CN107201343B - Goat cell clone embryo culture solution and culture method - Google Patents
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- CN107201343B CN107201343B CN201710574034.5A CN201710574034A CN107201343B CN 107201343 B CN107201343 B CN 107201343B CN 201710574034 A CN201710574034 A CN 201710574034A CN 107201343 B CN107201343 B CN 107201343B
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Abstract
The invention belongs to the technical field of bioengineering and discloses a goat cell cloned embryo culture solution and a culture method thereof, wherein the goat cell cloned embryo culture solution comprises a solution A and a solution B, and each liter of the solution A contains 0.2mg of ferrous nitrate, 350mg of potassium chloride, 100-120mg of citric acid, 100mg of sodium dihydrogen phosphate and the balance of DMEM high-sugar type liquid culture medium. The culture solution is used for improving a DMEM culture medium and an M16 culture medium in the prior art to prepare a solution A and a solution B of the culture solution, then cells are cultured in stages, the number of two-cell embryos, the number of four-cell embryos, the number of mulberry embryos, the number of blastula and the survival number of blastula after being implanted into a matrix are all higher, and the in vitro development rate of goat zygotes is generally higher than that of the DMEM culture medium and the M16 culture medium.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a goat cell cloned embryo culture solution and a culture method.
Background
The goat is an important economic animal, produces economic products such as meat, cashmere, milk and the like, particularly the goat milk has huge market potential, and if high-quality goat variety genetic resources are obtained and maintained, the existing population is improved, and the method becomes an urgent problem for large-scale breeding of the goat.
The most commonly used method for cloning embryo is somatic cell cloning technology, which is a process of adopting a nuclear transfer method, utilizing cell disassembly or cell recombination technology, enucleating an oocyte as a nuclear receptor, taking a somatic cell or a nuclear plastid containing a small amount of cytoplasm as a nuclear donor, transferring the nuclear donor into the nuclear receptor to construct a recombinant embryo (fertilized egg), and then reprogramming and developing the nuclear donor in the inclusion of the enucleated oocyte. The somatic cell cloning technology has many successful cases in the fields of biology and medicine, and has important application values in the aspects of researching animal embryonic development, researching embryonic development mechanism, treating drug action, cloning animals and the like.
The reasonable utilization of the cloned embryo technology and the construction of the goat cell cloned embryo are key means for maintaining the high-quality genetic resources of the goat, and with the continuous development of the biological science technology, the cloned embryo technology is gradually mature and popularized and applied. In the prior art, an M16 culture medium is a common culture medium used for culturing embryos before embryo transplantation, but the culture medium is a basic universal culture medium, has wide applicability but weak pertinence, and generally needs low clone embryo development rate.
Disclosure of Invention
The goat cell cloned embryo culture solution provided by the invention has strong pertinence, and solves the problem that the cloned embryo development rate generally required by an M16 culture medium is low.
The goat cell clone embryo culture solution provided by the invention comprises a solution A and a solution B, wherein each liter of the solution A contains 0.2mg of ferrous nitrate, 350mg of potassium chloride, 100-120mg of citric acid, 100mg of sodium dihydrogen phosphate and the balance of DMEM high-sugar type liquid culture medium;
each liter of the solution B contains 10-20mg of fetal calf serum, 120-150mg of malic acid, 0.2g of sodium chloride and the balance of M16 liquid culture medium.
The invention also provides a culture method of the goat cell cloned embryo, which comprises the following steps:
step 1, placing the recombined goat zygote into the A solution of claim 1 for isolated culture until the embryo develops to the two-cell stage;
step 2, washing the embryos which develop to the two-cell stage with 0.85g/100ml sodium chloride solution, and then placing the embryos in the solution B of claim 1 for in vitro culture until the cloned embryos develop to blastocysts.
Compared with the prior art, the goat cell cloned embryo culture solution provided by the invention has the following beneficial effects:
(1) the method comprises the steps of improving a DMEM medium and an M16 medium in the prior art to prepare a solution A and a solution B of a culture solution, and then culturing cells in stages, wherein the number of two-cell embryos, the number of four-cell embryos, the number of mulberry embryos, the number of blastula and the survival rate of blastula after being implanted into a mother body are all higher, and the in vitro development rate of goat zygotes is generally higher than that of the DMEM medium and the M16 medium.
(2) In a specific embodiment, comparing A, F, G, H groups, the addition of citric acid and malic acid is found, and the number of blastocysts obtained finally is higher than that of F, G, H group without addition of citric acid, malic acid or both citric acid and malic acid, which shows that citric acid and malic acid are helpful for improving the in vitro development rate of goat fertilized eggs and the survival number of blastocysts implanted into a mother body.
Detailed Description
The present invention is described in detail below with reference to specific examples, but the present invention should not be construed as being limited thereto. The test methods not specified in the following examples are generally carried out under conventional conditions, and reagents used are commercially available, and the steps thereof will not be described in detail since they do not relate to the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
The goat cell clone embryo culture solution provided by the invention comprises a solution A and a solution B, wherein each liter of the solution A contains 0.2mg of ferrous nitrate, 350mg of potassium chloride, 100-120mg of citric acid, 100mg of sodium dihydrogen phosphate and the balance of DMEM high-sugar type liquid culture medium;
each liter of the solution B contains 10-20mg of fetal calf serum, 120-150mg of malic acid, 0.2g of sodium chloride and the balance of M16 liquid culture medium.
Wherein the DMEM high-glucose liquid culture medium and the M16 liquid culture medium are commercially available DMEM high-glucose liquid culture medium and M16 liquid culture medium, respectively, and are prepared according to the instructions.
Wherein the malic acid is L-malic acid, D-malic acid or mixture DL-malic acid.
Example 1
A goat cell clone embryo culture solution comprises solution A and solution B, wherein each liter of the solution A contains 0.2mg of ferrous nitrate, 350mg of potassium chloride, 100mg of citric acid, 100mg of sodium dihydrogen phosphate and the balance of DMEM high-glucose type liquid culture medium;
each liter of the solution B contains 15mg of fetal calf serum, 150mg of malic acid, 0.2g of sodium chloride and the balance of M16 liquid culture medium.
The malic acid is L-malic acid.
Example 2
A goat cell clone embryo culture solution comprises solution A and solution B, wherein each liter of the solution A contains 0.2mg of ferrous nitrate, 350mg of potassium chloride, 120mg of citric acid, 100mg of sodium dihydrogen phosphate and the balance of DMEM high-glucose type liquid culture medium;
the solution B contains 17mg of fetal calf serum, 120mg of malic acid, 0.2g of sodium chloride and the balance of M16 liquid culture medium per liter.
The malic acid is D-malic acid.
Example 3
A goat cell clone embryo culture solution comprises solution A and solution B, wherein each liter of the solution A contains 0.2mg of ferrous nitrate, 350mg of potassium chloride, 100mg of citric acid, 110mg of sodium dihydrogen phosphate and the balance of DMEM high-glucose type liquid culture medium;
the solution B contains 10mg of fetal calf serum, 130mg of malic acid, 0.2g of sodium chloride and the balance of M16 liquid culture medium per liter.
The malic acid is a mixture DL-malic acid.
Example 4
A goat cell clone embryo culture solution comprises solution A and solution B, wherein each liter of the solution A contains 0.2mg of ferrous nitrate, 350mg of potassium chloride, 100mg of citric acid, 115mg of sodium dihydrogen phosphate and the balance of DMEM high-glucose type liquid culture medium;
the solution B contains 20mg of fetal calf serum, 140mg of malic acid, 0.2g of sodium chloride and the balance of M16 liquid culture medium per liter.
The malic acid is L-malic acid, D-malic acid or mixture DL-malic acid.
When the culture solution of the embodiment 1-4 is prepared, the DMEM high-glucose type liquid culture medium is prepared, and then the components are weighed according to the formula, mixed evenly and fully dissolved.
The present invention also provides a method for culturing goat cell cloned embryos, which comprises the steps of preparing recombined goat zygotes according to a conventional method, developing the zygotes by using the method of the following embodiment 5, and culturing the fertilized eggs until blastocysts of the cell cloned embryos are obtained. The procedure of example 5 will be described below by taking the culture medium formulation of example 1 as an example.
Example 5
A method for culturing goat cell cloned embryos comprises the following steps:
step 1, placing the recombined goat zygote in the solution A described in example 1 for isolated culture until the embryo develops to the two-cell stage;
step 2, the embryos developed to the two-cell stage are washed by 0.85g/100ml sodium chloride solution and then placed in the solution B described in example 1 for in vitro culture until the cloned embryos develop to blastocysts.
After the blastocyst is developed, the uterus of the plant parent can be differentiated and developed according to the conventional method.
Comparison of one and different culture solutions
The recombinant goat zygotes were cultured in different culture media according to the same method as in example 5, except that the culture media used at different stages were different, and the following experiments were designed:
group A: embryonic development culture was carried out using the culture solution of example 1 and the culture method of example 5.
Group B: the recombined goat zygote is placed in the A liquid described in example 1 for isolated culture until the cloned embryo develops to a blastocyst.
Group C: the recombined goat zygote is placed in the liquid B described in example 1 for isolated culture until the cloned embryo develops to a blastocyst.
Group D: and (3) placing the recombined goat zygotes in a DMEM high-sugar liquid culture medium for isolated culture until the cloned embryos develop to blastocysts.
Group E: and (3) placing the recombined goat zygotes in an M16 liquid culture medium for isolated culture until the cloned embryos develop to blastocysts.
And F group: the operation was substantially the same as that of group a except that citric acid was removed from solution a and malic acid was removed from solution B in example 1.
Group G: the operation was essentially the same as that of group a except that the citric acid was removed from solution a of example 1.
Group H: the operation was substantially the same as that of group A except that malic acid was removed from solution B of example 1.
Observing and recording the in vitro development rate of the fertilized eggs of the middle goats in the group. The results are shown in Table 1. From the results in table 1, it can be seen that compared with the two existing culture media of group D and group E, the number of two-cell embryos, the number of four-cell embryos, the number of morula, the number of blastocysts, and the survival number of blastocysts implanted in the mother body obtained in group a are all higher, and the in vitro developmental rate of goat zygotes and the survival rate of the blastocysts implanted in the mother body are overall higher;
comparing A, B, C, D, E groups, finding out that the group B using the liquid A alone or the group C using the liquid B alone finally obtains the number of blastula, and the survival number of the blastula after being implanted into a mother is lower than that of the group A and higher than that of the group D, E;
comparing A, F, G, H groups, finding that the addition of citric acid and malic acid results in that the number of blastocysts and the survival number of the blastocysts implanted in the mother body are higher than those of F, G, H groups without the addition of citric acid or malic acid or both citric acid and malic acid, which shows that citric acid and malic acid are helpful for improving the in vitro development rate of goat zygotes and the survival rate of the blastocysts implanted in the mother body.
TABLE 1100 number of cloned embryos developed in different culture methods
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (3)
1. A method for culturing goat cell cloned embryos is characterized by comprising the following steps:
step 1, placing the recombined goat zygotes in A liquid for isolated culture until embryos develop to a two-cell stage;
step 2, cleaning the embryos which develop to the two-cell stage by using 0.85g/100ml sodium chloride solution, and then placing the embryos in the solution B for isolated culture until the cloned embryos develop to blastocysts;
each liter of the solution A contains 0.2mg of ferrous nitrate, 350mg of potassium chloride, 100-120mg of citric acid, 100mg of sodium dihydrogen phosphate and the balance of DMEM high-sugar type liquid culture medium;
each liter of the solution B contains 10-20mg of fetal calf serum, 120-150mg of malic acid, 0.2g of sodium chloride and the balance of M16 liquid culture medium.
2. The method for culturing a goat cell cloned embryo as claimed in claim 1, wherein each liter of the solution A contains 0.2mg of ferrous nitrate, 350mg of potassium chloride, 100mg of citric acid, 100mg of sodium dihydrogen phosphate, and the balance of DMEM high-sugar type liquid medium;
each liter of the solution B contains 15mg of fetal calf serum, 150mg of malic acid, 0.2g of sodium chloride and the balance of M16 liquid culture medium.
3. The method for culturing a goat cell cloned embryo as claimed in claim 1 or 2, wherein said malic acid is L-malic acid, D-malic acid or mixture DL-malic acid.
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