TWI458825B - Non-human mammal chimeric embryos and method for preparing the same - Google Patents

Description

Non-human mammalian chimeric embryo and production method thereof

The present invention relates to a method for producing a chimeric embryo, and more particularly to a method for producing a chimeric embryo by microinjection.

The establishment of transgenic animals can help to understand the function of specific genes in animals. Since the establishment of large gene-transferred mammals is time-consuming and labor-intensive, the earliest successful gene-transforming animals are mice, and so far, gene-transferred mice have been widely used in the field of life science research.

The current method for establishing gene-transforming mice is micronucleus microinjection, which has the disadvantage that exogenously constructed DNA sequences are inserted into chromosomal DNA in a random multipoint or single-point manner, so the same DNA sequence usually must More than 3 to 5 gene-transferred mouse lines are more sure to determine their function. This method of production is relatively simple, but the subsequent breeding, maintenance and research are too laborious and laborious. Therefore, some of these studies have gradually turned into embryo targeting stem cells (ES, cells) for gene targeting. After being selected and confirmed, the chimeric embryo (the embryo contains the non-self cell) is used to produce the chimeric embryo, and the gene is transferred to the chimeric mouse. This method can be followed by post-breeding as long as there is a gemline transmission gene-targeted transgenic mouse. This method has been confirmed to understand the physiological significance of most DNA sequences in vivo . And the mechanism of action is very efficient.

The acquisition of chimeric embryos is currently dominated by microinjection and aggregation (Bradley, 1987; Hogan et al., 1994; Nagy et al., 2003; Wood et al. , 1993b), coculture (Lee et al., 2007; Shimada et al., 1999; Suzuki et al., 1994; Ueda et al., 1995; Wood et al., 1993a) .

The operation of the polymerization method and the co-cultivation method is relatively simple and does not require expensive instruments and equipment. The principle is to use a denuded embryo (naked embryo, zona pellucida-free embryo) (usually 4-cell stage embryo ~ mulberry Embryonic and pluripotent stem cells, such as embryonic stem cells (ESC) or induced pluripotent stems (iPS, cells), which remove the zona pellucida and the pluripotent stem cells Mutual adhesions form embryo-cell aggregates, and the embryo-cell aggregates are further cultured to obtain chimeric embryos. However, both the polymerization method and the co-cultivation method have the disadvantages of being too time consuming to process and recover, or the recovery rate is not ideal; more importantly, the number of embryonic stem cells bound by the chimeric embryo without the zona pellucida obtained by the polymerization method and the co-culture method is often not First, the chimeric mouse thus obtained has a large variation rate of gonad genetic ability.

The blastocyst microinjection method directly injects a specific number of pluripotent stem cells into 3.5 days after the microinjection micropipette containing pluripotent stem cells pierces the trophectoderm of the blastocyst. Blastocyst cavity (3.5-day blastocyst microinjection); this method is the mainstream practice of microinjection. Technically skilled individuals can complete 20 to 40 mouse blastocyst injections per hour (Bradley, 1987; Hogan et al. , 1994). When the method is applied to porcine blastocysts, the rate of application is slower due to the greater toughness of the trophoblast cells of the porcine blastocyst. The technical aspect of this method is very mature, and the efficiency of the chimeric mouse is stable; however, the genetic ability of the chimeric mouse gonad still needs time to confirm the breeding. Disadvantages of microinjection are the need for expensive microscopy operating systems and the need for long training sessions by operators.

Another microinjection method directly involves embryonic stem cells into 2.5-day 4-~8-cell stage embryos after breeding. This result is different: the results may be comparable to the 3.5-day blastocyst microinjection method ( Papaioannou and Johnson, 1993; Papaioannou and Johnson, 2000), or a ratio of chimeric mice that can improve gonad inheritance (Tokunaga and Tsunoda, 1992), or chimeric mice obtained from embryonic stem cells that are not genetically competent. Gonadal inheritance (Tokunaga and Tsunoda, 1992). This method is more likely to produce chimeric mice with gonad genetic ability than the 3.5-day blastocyst microinjection method, however, due to the transparent band cavity between the 4--8-cell stage blastomere and the zona pellucida ( The subzonal cavity is extremely small, and it is easy to injure the embryonic leaf cells when microinjected cells. The technical surface is much more difficult than the 3.5-day blastocyst microinjection method, so there are few adopters. The chimeric portion of the chimeric mouse obtained by this method is even 100% chimeric (Tokunaga and Tsunoda, 1992), which is significantly better than the 3.5-day blastocyst microinjection method. Therefore, in recent years, many studies have attempted to improve the production method.

Two sets of chromosomes (2n) pluripotent stem cells (microinjection, polymerization) 4n embryos can be obtained essentially by "embryonic stem cells (Nagy et al., 1993; Schoonjans et al., 2003) or induced pluripotent stem cells (Boland). Et al., 2009; Kang et al., 2009; Zhao et al., 2009) formed a mouse (ESC-, iPSC-derived mouse) with gem-line transmission. Recent studies have shown that mouse embryonic stem cells can be directly injected into 2n of 8-cell stage embryos to obtain F0 mice which are basically formed by "embryonic stem cells" (in F0 mice, only primordial blasts account for less than 0.1%) And has normal glandular inheritance ability (Poueymirou et al., 2007). The results showed that embryonic stem cells have a better chance of forming inner cell mass (ICM) cells in 8-cell stage embryos than in blastocysts, and even replace embryonic inner cell population cells to obtain essentially "embryo F0 mice formed by stem cells." The report subverts the notion that 4n embryos are basically made up of "embryos formed by embryonic stem cells", but the study uses "laser" (most laboratories have difficulty) to punch holes in the zona pellucida, and then the embryonic stem cells From the hole into the transparent belt, there is still much room for improvement in practice; and the production efficiency is not more than 10 due to the number of embryonic stem cell cells (too many embryonic stem cells will flow out from the rigid hole of the laser) ), it seems to be able to improve again. In 2008, Huang et al. changed the embryonic stem cells of 4-8-cell stage embryos with "Piezo drill" (Huang et al., 2008); the results and advantages and disadvantages are similar to Poueymirou et Al. (2007). The 2n embryo can be used to produce mice formed by "embryonic stem cells", but this method cannot obtain chimeric mice with 100% body color (Huang et al., 2008; Lee et al., 2007; Poueymirou et Al., 2007; Ramirez et al., 2009; Tokunaga and Tsunoda, 1992), therefore, fewer use in the production of mice formed by "embryonic stem cells"; instead, due to significantly improved gonad genetic ability The ratio of rats, which was accepted by the production of "chimeric mice" (De and Kothary, 2010; Huang et al., 2008; Kraus et al., 2010; Ramirez et al., 2009; Tanimoto et Al., 2008). However, the use of "laser" and "piezoelectric drill" to drill holes, and then microinjection requires extra expensive equipment and technology; piezoelectric drills must also use "mercury" (which is highly volatile and safe for workers) There is a great concern to microinject microtubes to control the rate, so both have not been widely used to date. Obviously, there is no need for extra expensive equipment, or more simple and effective technology has practical needs.

In summary, the traditional method of microinjection to produce chimeric embryos is still not perfect. Therefore, in the development of research, it is easy to operate and efficiently produce gonad inheritance without additional expensive equipment. The chimeric embryo production method of chimeric mice of ability has its needs.

The main object of the present invention is to provide a method for producing a chimeric embryo of a non-human mammal, which belongs to a microinjection method in which the fixed microtubule can be directly in any direction and angle. The embryo is incubated to microinject the cells, thus greatly increasing the rate of microinjection.

It is still another object of the present invention to provide a method for producing a chimeric embryo of a non-human mammal, which belongs to a microinjection method in which a microinjection microtubule of a smaller diameter can be selected, thereby The embryonic cells are hardly damaged during the injection and are easier to handle.

A further object of the present invention is to provide a method for producing chimeric embryos of non-human mammals which can efficiently produce non-human chimeric animals having high glandular inheritance without the need for additional expensive equipment.

To achieve the above object, the present invention provides a method for producing a non-human mammalian chimeric embryo comprising the steps of: providing a cell; providing a non-human embryo, the line being in a 1-cell stage embryo To the mulberry embryo; the aforementioned cells are microinjected into the aforementioned non-human in a hypertonic solution to obtain an embryo-cell aggregate; and the embryo-cell polymerization is cultured in the culture solution. The body obtains a non-human chimeric embryo.

Preferably, the high-tension solution refers to a solution having an osmotic pressure greater than the osmotic pressure of the isotonic solution of the aforementioned cells and/or the aforementioned non-human embryo.

Preferably, the osmotic pressure of the high-tension solution is 350 to 850 mOsm/kg H ₂ O.

Preferably, the high-tension solution is a culture solution containing 0.05 M to 0.5 M of sugar.

Preferably, the high-tension solution and/or the culture solution may further be added with serum, synthetic serum substitute or a combination thereof.

Preferably, the aforementioned cells and the source of the aforementioned non-human embryo are non-human mammals of the same species or different species.

Preferably, the aforementioned cells are either enriched or purified or unpurified.

Preferably, the cell line is an embryonic source, a fetal source, a placental source, a umbilical cord source, an adult source, a primary cell, a cell line cell, a stem cell, A pluripotent stem cell or an induced pluripotent stem (iPS, cell) or a protein-induced pluripotent stem cell (piPSC).

Preferably, the aforementioned cell line is an embryonic stem cell line.

Preferably, the embryonic stem cell line is established by dividing, but not dividing, cells of a fertilized egg, a 2-cell stage embryo to a morula, or a dissociated embryo leaf cell, a blastocyst or an inner cell group of a non-human mammal.

Preferably, the genetic material of the aforementioned cells is unchanged or altered.

Preferably, the aforementioned non-human germline contains two or more sets of chromosomes.

Preferably, the culture solution is EHK culture solution, TL-Hepes culture solution, HK culture solution, EK culture solution, KSOM-AA culture solution, EP culture solution or PZM-3 culture solution.

The invention further provides a method of producing a non-human chimera comprising the steps of: transferring the aforementioned embryonic embryo transfer into a non-human recipient; and The aforementioned chimeric embryos are developed in the aforementioned non-human embryonated animals to obtain non-human chimeric animals.

Preferably, the aforementioned non-human recipients comprise a fallopian tube, a uterus or a uterine horn.

Preferably, the aforementioned non-human chimeric animal may be a pluripotent stem cell-derived animal.

The invention further provides a non-human mammalian chimeric embryo produced by the method described above.

In summary, the present invention mainly provides a method for producing a chimeric embryo of a non-human mammal by microinjection in a high-tension solution to improve the efficiency of the conventional microinjection method - the average skilled person per hour 20 to 40 traditional mouse blastocyst microinjections can be completed (Bradley, 1987; Hogan et al., 1994). The present invention can complete 50-80 mouse 2- to 8-cell stage embryo display per hour on average. Microinjection and production of non-human chimeric animals with high glandular inheritance.

Embryos contain a non-genus cell called a chimeric embryo. The method of the present invention is a method for producing chimeric embryos by microinjecting cells in a high-tension solution. First, a cell and a non-human embryo are provided. The aforementioned cell line is an embryonic source, a fetal source, a placental source, a umbilical cord source, an adult source, a primary cell, a cell line cell, a stem cell, and a pluripotent stem cell ( Pluripotent stem cell) or induced pluripotent stem (iPS, cell) (Boland et al., 2009; Kang et al., 2009; Okita et al., 2007; Takahashi and Yamanaka, 2006; Wernig et al , 2007; Zhao et al., 2009), protein-induced pluripotent stem cell (piPSC) (Kim et al., 2009; Zhou et al., 2009); the aforementioned non-human germline is at 1-cell stage embryo to morula. The aforementioned cells and the source of the aforementioned non-human embryo are non-human mammals of the same species or different species. Preferably, the aforementioned cells have a foreign gene, and the foreign gene refers to a genetic material not belonging to the aforementioned cells or the aforementioned non-human embryo.

Next, the osmotic pressure of the aforementioned cells and the culture medium of the aforementioned non-human embryos is increased until it is suitable for the operation of the method of the present invention. After piercing the aforementioned cells into the lower subzonal cavity of the aforementioned non-human embryo by microinjection microtubules in a high-tension solution having an osmotic pressure suitable for the operation of the method of the present invention, the aforementioned microinjection A non-human embryo-cell aggregate is cultured in a culture medium to obtain a chimeric embryo.

The "high-tension solution" as used in the present invention refers to a solution having an osmotic pressure greater than the osmotic pressure of the isotonic solution of the aforementioned cells and/or the aforementioned non-human embryo. The "high-tension solution" of the present invention is prepared by adding an appropriate concentration of a solute to a suitable culture solution so that the osmotic pressure of the culture solution is greater than the isotonic solution osmotic pressure of the cells and/or the non-human embryo; preferably, The high tensile solution of the present invention has an osmotic pressure of 350 to 850 mOsm/kg H ₂ O. The aforementioned culture fluids are selected and formulated according to the prior art in the art without limitation.

For example, the high-tension solution of the present invention is prepared by dissolving 0.05 M to 0.5 M of a saccharide in a culture solution; for example, but not limited to: EHK culture solution, TL-Hepes culture solution, HK culture solution, EK culture solution, KSOM-AA culture solution, EP culture solution, or PZM-3 culture solution; wherein the higher the concentration of the aforementioned sugar, the higher the osmotic pressure of the prepared high-tension solution; Classes are for example, but not limited to, sucrose, glucose or a combination thereof.

Accordingly, the formulation and formulation method of the "high-tension solution" of the present invention need not be limited, and those skilled in the art can prepare a high-tension solution having an osmotic pressure in accordance with the spirit of the present invention when the experimental design and the culture solution used can be combined.

[embryo and cells]

The "non-human embryo" described in the present invention is a non-human mammalian embryo, and more specifically, the aforementioned non-human germline is in a 1-cell stage embryo to a mulberry embryo. The "embryonic stem cell line" of the present invention is a fertilized egg, a 2-cell stage embryo, a morula or a dissociated embryo leaf cell, a blastocyst or an inner cell mass (ICM) of a non-human mammal. Cell division but not differentiation, such as, but not limited to, mouse embryonic stem cell line ESC 26, ESC 26GJ9012-8-2, ESC98B33, ESC98B34, ESC98B37 or porcine embryonic cell line pESL-23, pESL-23 1-22. The aforementioned cells and the aforementioned non-human embryos may be of the same or different species of non-human mammals such as, but not limited to, mice or pigs.

The aforementioned mouse embryonic stem cell line ESC 26 is a super-breasted (albino, cc) inbred BALB/c female mouse and the body surface color is mainly white (cc). Or the light chinchilla (cc ^ch ) inbred is purely established by the cells of the 3.5 day blastocyst of 129/SvJ male mouse natural breeding. The aforementioned mouse embryonic stem cell line ESC 26GJ9012-8-2 is a cell line having green fluorescent characteristics obtained by further transfecting and selecting the mouse embryonic stem cell line ESC 26 by pCX-EGFP (Lee et al., 2003; Lee et al., 2007).

The aforementioned mouse embryonic stem cell lines ESC98B33, ESC98B34 and ESC98B37 are 0.5-day fertilized eggs (ESC98B33, which are naturally bred by wild-type C57BL/6 (B6) female mice and B6 male mice, which are superovulated and black in color. ESC98B34) or 2.5 days of morula (ESC98B37) cells were established.

The aforementioned porcine embryonic cell line pESL-23 was established by cells of hatched blastocysts seven days after the Yorkshire sows and Yorkshire boars were bred (Lee et al., 1992). The aforementioned pig embryo stem cell line pESL-23 1-22 is a cell line having green fluorescent characteristics obtained by further transfecting and culturing the porcine embryo-derived cell line pESL-23 with pCX-EGFP.

[embryo and cell culture conditions]

The culture solution of the present invention is selected according to the conventional knowledge in the art, and is directed to experimental subjects such as mice or pigs, culture environment, experimental purpose and the like.

The "embryonic stem cell culture solution (ESC M.)" of the present invention is prepared according to the formulation of a conventional embryonic stem cell culture solution; more specifically, the embryonic stem cell culture solution used in the present invention comprises a DMEM culture solution and a leukemia inhibitory factor (leukemia inhibitory factor). , LIF) and fetal bovine serum (FBS), synthetic serum replacement (KSR) or a combination thereof; wherein the fetal calf serum and the synthetic serum substitute are used as an alternative The embryonic stem cell culture solution using fetal bovine serum is abbreviated as FBS ESC M.; the embryonic stem cell culture solution using artificial synthetic serum substitute is abbreviated as KSR ESC M. More specifically, the aforementioned FBS ESC M. and the aforementioned KSR ESC M. respectively contain 10-20% fetal calf serum or 10-20% synthetic serum substitute.

Embryonic stem cells are typically cultured in a feeder layer to maintain their undifferentiated state. The preparation of the STO cell feeder layer allows STO cells to be inactivated by mitomycin C, and then made into a single cell suspension in STO medium (STO M.), and then into the gelatin. Dispose of the culture dish for use. The preparation method of the human foreskin fibroblast (Hs68, BCRC number: 60038; ATCC number: CRL-1635) is the same as the preparation method of the aforementioned STO cell feeder layer. The aforementioned STO culture solution contains DMEM culture solution and 10% fetal calf serum.

Mouse embryos at 37 ℃, 5% CO ₂ incubator lines were cultured in KSOM-AA broth; at 37 ℃, 5% CO ₂ incubator containing outer operating HEPES (molar concentration of 20.85 mM; Sigma H6147) of KSOM-AA medium (abbreviated as HK medium) and either fetal fetal serum, synthetic serum substitutes or combinations thereof to provide additional nutrients.

Pig embryos were cultured in PZM-3 medium (Yoshioka et al., 2002) in a 38.5~39.0 °C, 5% CO ₂ incubator; HEPES (20.85 millimolar concentration) was used outside the CO ₂ incubator. TL medium (abbreviated as TL-Hepes medium) and either fetal fetal serum, synthetic serum substitutes or combinations thereof to provide additional nutrients.

The "EHK culture solution" according to the present invention comprises 25 to 75% of embryonic stem cell culture solution, which comprises DMEM culture solution, leukemia inhibitory factor, fetal bovine serum, synthetic serum substitute or a combination thereof; and 25 to 75%. The HK culture solution; more specifically, the EHK culture solution contains 25 to 75% of the aforementioned FBS ESC M. or the aforementioned KSR ESC M. and 25 to 75% of the HK culture solution.

The "EK culture solution" according to the present invention comprises 25 to 75% of embryonic stem cell culture solution, which comprises DMEM culture solution, leukemia inhibitory factor, fetal bovine serum, synthetic serum substitute or a combination thereof; and 25 to 75%. The KSOM-AA culture solution; more specifically, the EK culture solution contains 25 to 75% of the aforementioned FBS ESC M. or the aforementioned KSR ESC M. and 25 to 75% of the KSOM-AA culture solution.

The "EP culture solution" according to the present invention comprises 25 to 75% of embryonic stem cell culture solution, which comprises DMEM culture solution, leukemia inhibitory factor, fetal bovine serum, synthetic serum substitute or a combination thereof; and 25 to 75%. The PZM-3 culture solution; more specifically, the EP culture solution contains 25 to 75% of the aforementioned FBS ESC M. or the aforementioned KSR ESC M. and 25 to 75% of the aforementioned PZM-3 culture solution.

[enriched or purified mouse embryonic stem cells]

First, mouse embryonic stem cells (such as the aforementioned mouse embryonic stem cell line ESC 26, ESC 26GJ9012-8-2, ESC98B33, ESC98B34, ESC98B37) were cultured in a Petri dish (35 mm) over the feeder cells, and subcultured. 1.5 ± 0.5 days, trypsin (trypsin) or ^{TrypLE TM Express (Gibco Cat. #} 12605) and the like in the culture from the cells attached to each other, and then to release manner pipette (Pipetting) the cells were broken as a single Cell suspension. The single cell suspension was then transferred to a blank petri dish containing embryonic stem cell culture medium, and the cells were allowed to stand in a 5% CO ₂ incubator at 37 ° C for about 80 minutes. Then, the unattached upper cell suspension is aspirated (most of which are poorly viable or dead cells, debris, impurities), and then about 2 ml of embryonic stem cell culture solution is added and attached to the aforementioned culture dish. The cells in the cells are washed and recovered as cell suspensions, and the cell suspensions are mostly viable embryonic stem cells. The cell suspension was then transferred to a blank culture dish, and the cells were allowed to stand in a 5% CO ₂ incubator at 37 ° C for about 20 minutes. Then, the unattached upper layer cell suspension was aspirated into a centrifuge tube and centrifuged at 170 x g for three minutes to collect the cells in the cell suspension. After centrifugation, the supernatant was aspirated and the pellet was retained, and then the cells of the cell pellet were resuspended in EHK medium (FBS ESC M.+HK or KSR ESC M.+HK) and the cell concentration was adjusted. The purity of the embryonic stem cells purified by the foregoing method can reach 97% (Lee et al., 2007).

In addition to the foregoing methods, those of ordinary skill in the art may use other purification methods as appropriate, such as purification of freshly thawed mouse embryonic stem cells by continuous secondary resting (100 minutes and 30 minutes) (Lee et al., 2007).

[microscope]

The experimental operation of the present invention is to observe and photograph green fluorescent cells and embryos with a Zeiss Axiovert 35 inverted microscope fluorescent system. The light source is OSRAM HBO 50 W/AC, 200 V high pressure mercury arc lamp. The best excitation wavelength of EGFP is 488~490nm (nm), the best divergence wavelength is 507~509nm (the optimal wavelength range of green light is about 520~530nm); the filter set used (filter set 09) , Cat. No. 487909) contains BP 450~490 excitation (source) filter (exciter filter), FT 510 dichromatic beam splitter and LP 520 barrier filter. UVG-50 (Spectronics Co., Westbury, NY, USA) UV goggles were used for the observation of fluorescence.

Embryos and cells were photographed on a microscope or green fluorescent mouse using a Nikon D1 digital camera. When photographing green fluorescent mice, UVP (http://www.uvp.com; Upland, CA, USA) long-wave (365 nm) ultraviolet light (several light intensity can be used simultaneously) is used as the light source ( Passing blue glass); using a yellow absorbing filter (yellow complementary color blue is absorbed, 520 nm or more light wave passing) is a light blocking filter. The resulting image file is fully imaged, brightnessed, contrasted, and tinted with Photoshop 7.1 image software (without basic changes).

The following examples are intended to further understand the advantages of the present invention and are not intended to limit the scope of the invention.

Example 1: Obtaining the non-human embryo required for the method of the present invention

In this example, an outbred ICR female mouse whose body color is white (albino, cc) was used, and was naturally mated with ICR male mice after superovulation. 2~8-cell stage embryos and/or morulae embryos were recovered after 1.5 days and 2.5 days of natural breeding; wherein the 2-~8-cell stage embryos were washed into HK culture medium and left at room temperature for use; The morula was placed in a phosphate buffer solution (PBS) containing no calcium ions (Ca ²⁺ ) and magnesium ions (Mg ²⁺ ) for about 1 hour to expose cells in the morula.

Example 2: Observing the morphology of the aforementioned non-human embryo and the aforementioned embryonic stem cell in a high-tension solution

The 3-cell stage embryo-morulae embryo and the mouse embryonic stem cell line cell ESC 26GJ9012-8-2 (P20) obtained in Example 1 were placed in HK culture medium containing different concentrations of sucrose or glucose, and the embryos and cells were observed. Morphological changes and subsequent development.

The first panel a~e shows the embryo morphology of the 4-cell stage embryo-mulberry embryo obtained in ICR×ICR mice 2.5 days after mating in different concentrations of sucrose in HK culture medium; among them, the first figure a~e HK The sucrose concentration and osmotic pressure of the culture medium are listed in Table 1 below:

The HK culture medium with high sucrose concentration has a high osmotic pressure. Comparing the first graph a~e, it can be seen that the embryo body is reduced in the 4-cell stage embryo-mulberry embryo in the HK culture medium with higher osmotic pressure. The smaller; in other words, the larger the subzonal cavity between the zona pellucida and the embryo body, which facilitates the microinjection of cells and reduces possible damage to the blast cells.

The second panel a, b, c shows the embryo morphology and subsequent development of the 3-cell stage embryo ~ mulberry embryo obtained in ICR × ICR mice 2.5 days after mating in different concentrations of glucose for 2 hours; The glucose concentration and osmotic pressure of the HK culture solution of Figures a, b and c are listed in Table 2 below:

Comparing the second graphs a, b, and c, it is known that the higher the osmotic pressure of the HK medium (i.e., the higher concentration of glucose) is between the zona pellucida-mulberry embryo and the embryonic body. The larger the cavity; this result is consistent with the experimental results obtained in the first graph a~e.

Next, the embryo in the second panel a is washed into a KSOM-AA culture solution having an osmotic pressure of about 285 mOsm/kg H ₂ O. Please refer to the second panel a1, a2, and a3 for 1 day and 2 respectively. As a result of days and 3 days, the results show that the embryo still develops to the blastocyst stage, and it is apparent that the high-tension solution of the present invention does not significantly impair the subsequent development of the embryo. Similarly, please refer to the second figure b1, b2, b3 to show that the embryo of the second figure b is transferred to the KSOM-AA culture solution with an osmotic pressure of about 285 mOsm/kg H ₂ O for 1 to 3 days; and the second Figures c1, c2, and c3 show that the embryos of the second panel c were transferred to the KSOM-AA culture solution having an osmotic pressure of about 285 mOsm/kg H ₂ O for 1 to 3 days, and all of them were shown to be cultured in the high-tension solution of the present invention. The embryo still retains normal vitality and developmental function.

Finally, the cell morphology of mouse embryonic stem cell ESC 26GJ9012-8-2 in high-tension solution was observed. Referring to the third panel a, b and c, the mouse embryonic stem cell ESC 26GJ9012-8-2 is cultured in HK culture medium (containing 1% fetal bovine serum) containing different sucrose concentrations; wherein, the third figure a, The sucrose concentration and osmotic pressure of HK culture fluids of b and c are listed in Table 3 below:

As is clear from the results in the figure, the embryonic stem cells in the HK culture solution (i.e., the higher the sucrose concentration) having a higher osmotic pressure contracted more, that is, the smaller the diameter. In the manipulation of microinjected cells, the smaller embryonic stem cells represent microinjection micropipettes that can be used to make smaller diameters of the device, thus helping to reduce possible damage to the embryo and thereby enhancing microinjection. s efficiency. In addition, since the mouse embryonic stem cell ESC 26GJ9012-8-2 has green fluorescence characteristics, please refer to the third image a1, b1 and c1 for the fluorescence images of the third figure a, b and c, respectively, which are shown in the present invention. Culture in high-tension solution did not affect the green fluorescence characteristics of mouse embryonic stem cell ESC 26GJ9012-8-2.

Example 3: Production of mouse chimeric embryos according to the method of the present invention

The main experimental parameters of the seven experimental groups in this example are organized as follows:

[Experimental Group 1]

First, the 4-cell stage embryo to morula of 2.5 days after ICR×C57BL/6 mouse breeding was obtained by the method described in Example 1, and the EHK solution containing 0.05 M sucrose (50% FBS ESC M. and 50% HK). In the osmotic pressure of about 354 mOsm/kg H ₂ O), about 16 green-fluorescent embryonic stem cells ESC 26GJ9012-8-2 (P16) were injected by microinjection into the aforementioned 4-cell stage embryo ~ mulberry A subzonal cavity under the embryo to produce a chimeric embryo. Next, the aforementioned 4-cell stage embryo-morula (embryo-cell aggregate) completed by microinjection was placed in EK medium (50% FBS ESC M. and 50% KSOM-AA, and the osmotic pressure was about 285 mOsm/ Incubate for two days in kg H ₂ O). The fourth figure a is the aforementioned 4-cell stage embryo-morula (embryo-cell aggregate) which has just completed microinjection, in which the embryonic stem cells are clearly visible and have not yet formed into a chimeric embryo with the embryo; a culture is carried out in the EK medium. The aforementioned embryo-cell aggregates at night have developed into chimeric embryos (Fig. 4b); the fourth panel c shows the aforementioned chimeric embryos cultured in EK medium for two nights. As can be seen from the figure, the chimeric embryo of the experimental group continued to develop into the blastocyst in hatching (Fig. 4c), and it is apparent that the method of the present invention can successfully produce mouse chimeric embryos.

Further, from the fourth map b1 (the fluorescent image of the fourth graph b) and the fourth graph c1 (the fluorescent image of the fourth graph c), it can be seen that the aforementioned green fluorescent embryonic stem cells (ESC) are present in the chimeric embryo of the experimental group. 26GJ9012-8-2) continues to proliferate and mainly enters the embryonic cell population (Fig. 4c).

[Experimental Group 2]

The 4-cell stage embryo to morula of 2.5 days after ICR×ICR mouse breeding was obtained according to the method of Example 1 in EHK medium containing 0.2 M sucrose (37.5% KSR ESC M. and 62.5% HK, about 500 mOsm). /kg H ₂ O), about 20 green fluorescent stem cells ESC 26GJ9012-8-2 (P16) were injected by microinjection into the transparent zone of the 4-cell stage embryo to the mulberry embryo ( Subzonal cavity) to produce chimeric embryos. Next, the aforementioned 4-cell stage embryo-morula (embryo-cell aggregate) completed by microinjection was placed in EK medium (37.5% KSR ESC M. and 62.5% KSOM-AA, osmotic pressure was about 285 mOsm/ Incubate for two days in kg H ₂ O).

Please refer to the fifth figure a, b, c, d for the chimeric embryo produced by the embryo-cell polymer, wherein, the fifth figure a: the 8-cell stage embryo after microinjection; the fifth figure b: sham control, that is, an embryo with a zona pellucida penetrating through microinjected microtubules but not injecting any cells; fifth panel c: 4-cell stage embryo after microinjection; d: larger than 8-cell stage embryo after microinjection (ie, morula, immersed in phosphate buffer containing no Ca ²⁺ and Mg ²⁺ before microinjection to visualize blast cells, please refer to the examples One). The fifth graphs a1 and a2 are the chimeric embryos of the fifth graph a of one night and two nights in the EK medium, respectively; the fifth graphs b1 and b2 are cultured one night and two in the EK medium, respectively. The embryo of the fifth graph b of the night; the fifth graphs c1 and c2 are the chimeric embryos of the fifth graph c of one night and two nights in the EK culture solution; the fifth graphs d1 and d2 are respectively The chimeric embryo of the fifth panel d of one night and two nights was cultured in the aforementioned EK medium. The results showed that the chimeric embryos of the experimental group 2 continued to develop into the blastocysts in hatching (fifth panels a2, b2, c2 and d2), meaning that the method of the present invention successfully produced mouse chimeric embryos.

In addition, except for the fifth diagrams b, b1, and b2, which are brightfields, the fifth diagrams a, a1, a2, c, c1, c2, d, d1, and d2 are images of bright field and fluorescent exposure simultaneously, which can be observed. The chimeric embryos of the experimental group 2 retained the green fluorescent characteristics of the aforementioned embryonic stem cells (ESC 26GJ9012-8-2).

[Experimental Group 3]

First, the 8-cell stage embryo of 2.5 days after ICR×ICR mouse breeding was obtained by the method described in Example 1, and the osmotic pressure was about 50% KSR ESC M. and 50% HK in 0.2% sucrose-containing EHK solution. In 500 mOsm/kg H ₂ O), about 20 green-fluorescent embryonic stem cells ESC 26GJ9012-8-2 (P15) were injected into the aforementioned 8-cell stage embryo by microinjection. Next, the 8-cell stage embryo (embryo-cell polymer) completed by microinjection was placed in a KSOM-AA medium containing 5% fetal bovine serum (osmotic pressure of about 285 mOsm/kg H ₂ O). Three days.

Figure 6 is a picture during microinjection; the sixth picture a1 is the chimeric embryo produced by microinjection of the aforementioned 8-cell stage embryo; the sixth figure b, b1 is containing 5% fetal bovine serum. The KSOM-AA culture solution was cultured for one night of the chimeric embryo; the sixth figure c, c1 was the incubated embryo of KSOM-AA containing 5% fetal bovine serum for two nights; the sixth figure d, D1 is the aforementioned chimeric embryo cultured for three nights in KSOM-AA medium containing 5% fetal bovine serum; wherein, the sixth figure a, a1, b1 (corresponding to b), c1 (corresponding to c), d1 (corresponding to d ) An image that is simultaneously exposed to bright field and fluorescent light. The results showed that the chimeric embryos of the experimental group had good activity and continued to develop into the blastocysts in hatching (sixth figure b, b1, c, c1, d, d1), which means that the method of the present invention successfully produced mouse inlays. Embryo.

In addition, the chimeric embryos of the experimental group three overnight cultures were transferred into the uterine horn of the embryonic mouse of ICR pseudopregnant 2.5 days, and the chimeric mice which were sexually matured after delivery were returned to the ICR. The mother mouse produces the next generation of mice (see Figure 6 e, an image of the mouse's bright field and fluorescent exposure), the mouse still retains the green fluorescent characteristics, meaning the method of the present invention The chimeric mice produced have gonad inheritance.

[Experimental group four to six]

First, the 4- to 8-cell stage embryos of ICR×ICR mice were incubated 2.5 days later, followed by EHK solution containing 0.2 M sucrose (37.5% KSR ESC M. and 62.5% HK, osmotic pressure of about 500 mOsm/kg). In H ₂ O), about 15 embryonic stem cells ESC98B34 (P5, experimental group 4, seventh panel a) established from C57BL/6J×C57BL/6J fertilized eggs were injected into the aforementioned 8-cell stage embryo, and the resulting embryos were obtained. - The cell aggregate was transferred to the KSOM-AA culture medium; about 15 embryonic stem cells ESC98B33 (P5, experimental group 5, panel 7b) established from the C57BL/6J×C57BL/6J fertilized egg were injected into the aforementioned 4-cell. After the embryo, the resulting embryo-cell aggregate was transferred to EK (37.5% KSR ESC M.+62.5% KSOM-AA; ~285 mOsm) culture medium; and about 20 were established from C57BL/6J×C57BL/6J The embryonic stem cell ESC98B37 (P5, experimental group 6, seventh panel c) of the morula was injected into the aforementioned 8-cell stage embryo, and the resulting embryo-cell aggregate was transferred into the KSOM-AA culture medium for cultivation.

Next, after culturing the embryonic-cell aggregates of the experimental group of four to six in the seventh panel a, b, and c overnight, the obtained chimeric embryos were transferred into the uterine horn of the embryonic mouse of ICR pseudopregnant 2.5 days, and delivered. The obtained mice were respectively shown in the seventh panel a1, b1, and c1; among them, the chimeric embryos transferred into 12 experimental groups were symbiotically derived from 7 mice; the chimeric embryos transferred into 13 experimental groups were co-produced 4 small. Rats; 6 mice were transferred to chimeric embryos of 12 experimental groups. As can be seen from the figure, the ratio of the specific epidermal chimeric mice is high, and the coat color mosaic ratio may even be 100%; this shows the effectiveness of the chimeric mouse produced by the present method.

[Experimental Group 7]

First, the 2-cell stage embryos of ICR×ICR mice were obtained, and the number of chromosome sets was 2 (2n) (Fig. 8). After electrofusion, the number of chromosome sets was 4 (4n). Cell stage embryos (Fig. 8b), the 1-cell stage embryos were placed overnight in KSOM-AA medium to obtain 4n 2-~4-cell stage embryos (Fig. 8c). The eighth panels d, e, and f show that 2n 2-cell stage embryos were cultured in KSOM-AA culture medium for 1 to 3 days, respectively, without microinjection as a control group.

In an EHK solution containing 0.2 M sucrose (37.5% KSR ESC M. and 62.5% HK, osmotic pressure approx. 500 mOsm/kg H ₂ O), 25 to 30 green embryonic mouse embryonic stem cells ESC26GJ9012- 8-2 (P14) was injected into the aforementioned 4n 2-~4-cell stage embryo by microinjection, and the resulting embryo-cell aggregate was transferred into KSOM-AA medium for three nights (represented as the eighth figure g). , h, i) or EK (37.5% KSR ESC M. + 62.5% KSOM-AA; ~ 285 mOsm) culture medium for three nights (represented as the eighth figure j, k, l). The eighth figure g, h, i, j, k, l are images of common field of view and fluorescent exposure. The results showed that the chimeric embryos of the experimental group 7 could continue to develop into the blastocysts in the hatching and showed green fluorescence.

Example 4: Production of a pig chimeric embryo according to the method of the present invention

The main experimental parameters of the three experimental groups in this example are summarized in Table 5 below:

[Experimental Group 8]

First, the 4-cell stage embryo to mulberry embryos of Duroc × Duroc pigs were obtained five days after breeding. Green fluorescent porcine embryo-derived cells pESL-23GJ in TL-Hepes medium (osmotic pressure approximately 500 mOsm/kg H ₂ O) containing 0.2 M sucrose 1-22 (P10) microinjection into the aforementioned 4-cell stage embryo-morulae, and then the resulting embryo-cell aggregate was transferred to PZM-3 medium for overnight culture. The ninth panel a shows the situation at the time of microinjection; the ninth panel b shows the aforementioned 4-cell stage embryo to morula; and the ninth panel c shows the aforementioned chimeric embryo after overnight culture. . As can be seen from Figure 9c, the chimeric embryo of the experimental group 8 can continue to develop into the blastocyst, meaning that the method of the present invention successfully produces a pig chimeric embryo.

[Experimental Group Nine]

The mature porcine eggs cultured in vitro were first obtained, and the embryos were developed for three days after electroactivation. Next, the green-fluorescent porcine embryo-derived cells pESL-23GJ will be expressed in EHK medium (50% KSR ESC M. and 50% HK, about 500 mOsm/kg H ₂ O) containing 0.2 M sucrose. 1-22 (P11) microinjection into the aforementioned parthenogenetic embryo; then, transfer the resulting embryo-cell aggregate into PZM-3 medium (Fig. a, b, c and d) or EP medium (50% FBS) ESC M. and 50% PZM-3, the tenth figure e, f, g and h) were cultured for three nights.

Figure 11 a and e show the aforementioned parthenogenetic embryos completed by microinjection; the tenth panels b and f show chimeric embryos cultured overnight in PZM-3 medium or EP medium, respectively; The g lines are respectively displayed in PZM-3 culture solution or EP culture medium for three nights of chimeric embryos; the tenth picture c and d have the same field of view, but the tenth figure c is an image of simultaneous exposure of bright field and fluorescence; The ten map g is the same as the field of view of h, but the tenth graph g is an image in which the bright field and the fluorescent light are simultaneously exposed. As can be seen from Figures c and g, the chimeric embryo of the experimental group 9 can continue to develop to the blastocyst and exhibit green fluorescence, meaning that the method of the present invention successfully produces a pig chimeric embryo.

[Experimental Group 10]

First, the mature pig eggs cultured in vitro were obtained, and the parthenogenetic embryos developed for three days were obtained after electroactivation. Green fluorescent mouse embryonic stem cell ESC 26GJ9012-8-2 (P14) will be expressed in EHK medium (50% KSR ESC M. and 50% HK, about 500 mOsm/kg H ₂ O) containing 0.2 M sucrose. Microscopic injection of the aforementioned parthenogenetic embryos to produce mixed chimeric embryos (Fig. 11 a~f); then, the resulting embryo-cell aggregates were transferred to PZM-3 medium (osmotic pressure of about 285 mOsm/ Kg H ₂ O, eleventh and eleventh i) or EP medium (50% FBS ESC M. and 50% PZM-3, osmotic pressure approx. 285 mOsm/kg H ₂ O, eleventh h And train three nights in j).

The above-mentioned eleventh image a~f is an image when microinjection is performed, wherein e, f are images of simultaneous exposure of bright field and fluorescent image; and eleventh images i and j are pseudo-processing groups, that is, transparent bands Parthenogenetic embryos that were microinjected into microtubules but not injected into any cells. As can be seen, the chimeric embryos of the experimental group 10 can continue to develop into blastocysts, showing that the method of the present invention also successfully produces chimeric embryos using embryos and cells of different non-human mammalian origin.

It will be apparent to those skilled in the art that various changes can be made in accordance with the embodiments of the present invention without departing from the spirit of the invention. Therefore, it is to be understood that the invention is not limited by the scope of the invention, and is intended to cover the modifications of the spirit and scope of the invention.

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The first figure shows the embryo morphology of mouse 4-cell stage embryo-morulae in HK culture medium containing different concentrations of sucrose, wherein the sucrose concentrations are (a) 0 M; (b) 0.1 M; (c) 0.2 M; (d) 0.3 M; (e) 0.4 M.

The second panel shows the embryo morphology of mouse 3-cell stage embryo-mulberry embryos in HK culture medium containing different concentrations of glucose, wherein the glucose concentrations are (a) 0.5 M; (b) 0.25 M; (c) 0.125 M; (a1) is to transfer the embryo of (a) to a KSOM-AA culture solution having an osmotic pressure of about 285 mOsm/kg H ₂ O for one day; (a2) to transfer the embryo of (a) to an osmotic pressure of about 285 mOsm/KgH ₂ O in KSOM-AA culture solution for 2 days; (a3) is to transfer the embryo of (a) into KSOM-AA culture solution with an osmotic pressure of about 285 mOsm/kg H ₂ O for 3 days; (b1) for culturing the embryo of (b) into a KSOM-AA culture solution having an osmotic pressure of about 285 mOsm/kg H ₂ O for one day; (b2) for moving the embryo of (b) to an osmotic pressure of about 285 mOsm /kg H2O in KSOM-AA culture solution for 2 days; (b3) to transfer embryo of (b) to KSOM-AA culture solution with osmotic pressure of about 285 mOsm/kg H ₂ O for 3 days; (c1) To transfer the embryo of (c) to a KSOM-AA culture solution having an osmotic pressure of about 285 mOsm/kg H ₂ O for 1 day; (c2) to move the embryo of (c) to an osmotic pressure of about 285 mOsm/kg H KSOM-AA ₂ O in the broth for 2 days; (C3) to (c) to the embryo into an osmotic pressure of about 285 mOsm / kg KSOM-AA broth of H ₂ O for 3 days

The third panel shows the cell morphology of mouse embryonic stem cell line ESC 26GJ9012-8-2 (P20) in HK culture medium (containing 1% FBS) containing different concentrations of sucrose, wherein the sucrose concentration is (a) 0 M (b) 0.2 M; (c) 0.3 M; (a1) is a fluorescent image of (a); (b1) is a fluorescent image of (b); (c1) is a fluorescent image of (c).

The fourth panel shows the results of microscopic injection of embryonic stem cells ESC 26GJ9012-8-2 in mouse embryos of Example 3 of the present invention, wherein (a) is a mouse embryo that has just completed microinjection; (b) One night of chimeric embryos were cultured in EK medium; (c) two nights of chimeric embryos were cultured in EK medium; (b1) was a fluorescent image of (b); (c1) was (c) Fluorescent image.

The fifth panel shows the results of microinjection of mouse embryos in the third experimental group of the present invention, wherein (a) is a microinjected 8-cell stage embryo; (b) is a sham control group. , that is, an embryo with a zona pellucida penetrating through microinjected microtubules but not implanted with any cells; (c) a microinjected 4-cell stage embryo; (d) a microinjected greater than 8-cell stage Embryo; (a1) is a chimeric embryo of (a) cultured overnight in EK (37.5% KSR ESC M.+62.5% KSOM-AA; ~285mOsm/kg H ₂ O); (a2) Two nights of (a) chimeric embryos were cultured in EK medium; (b1) one (b) chimeric embryo cultured in EK medium for one night; (b2) two cultures in EK medium (b) chimeric embryo in the evening (c1) is a chimeric embryo of (c) cultured in EK medium for one night; (c2) is embedded in EK medium for two nights (c) Embryo; (d1) is a chimeric embryo of (d) cultured overnight in EK medium; (d2) is a chimeric embryo of (d) cultured in EK medium for two nights.

Figure 6 is a graph showing the results of microinjection of mouse embryos in the third experimental group of the present invention, wherein (a) shows the process of microinjection; (a1) is produced by microinjection of 8-cell stage embryos. Embryo-cell aggregate; (b, b1) is a chimeric embryo cultured in KSOM-AA medium containing 5% fetal bovine serum for one night, (b1) is (b) clear field of view and fluorescence of the same field of view Simultaneously exposed images; (c, c1) are chimeric embryos cultured in KSOM-AA medium containing 5% fetal bovine serum for two nights, (c1) for (c) simultaneous field of view and fluorescence The exposed image; (d, d1) is the chimeric embryo after three nights of incubation in the KSOM-AA medium containing 5% FBS, (d1) is (d) simultaneous field of view and fluorescence exposure. Image; (e) an image of a simultaneous field of view and fluorescence of a chimeric mouse.

The seventh panel shows the results of microinjection of mouse embryos of the experimental group 4, 5 and 6 of the third embodiment of the present invention, wherein (a) is a chimeric embryo of the experimental group 4; (b) is a chimeric embryo of the experimental group 5. (c) is a chimeric embryo of the experimental group 6; (a1) is a chimeric mouse of the experimental group 4; (b1) is a chimeric mouse of the experimental group 5; (c1) is a small chimeric small of the experimental group 6. mouse.

The eighth panel shows the results of microinjection of mouse embryos in the third experimental group of the present invention, wherein (a) is a 2-cell stage embryo with 2 sets of chromosomes (2n); (b) is electrofusion. 4-cell stage embryo with 4 sets of chromosomes (4n); (c) 4n 2-~4-cell stage embryo; (d) 2n embryos cultured on KSOM-AA medium for the first day; (e) 2n embryos were cultured in the KSOM-AA medium for the second day; (f) 2n embryos were cultured in the KSOM-AA medium for the third day; (g) ICR 4n 2-~4-cell stage embryos After microinjection, the first day of chimeric embryos were cultured in KSOM-AA medium; (h) was (g) embryos, and the next day of chimeric embryos were cultured in KSOM-AA medium; (i) For the embryo of (g), the chimeric embryo of the third day was cultured in KSOM-AA culture solution; (j) was ICR×ICR 4n 2-~4-cell stage embryo after microinjection, at EK (37.5%) KSR ESC M.+62.5% KSOM-AA; ~285 mOsm/kg H ₂ O) Chimeric embryos cultured on the first day in culture medium; (k) embryos of (j), cultured in EK medium The chimeric embryo of day (l) is the embryo of (j), and the chimeric embryo of the third day is cultured in EK medium.

The ninth figure shows the micro-injection of porcine embryo-derived cells pESL-23GJ in pig embryos of the fourth experimental group of the present invention. Results 1-22, wherein (a) shows the process of microinjection; (b) 4-cell stage embryo to morula after microinjection; (c) (b) chimeric embryo after overnight culture .

The tenth figure shows that the pig parthenogenetic embryo of the experimental group N in the fourth embodiment of the present invention is microinjected into the porcine embryo source to express the green fluorescent cell pESL-23GJ. 1-22 results, wherein (a) is a parthenogenetic embryo that completes microinjection; (b) is a chimeric embryo of (a) cultured in a medium of PZM-3 for one day; (c, d) is a PZM- 3 (3) chimeric embryos of (a) cultured in culture medium, (c) images of (d) bright field and fluorescent exposure; (e) parthenogenetic embryos for microinjection; (f) For one day (e) chimeric embryos were cultured in EP (50% FBS ESC M. +50% PZM-3) medium; (g, h) was cultured in EP medium for three days (e) The chimeric embryo, (g) is an image of the (h) bright field and the fluorescent exposure.

The eleventh figure shows the results of microinjection of mouse embryonic stem cells ESC 26GJ9012-8-2 (P14) in pig embryos of the experimental group of the fourth embodiment of the present invention, wherein (a)~(f) are microinjected. (e), (f) are images of simultaneous exposure of bright field and fluorescent light; (g) mixed three-night mixed chimeric embryos in PZM-3 medium; (h) in EP ( 50% FBS ESC M. +50% PZM-3) Three-night mixed chimeric embryos were cultured in culture medium; (i) and (j) were sham control, ie, the zona pellucida was injected by microinjection Parthenogenetic embryos with microtubules penetrating but not infused with any cells; (i) cultured in PZM-3 medium for three nights; (j) cultured in EP medium for three nights.

Claims (11)

A method for producing a non-human mammalian chimeric embryo in vitro comprises the steps of: providing a cell which is a germ source, a fetal source, a placental source, a umbilical cord source, an adult source, a non-synthetic primary cell, Cell line cells, stem cells, pluripotent stem cells or induced pluripotent stem cells, protein-induced pluripotent stem cells; providing a non-human embryo, which is in the 1-cell stage embryo to the morula; the cells are exposed in a high-tension solution Microinjection into the aforementioned non-human embryo to obtain an embryo-cell aggregate; and culturing the embryo-cell aggregate in the culture medium to obtain a non-human chimeric embryo; wherein the osmotic pressure of the high-tension solution is 350-850 mOsm/kgH ₂ O. The method of claim 1, wherein the high-tension solution is a culture solution containing 0.05M to 0.5M of sugar. The method of claim 1, wherein the high-tension solution and/or the culture solution may further be added with serum, synthetic serum substitute or a combination thereof. The method of claim 1, wherein the cells and the source of the non-human embryo are non-human mammals of the same species or different species. The method of claim 1, wherein the aforementioned cells are purified or unpurified. The method of claim 1, wherein the cell line is an embryonic stem cell line. The method of claim 6, wherein the embryonic stem cell line is a fertilized egg or a 2-cell stage embryo of a non-human mammal. The cells of the morula or dissociated embryonic leaf cells, blastocysts or inner cell mass are divided but not differentiated. The method of claim 1, wherein the genetic material of the aforementioned cells is unchanged or altered. The method of claim 1, wherein the aforementioned non-human germline contains two or more sets of chromosomes. The method according to claim 1, wherein the culture solution is EHK culture solution, TL-Hepes culture solution, HK culture solution, EK culture solution, KSOM-AA culture solution, EP culture solution, or PZM-3 culture. liquid. The method according to claim 2, wherein the culture solution is EHK culture solution, TL-Hepes culture solution, HK culture solution, EK culture solution, KSOM-AA culture solution, EP culture solution, or PZM-3 culture. liquid.