mode that the blunt end is placed upwards makes it more convenient to get the embryo through the air chamber when getting the donor, and the success rate is higher. (since the donor is a GFP egg, the GFP egg develops slightly more slowly than a normal egg, and therefore the donor incubation time is slightly longer in this example than a normal fertilized egg of the recipient)
(1-2) recipient embryo preparation: hatching 20 common fertilized eggs without GFP, horizontally placing, starting hatching at 1 PM on the same day, turning the eggs once every 30 minutes at the temperature of 37.5 ℃ and the humidity of 60 percent, and hatching to an H & H stage 11/12, wherein the development state of the chick embryo is 13-15 somites, the time required by the development is about 40-44 hours, namely hatching to about 8 AM on the third day and taking out; the adoption of the horizontal placement mode can lead the chick embryo to avoid the air chamber of the blunt end, thereby leading the chick embryo to be closer to the eggshell, leading the operation to be more convenient when the chick embryo is implanted into the donor and leading the success rate to be higher.
The surfaces of the hatched donor fertilized eggs and the hatched recipient fertilized eggs are cleaned and sterilized with 75% alcohol until they are completely dried.
(2) Reagent and material preparation
Preparing a 3% agarose/indian ink gel plate 2 block by mass, wherein the 3% agarose/indian ink volume ratio is 10:1, and fixing the donor chick embryo.
Preparing 100ml of a buffer solution containing penicillin-streptomycin (1 ×) -PBS; in the buffer solution, the concentration of the penicillin is 100U/ml, and the concentration of the streptomycin is 100 mu g/ml;
preparing 50. mu.l of penicillin-streptomycin (1X) -PBS buffer containing India ink in a volume ratio of 1:4 to penicillin-streptomycin (1X) -PBS buffer;
1ml of carbon dioxide-Independent medium/10% Chicken serum medium (C-C medium, CO2-Independent medium/10% Chicken serum) was prepared
EXAMPLE 2 Donor tissue isolation
(2-1) removing chick embryo
Opening a small window from the blunt end of the donor chick embryo prepared in the step (1.1), checking the state of the embryo under a GFP microscope, and confirming that the donor chick embryo shows fluorescence and has 13-15 somites; i.e., at the H & H stage 11/12, if the chick embryos are grown over 15 somites, the chick embryos are discarded; if the chick embryo grows less than 13 somites, the donor embryo egg is resealed and continued to hatch (1 somite grows about every 1.5 hours) to 13-15 somites;
the embryos were cut along the periphery of the blood island and donor chick embryos were carefully removed;
donor chick embryos were placed face up on a 3% agarose/indian ink gel plate with streptomycin-containing PBS buffer to facilitate visualization of the chick embryos and fixed with pins.
(2-2) isolation of donor tissue
Under stereomicroscope, the left side of the donor chick embryo spine (right side of the observer) is predicted to develop into the 16 th-23 rd somite position. The method for predicting (developing into) the 16 th-23 th body segment of the donor chick embryo spine comprises the following steps: the approximate length of each body segment is observed under a microscope, then the calculation is carried out on the basis of the approximate length, if 13 body segments exist, 10 body segments are calculated downwards (the position is at the tail part of the spine), and if 14 and 15 body segments exist, 9 or 8 body segments are calculated downwards. If the donor chick embryo currently has 13 segments, the 23 rd segment will be located at the tail of the spine.
Cutting a tissue strip with the length of 8 body sections and the width of 2 body sections; two rows of holes are carefully punched longitudinally along the left side of the body segment along the predicted 16 th-23 th body segment position by cutting with a micromanipulator needle, the transverse distance between the two rows of holes being 2 body segment widths. The junction between the small holes was cut with a micromanipulator needle to form a tissue band of 8-body nodes in length and 2-body nodes in width (the upper part of the band was ectoderm, and the lower part of the band was mesoderm and endoderm).
The tissue strip was cut into tissue pieces of 2 body segments each in length and width to obtain donor tissue. Note that the tissue blocks are differentiated in the positive and negative directions (ectoderm and mesoderm endoderm are clearly defined, and ectoderm is similar to a membranous form, is slightly transparent, has viscosity, and is easy to form a concave surface).
(2-3) collecting donor tissue
The donor tissue was transferred to a small petri dish containing carbon dioxide independent medium/10% chicken serum medium (C-C medium) using a mouth pipette and a micropipette of 100 μm caliber, and the tissue was transplanted.
Care was taken to cover the lid of the dish to avoid evaporation of the medium.
3-4 donor tissues can be removed per chick embryo that develops to 13-15 somites. A total of 13 donor tissues (4 each for donor 1 and donor 2, three for donor 3, and two for donor 4) were removed from 4 chicken embryos. Donor tissues from different donors were transferred separately to small petri dishes containing C-C medium, awaiting tissue transplantation. The remaining tissue after the tissue band of the 4 chick embryos with the length of 8 body segments and the width of 2 body segments was retained for sex determination. (see attached FIG. 2)
Example 3 recipient embryo preparation
(3-1) windowing the embryo egg
Recipient embryos incubated approximately to 13-15 somites were placed horizontally.
Pricking a small hole at the blunt end (air chamber part) with a syringe needle, and then carefully sticking adhesive tapes (each having a length and a width of 1.5cm) on the topmost part of the horizontally placed egg.
A small window with a diameter of 1cm is arranged at the topmost part of the rubberized fabric.
(3-2) display of embryos
The penicillin PBS buffer was carefully added on top of the embryos through the small window opened in step (3-1) to keep the embryos moist and to allow the embryos to rise slightly close to the window.
Using a pipette tip, a micropipette (previously drawn at 90 ℃ right angle) with a diameter of about 50 μm was attached, and India ink diluted in penicillin PBS buffer was aspirated and injected under the embryo to make the embryo clearly visible.
The stage of development of the chick embryos (which should have 13-15 somites) was confirmed under the microscope.
(3-3) removal of recipient tissue
The yolk membrane of the corresponding portion of the donor tissue was carefully lifted using a micromanipulation needle to expose the ectoderm tissue.
The ectodermal tissue corresponding to the 21 st to 23 rd body segment was gently dissected with a micromanipulator and turned to one side, exposing the mesoderm.
Mesodermal tissue (approximately 1.5-2 knots in length and width) was carefully removed, taking care not to disrupt the endoderm. To this end, the remaining recipient embryos have been used for embryo transfer.
13 recipient embryos are prepared for development to the appropriate stage (13-15 somites). (see attached FIG. 3)
Example 4 embryo transfer
(4-1) transfer of donor tissue
Transfer of donor tissue into recipient embryos using a mouth pipette. A small amount of medium is aspirated simultaneously during the transfer process, which aids in adhesion of the donor tissue.
The donor tissue was placed in the position corresponding to the recipient embryo in the direction of the ectoderm, and the donor tissue was bonded as tightly as possible to the recipient embryo using a micromanipulation needle.
The turned-up ectoderm and yolk membrane were re-overlaid on top of the donor tissue.
(4-2) resealing
Carefully sucking 0.5-1.0ml of albumen from the needle hole of the blunt end of the recipient embryo egg by using a syringe to ensure that the position of the embryo is sunk to be lower than the opening on the egg shell. Embryos were observed under GFP microscope to ensure donor tissue was still in the corresponding position. The opening in the egg shell and the blunt-ended needle hole are tightly sealed with adhesive tape.
(4-3) Re-incubation
The eggs were placed horizontally in an incubator at 37.5 ℃ and 60% humidity, and were not turned on the first day of re-incubation. Care is taken during the sealing and incubation of the eggs so as not to cause separation of donor tissue from the recipient.
13 transplanted embryonated eggs are obtained. The donor source of each embryonated egg was accurately recorded (see FIG. 4, recipients 1-4 from donor 1, recipients 5-8 from donor 2, recipients 9-11 from donor 3, and recipients 12-13 from donor 4).
Example 5 examination of transplant Effect
Dissecting and gonad examination of partial chick embryo at 7 th day of embryo stage, 20 th day of embryo stage and 24 weeks after hatching. The results of the examination are shown in Table 1.
TABLE 1 attached hereto examination of embryo transfer tests in chickens
Note: e7 denotes day 7 of embryonic stage, E20 denotes day 20 of embryonic stage, and W24 denotes 24 weeks after hatching.
The chick embryos and gonads were observed at different time points, and the results are shown in FIG. 5, in which the highlighted portions indicated by arrows are fluorescence signal regions. As can be seen in FIG. 5a, both the 7-day-old embryo and the hen 24 weeks after hatching had a portion of tissue with green fluorescent signal, indicating that the embryo or individual was a chimera. From FIG. 5b, it can be seen that there are some tissues with green fluorescence signals in the gonads incubated for 20 days and the adult gonads 24 weeks after hatching, indicating that the construction of the chimaeric gonads was successful.
The section is taken from the gonad tissue of the chimera at the embryonic stage of 20 days, the gonad of the chimera is further subjected to tissue section and immunostaining, a GFP signal (green) represents a male donor tissue, an Aromatase (Aromatase) antibody is used for marking a female tissue (red), a Hoechst antibody is used for marking a cell nucleus (blue), and whether the gonad of the chimera with the cross sex is judged through the tissue morphology and the marker gene, and the result is shown in figure 6. As can be seen from the figure, the left gonad exhibits the morphology of an ovary with a cortical layer, while the right gonad exhibits the morphology of a testis with a spermatic structure. By using aromatase as a marker gene (red) of female tissues, it can be seen that the gonad simultaneously contains male donor cells with green fluorescence gonads and female tissues expressing aromatase genes, which indicates that the construction of the crossed sex chimera gonad is successful.
In this example, 13 transplanted embryonated eggs were successfully constructed with 5 chimera gonads, with a success rate of 38%.
Claims (7)
1. A method for constructing a chimeric gonad chick embryo, comprising the following steps:
(1) preparing chicken embryos: taking a fertilized egg with green fluorescent protein as a donor, taking a normal fertilized egg as an acceptor, and incubating the donor and the acceptor until a chicken embryo develops into 13-15 somites;
(2) donor tissue isolation: taking out a donor chick embryo from a donor, predicting to develop into the 16 th to 23 th body sections of the spine of the donor chick embryo, and separating mesoderm from the left tissue of the 16 th to 23 th body sections of the spine of the donor chick embryo predicted to develop into small sections with the length and the width of each 1 to 3 body sections, thereby obtaining donor tissue; preferably, cutting into small sections of 2 body sections with length and width;
(3) preparing a receptor embryo: removing mesoderm of left tissues of 21 st to 23 rd body sections of the spinal column predicted to develop from the receptor chick embryo, and reserving the residual receptor embryo to be transplanted;
(4) embryo transplantation: taking donor tissue and transplanting the donor tissue into a receptor according to the epiblast orientation; continuously incubating until the embryo age or the day age to be detected is reached after the sealing; determining the sex of the donor and the acceptor by performing genotype measurement on donor and acceptor tissues, and selecting a sex combination of the donor and the acceptor according to the required target sex to obtain the cross-sex chimeric gonadal chick embryo.
2. The method for constructing a chimeric gonadal chicken embryo according to claim 1, wherein in step (1), the fertilized egg as a donor is incubated with its blunt end facing upward; the fertilized eggs as the receptors are horizontally placed when being hatched; after incubation, the donor and recipient were sterilized.
3. The method for constructing chimeric gonadal chicken embryos of claim 1, wherein the step (2) is specifically performed by:
(2-a) removal of donor chick embryos: opening a small window at the blunt end of the donor, cutting off the embryo along the periphery of the blood island, and taking out the donor chick embryo;
(2-b) isolation of donor tissue bands: the donor chick embryo is arranged right side up, a donor tissue band with a length of 8 body segments and a width of 2 body segments is cut from the mesoderm predicted to develop into the 16 th to 23 rd body segments from the left side of the spine of the donor chick embryo through micromanipulation, and the remaining donor chick embryos are subjected to sex determination;
(2-c) isolation of donor tissue: dividing the tissue strip into small sections with length and width of each 2 body sections, namely donor tissue; the donor tissue was placed in carbon dioxide independent medium/10% chicken serum medium for use.
4. The method for constructing chimeric gonadal chicken embryos of claim 1, wherein the step (3) is specifically performed by:
(3-a) windowing the embryonated egg: horizontally placing a receptor, sticking an adhesive tape on the topmost part, and forming a small window in the middle of the adhesive tape;
(3-b) shows embryo: adding penicillin-streptomycin (1X) -PBS buffer above the recipient chick embryo through the small window opened in the step (3-a), and injecting penicillin-streptomycin (1X) -PBS buffer dissolved with Indian ink below the recipient chick embryo;
(3-c) removing recipient tissue: using a micromanipulation needle to open the yolk membrane of the left tissue of the donor tissue predicted to develop into the 16 th to 23 rd body segment, and exposing the ectoderm tissue; cutting the ectoderm tissue corresponding to the 21 st to 23 rd body segments with a micromanipulator and turning to one side to expose mesoderm; the mesoderm is removed and the endoderm is retained, leaving the remaining recipient embryo to be transplanted.
5. The method for constructing a chimeric gonadal chicken embryo according to claim 4, wherein the diameter of the small window in step (3-a) is 0.5-1 cm.
6. The method for constructing a chimeric gonadal chick embryo according to claim 4, wherein in said penicillin-streptomycin (1 ×) -PBS buffer in step (3-b), the concentration of penicillin is 100U/ml streptomycin and the concentration of penicillin is 100 μ g/ml.
7. The method for constructing chimeric gonadal chicken embryos of claim 1, wherein the step (4) is specifically performed by:
(4-a) transfer of donor tissue: transferring the donor tissue to the receptor embryo processed in the step (3) in the direction of the ectoderm, and re-covering the turned-up ectoderm and yolk membrane on the donor tissue;
(4-b) resealing: sucking 0.5-1.0ml of protein from the blunt end of the receptor to make the embryo sink to a position lower than the opening on the eggshell; after observing the embryos under a GFP microscope to ensure that the donor tissue is still in place, the openings in the shells of the recipient are tightly sealed with tape.
(4-c) Re-hatching: horizontally placing the receptor in an incubator, incubating again at 37.5 ℃ and 60% humidity until the receptor is incubated to the age of the embryo to be tested or the age of the embryo in days; determining the sex of the donor and the acceptor by performing genotype measurement on the donor and the acceptor, and selecting a sex combination of the donor and the acceptor according to the required target sex to obtain the cross-sex chimeric gonadal chick embryo.
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| US20030170888A1 (en) * | 2002-02-01 | 2003-09-11 | Origen Therapeutics | Chimeric bird from embryonic stem cells |
| CN101423818A (en) * | 2008-12-18 | 2009-05-06 | 中国农业科学院北京畜牧兽医研究所 | Method for culturing chicken embryonic germ cells and special nutrient fluid thereof |
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| US20030170888A1 (en) * | 2002-02-01 | 2003-09-11 | Origen Therapeutics | Chimeric bird from embryonic stem cells |
| CN101423818A (en) * | 2008-12-18 | 2009-05-06 | 中国农业科学院北京畜牧兽医研究所 | Method for culturing chicken embryonic germ cells and special nutrient fluid thereof |
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