Method for efficiently producing genetically modified rats using frozen eggs

Abstract

The purpose of the present invention is to provide a method for thawing cryopreserved rat eggs. The present invention provides a method for thawing cryopreserved rat eggs, the method being characterized in that the thawing is carried out in a thawing solution containing 0.05 M to 0.2 M of sucrose. The present invention also provides a method for producing pups from cryopreserved fertilized rat eggs, the method comprising: a step for thawing the cryopreserved fertilized rat eggs in a thawing solution containing about 0.05 M to about 0.2 M of sucrose and culturing the eggs; a step for selecting and culturing morphologically normal eggs if necessary; optionally, a step for genetically engineering the cultured fertilized eggs to create genetically modified fertilized eggs and culturing the eggs; and a step for transplanting the fertilized rat eggs into a recipient female rat to generate the pups.

Description

Efficient method for producing genetically modified rats using frozen eggs

[0001] The present invention relates to a method for freezing and thawing rat eggs, and a method for producing genetically modified rats using frozen and thawed eggs.

[0002] In recent years, the emergence of a new genome editing tool, CRISPR-Cas9, has enabled the creation of genetically modified animals in a variety of animal species. Rats, in particular, are the second most popular experimental animal after mice and are widely used in physiological, pharmacological, and toxicity tests, as well as behavioral analysis. Furthermore, due to their relatively simple surgical procedures and the ability to repeatedly collect large amounts of blood, urine, and other samples, genetically modified rats are being rapidly created as models for human diseases, drug discovery and toxicology in human metabolism, brain function and behavioral analysis, and regenerative medicine transplants. Rats are approximately 10 times larger than mice, and maintaining their lineages requires enormous amounts of breeding space and costs. Therefore, there is an urgent need to develop reproductive engineering technologies for the efficient creation, preservation, and breeding of genetically modified rats.

[0003] For example, efficient production of genetically modified rats using in vitro fertilized embryos has been reported (Non-Patent Document 1). Superovulation was induced by treating female rats with anti-inhibin serum (AIS) simultaneously with PMSG and hCG. Oocytes were obtained and then fertilized in vitro with sperm. Gene disruption (knockout) was then performed using the in vitro fertilized embryos. The disrupted embryos were then implanted into female rats to produce offspring. Gene disruption was performed on 224 in vitro fertilized eggs targeting the tyrosinase gene, and 171 embryos were implanted. 49 offspring were obtained (49 / 171 = 28.7%), and all of these individuals were gene-disrupted (albino individuals) (49 / 49 = 100%) (see Table 4).

[0004] However, the series of procedures, including collecting oocytes from female rats, performing in vitro fertilization using the collected oocytes and sperm, then performing genome editing (e.g., gene disruption or gene introduction) on the resulting fertilized eggs, and then implanting the genome-edited fertilized eggs into recipient female rats, takes an extremely long time, making it extremely difficult to complete these procedures during the day and placing a heavy burden on the workers.

[0005] Methods for cryopreserving unfertilized and fertilized rat eggs have also been reported. For example, Non-Patent Document 2 reports that oocytes collected from superovulated female rats were cryopreserved, thawed, and fertilized in vitro with sperm collected from male rats to produce offspring. Morphologically normal oocytes were obtained (630 / 958 = 65.1%) when oocytes transferred to 20-30 μL of DAP224 solution and frozen, and then thawed with 0.4 mL of 0.5 M sucrose solution. Furthermore, when normal frozen oocytes were fertilized with sperm, pronuclear oocytes and 2-cell embryos were obtained in 60.8% (152 / 250) and 29.8% (9 / 131) of the oocytes, respectively. Transfer of the fertilized eggs resulted in offspring in 18.7% (28 / 150) of the oocytes. However, neither the rate of morphologically normal eggs recovered nor the rate of hatched offspring are satisfactory.

[0006] Non-Cited Document 3 also discloses a method for cryopreserving rat one-cell embryos and a method for transferring the cryopreserved one-cell embryos to recipient female rats to obtain offspring. In this study, superovulated female rats were mated with male rats, and then the resulting one-cell embryos were vitrified in EFS10 medium containing 0.45 M sucrose and cryopreserved in cryotubes. The cryotubes containing the cryopreserved one-cell embryos were rapidly warmed and thawed, and then transferred to female rats. This resulted in offspring development rates comparable to those of non-cryopreserved one-cell embryos. Relatively good results were obtained with regard to the survival and developmental potential of thawed frozen fertilized eggs derived from in vivo fertilization. However, in the case of mating, females do not necessarily mate with males in a single mating. Furthermore, the number of males with the desired genetic phenotype is limited. Therefore, multiple matings are required to obtain the desired number of one-cell embryos, especially when the number of males is small. Furthermore, in the case of mating, fertilized eggs must be collected by perfusing the oviduct with culture medium under a stereomicroscope for each individual. Therefore, it is not suitable as a means of preparing fertilized eggs for genome editing. On the other hand, in vitro fertilization is efficient, producing more than 100 fertilized eggs per IVF using sperm collected from one male and oocytes collected from several females.

[0007] Methods for cryopreserving rat pronuclear stage fertilized eggs have been reported (Non-Patent Documents 4, 5, and 6). Non-Patent Document 4 describes the following: unfertilized eggs collected from the ampulla of the oviduct of superovulated 3- to 4-week-old immature female rats were fertilized in vitro with sperm collected from the cauda epididymis of a mature male rat; the resulting pronuclear stage fertilized eggs were transferred together with a small amount of culture medium to 20-30 μL of storage solution (DAP213) in a sampling tube and cryopreserved; the pronuclear stage fertilized eggs were then thawed in 37°C warm water by adding 0.3 mL of PB1 (modified phosphate-buffered saline) containing 0.3 M sucrose to the sampling tube. The thawed fertilized eggs were then subjected to morphological observation, and eggs that were determined to be morphologically normal were transplanted into recipient female rats and the development of offspring was examined.It was reported that 41% (40 / 97) of the retrieved eggs were found to be morphologically normal, and of these, 10% (4 / 40) developed into offspring.Neither the rate of morphologically normal fertilized eggs nor the rate of offspring that developed were sufficient.

[0008] Non-Patent Document 5 reports that pronuclear stage embryos of rats obtained by natural mating were cryopreserved, then thawed and implanted into female rats to produce offspring. Non-Patent Document 6 reports that pronuclear stage fertilized eggs of rats obtained by natural mating were cryopreserved in plastic straws, then thawed and used for genome editing. However, both reports use pronuclear stage fertilized eggs obtained by natural mating, which has the same problems as Non-Patent Document 3 and is not suitable as a means of preparing fertilized eggs for genome editing.

[0009] Although there have been reports of freezing and thawing unfertilized and fertilized rat eggs, the rate of morphologically normal eggs recovered and the rate of offspring developed from thawed eggs are both insufficient. Furthermore, there have been no reports of genome editing using frozen unfertilized or fertilized eggs to efficiently produce genetically modified rats.

[0010] PCT / JP2024 / 015563

[0011] A. Honda et al., Scientific Reports (2019) 9:11571N. Nakagata, Eep. Anim. (Experimental Animals) 41(4), 443-447 (1992)Fukuda et al., Biology of Reproduction, 2021, 105(1), 258-266Anzai et al., Exp. Anim. 43(2). 247-250, 1994Taketsuru et al., Cryobiology 84 (2018) 1-3Kaneko et al., Cryobiology 92 (2020) 231-234Kaneko, T. et al., Scientific Reports 4, 6382 (2014)Nakagata et al., Scientific Reports, (2020) 10:93. doi: 10.1038 / s41598-019-57090-7H. Kishi et al., The Journal of endocrinology 151(1): 65-75, 1996Nakagata, N. Embryo transfer through the wall of the fallopian tube in mice. Exp. Anim. (Experimental Animals) 41(3), 387-388 (1992)

[0012] An object of the present invention is to provide a method for thawing cryopreserved rat eggs. The present invention also provides a method for producing genetically modified rats using rat eggs thawed by the method.

[0013] The present inventors have conducted extensive research into the cryopreservation of rat eggs and the conditions for thawing them, and have found that thawing frozen rat eggs in the presence of a specific concentration of sucrose results in good offspring development from fertilized eggs after transplantation, leading to the completion of the present invention. The present invention includes the following: [1] A method for thawing cryopreserved rat eggs (unfertilized eggs or fertilized eggs), characterized in that the thawing is carried out in a thawing solution containing about 0.05 M to about 0.2 M (preferably about 0.075 M to about 0.15 M, more preferably about 0.09 M to about 0.11 M) of sucrose. [2] The method described in [1] above, wherein the rat eggs are in vitro fertilized eggs. [3] The method described in [2] above, wherein the in vitro fertilized eggs are in vitro fertilized eggs obtained from unfertilized eggs collected from superovulated female rats aged 6 to 7 weeks and sperm (fresh sperm or cryopreserved sperm). [4] The method according to the above-mentioned [1], wherein the rat eggs are oocytes collected from a 6- to 7-week-old female rat that has been subjected to superovulation induction. [5] The method according to the above-mentioned [3] or [4], wherein the superovulation induction is carried out by simultaneously administering an anti-inhibin antibody (preferably, an inhibin antiserum [preferably, 0.3 mL or less, more preferably 0.05 mL to 0.2 mL of inhibin antiserum]) and equine chorionic gonadotropin (eCG) (preferably, 5 IU to 40 IU) to a 6- to 7-week-old female rat, followed by administering human chorionic gonadotropin (hCG) (preferably, 5 IU to 40 IU).

[0014] [6] A method for producing offspring from cryopreserved rat eggs, comprising the following steps: (1) thawing and culturing cryopreserved rat eggs (unfertilized eggs or fertilized eggs) in a melting solution containing about 0.05 M to about 0.2 M (preferably about 0.075 M to about 0.15 M, more preferably about 0.09 M to about 0.11 M) sucrose, (2) selecting and culturing morphologically normal eggs as needed, (3) if the cryopreserved rat eggs are unfertilized, fertilizing the unfertilized eggs with sperm in vitro and culturing the fertilized eggs, and (4) implanting the cultured fertilized rat eggs into recipient female rats to generate offspring. [7] The method according to [6] above, comprising the following step before step (4): (A) genetically manipulating the fertilized eggs to produce and culture genetically modified fertilized eggs. [8] The method according to [7] above, wherein the genetic manipulation is selected from the group consisting of CRISPR-Cas9, TALEN, ZFN, RNAi, gene knockout, and gene knockin. [9] The method according to [6] to [8] above, wherein the rat egg is an in vitro fertilized egg.

[10] The method according to [9] above, wherein the in vitro fertilized egg is an in vitro fertilized egg of an unfertilized egg collected from a 6- to 7-week-old female rat that has been subjected to superovulation induction and sperm (fresh sperm or cryopreserved sperm).

[11] The method according to any one of [6] to [8] above, wherein the rat egg is an oocyte collected from a 6- to 7-week-old female rat that has been subjected to superovulation induction.

[12] The method according to

[10] or

[11] above, wherein the superovulation induction is carried out by simultaneously administering an anti-inhibin antibody (preferably, an inhibin antiserum [preferably, 0.3 mL or less, more preferably 0.05 mL or more and 0.2 mL or less of inhibin antiserum]) and equine chorionic gonadotropin (eCG) (preferably, 5 IU or more and 40 IU or less) to 6- to 7-week-old female rats, and then administering human chorionic gonadotropin (hCG) (preferably, 5 IU or more and 40 IU or less).

[0015] The present invention provides a new method for thawing frozen rat eggs. The rat eggs prepared using the thawing method of the present invention have a good ratio of morphologically normal eggs, and also produce good offspring following embryo transfer using the thawed eggs.

[0016] Figure 1 shows a simplified example of a method for thawing frozen fertilized rat eggs according to the present invention, along with an example of a method for freezing fertilized rat eggs. Figure 2 shows the results of genome editing using frozen fertilized rat eggs thawed using the method of the present invention. B shows offspring developed from fertilized eggs in which the tyrosinase gene was disrupted by genome editing. A shows the control. Figure 3 shows the results of genetic analysis of offspring (F0) obtained by genome editing using frozen-thawed fertilized rat eggs and embryo transfer. Using the genomic DNA of each offspring as a template, the target region of the Slc6a19 gene was amplified by PCR, and the results were electrophoresed on a 1.5% agarose gel. Numbers indicate each offspring, and W represents the wild-type PCR product. Gene deletions and insertions were confirmed in offspring 2, 3, 6, 9, and 16.

[0017] The present invention will be described below by way of illustrative embodiments, along with preferred methods and materials that can be used in carrying out the present invention, but the present invention is not limited to the embodiments described below. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. Furthermore, any materials and methods equivalent or similar to those described herein can also be used in carrying out the present invention. Furthermore, all publications and patents cited in this specification in connection with the present invention are incorporated herein by reference and constitute a part of this specification, for example, to describe methods, materials, and the like that can be used in the present invention.

[0018] In this specification, the expression "A to B" indicating a range of values ​​means a range of values ​​including the endpoints A and B. The same applies to "A to B." In addition, in this specification, the word "about" is used to mean that ±10% is acceptable.

[0019] As used herein, "unfertilized egg" refers to an egg (unfertilized egg) from ovulation to fertilization, and "fertilized egg" is used to include an egg in which a sperm has penetrated the egg cytoplasm (sperm-penetrated egg), an egg that has formed a pronucleus (pronuclear stage fertilized egg), and an egg just before developing into the two-cell stage (male-female pronuclear fusion egg). As used herein, "egg" or "egg" is used to include an egg from ovulation (unfertilized egg) to just before fertilization and becoming a two-cell stage embryo, and may be used to mean either an unfertilized egg or a fertilized egg, or both, depending on the context.

[0020] In one aspect, the present invention relates to a method for thawing cryopreserved rat eggs (unfertilized eggs or fertilized eggs), characterized by thawing the eggs in a thawing solution containing about 0.05 M to about 0.2 M sucrose. In another aspect, the present invention relates to a method for producing offspring by thawing cryopreserved fertilized rat eggs in a thawing solution containing about 0.05 M to about 0.2 M sucrose, followed by embryo transfer of the thawed fertilized eggs. In yet another aspect, the present invention relates to a method for producing offspring by thawing cryopreserved fertilized rat eggs in a thawing solution containing about 0.05 M to about 0.2 M sucrose, followed by embryo transfer of a genetically engineered, genetically modified fertilized egg. In yet another aspect, the present invention relates to a method for producing offspring by thawing cryopreserved unfertilized rat eggs in a thawing solution containing about 0.05 M to about 0.2 M sucrose, followed by in vitro fertilization of the thawed unfertilized eggs with sperm, followed by embryo transfer. In another aspect, the present invention provides a method for producing offspring by thawing cryopreserved unfertilized rat eggs in a thawing solution containing about 0.05 M to about 0.2 M sucrose, fertilizing the thawed unfertilized eggs with sperm in vitro, and then genetically manipulating the genetically modified fertilized eggs and implanting the resulting embryos.

[0021] 1. Thawing of Rat Eggs The rat eggs used in the present invention include both unfertilized and fertilized eggs. Preferably, the rat eggs used in the present invention are fertilized eggs. Fertilized eggs may be eggs in which sperm have penetrated the cytoplasm (sperm-penetrated eggs), eggs in which a pronucleus has formed (pronuclear stage fertilized eggs), or eggs immediately prior to development to the two-cell stage (male-female fusion eggs). In one embodiment, the present invention provides a method for thawing cryopreserved rat eggs (unfertilized or fertilized eggs), characterized in that the thawing is carried out in a thawing solution containing a specific concentration of sucrose. The lower limit of the sucrose concentration in the egg thawing solution is about 0.05 M, preferably about 0.075 M, more preferably about 0.08 M, and even more preferably about 0.09 M, while the upper limit is about 0.2 M, preferably about 0.15 M, more preferably about 0.13 M, and even more preferably about 0.11 M. The range of sucrose concentration in the thawing solution for fertilized eggs is about 0.05M to about 0.2M, preferably about 0.05M to about 0.15M, more preferably about 0.075M to about 0.15M, even more preferably about 0.08M to about 0.13M, and most preferably about 0.09M to about 0.11M.

[0022] The following description will use rat fertilized eggs as an example, but unfertilized rat eggs can also be used in a similar manner. Thawing can be performed by appropriately referring to known methods, but the thawing solution used during thawing must contain a predetermined concentration of sucrose. For example, but not limited to, thawing can be performed by removing a tube containing a small amount of cryopreservation solution containing cryopreserved rat fertilized eggs from liquid nitrogen, leaving it at room temperature for about 60 seconds, then immersing it in warm water at 37°C, and adding a large amount of thawing solution containing sucrose so that the desired concentration of sucrose is contained. The term "small amount (of cryopreservation solution containing cryopreserved rat fertilized eggs)" refers to, but is not limited to, about 20 μL to about 80 μL, preferably about 30 μL to about 70 μL, and more preferably about 50 μL. The term "large amount (of thawing solution containing sucrose)" refers to, but is not limited to, about 10 to about 30 times the amount, preferably about 15 to about 25 times the amount, and more preferably about 20 times the amount. For example, but not limited to, 50 μL of cryopreservation solution containing a small amount of cryopreserved rat fertilized eggs in a tube can be thawed by adding 0.9 mL of a thawing solution containing 0.05 M to 2.0 M sucrose, such as PB1 (modified phosphate-buffered saline) solution. In this case, the sucrose concentration at the time of thawing (thawing solution) is 0.0474 M to 0.189 M.

[0023] The "melting solution" (hereinafter sometimes simply referred to as the "melting solution") containing sucrose used for thawing can be used without any particular limitation, as long as it is a solution that can be used to thaw cryopreserved fertilized rat eggs. For example, solutions that can be used to thaw cryopreserved rat or mouse eggs in the present technical field include, but are not limited to, PBS, PB1, HEPES buffer (including, but not limited to, M2, KSOM-HEPES, H-CZB, and TL-HEPE), HTF, and mHTF, as long as they contain a predetermined concentration of sucrose. Furthermore, the melting solution can also contain additives other than sucrose, such as other sugars, such as glucose, fructose, lactose, raffinose, and trehalose.

[0024] The thawing step can be performed, for example, as follows, but is not limited to this. The cryotube is removed from liquid nitrogen and allowed to stand at room temperature for a short period of time, for example, several tens of seconds to several minutes, preferably 30 seconds to 2 or 3 minutes, more preferably about 60 seconds. The cryotube is then placed in a 37°C warm bath, for example, to maintain the temperature at about 37°C, and a thawing solution containing sucrose preheated to 37°C is added to thaw the frozen fertilized eggs. The thawed fertilized eggs are then processed according to the purpose.

[0025] After thawing frozen fertilized eggs using the thawing method of the present invention, the thawed solution containing the fertilized eggs is transferred to a Petri dish, and a culture medium, for example, HTF or mHTF culture medium, is added. The mixture is then incubated at 37°C and 5% CO ２ The eggs are cultured under these conditions for approximately 60 minutes, and morphologically normal fertilized eggs can be selected. Although rat fertilized eggs have been used as an example, the same procedure can be used with unfertilized rat eggs. An example of the thawing process through the selection process is shown in "Thawing Process" in Figure 1.

[0026] 2. Cryopreservation of Rat Fertilized Eggs Cryopreservation of rat fertilized eggs can be performed by appropriately referring to known methods. The freezing method for rat fertilized eggs is not particularly limited, and various methods can be selected and used, including, for example, the simple vitrification method, the slow method, and the two-step method. However, the simple vitrification method is preferred. The simple vitrification method can be, for example, the same method as that used for mice (see "Freezing of Pronuclear Stage Fertilized Eggs" in the Reproductive Engineering Technology Manual: https: / / card.medic.kumamoto-u.ac.jp / card / japanese / manual / index.html). For example, but not limited to, a rat fertilized egg is removed from a drop (approximately 100 to approximately 200 μL) of culture medium (e.g., HTF culture medium containing FCS) and then transferred to a drop (approximately 100 μL) of PB1 solution containing 1 M dimethyl sulfoxide (DMSO) at room temperature. The fertilized eggs are then transferred from the drop into a cryotube, for example, with about 5 μL of 1 M DMSO, and placed at 0°C for 5 minutes. Approximately 45 μL of a simple vitrification solution (e.g., DAP213; a phosphate buffer containing 2 M dimethyl sulfoxide, 1 M acetamide, and 3 M propylene glycol) is then added, and the mixture is placed at 0°C for 5 minutes, after which the mixture is immersed in liquid nitrogen to freeze and preserve the rat fertilized eggs. An example of the cryopreservation process is shown in the "Freezing Process" section of Figure 1. While rat fertilized eggs have been used as an example, the same process can be carried out with unfertilized rat eggs.

[0027] 3. Genetic Manipulation Using Thawed Fertilized Rat Eggs Genetic manipulation of thawed fertilized rat eggs can be performed with appropriate reference to known methods. Examples of genetic manipulation include, but are not limited to, genome editing such as CRISPR-Cas9, TALEN, and ZFN, as well as other genetic manipulations such as RNAi, gene knockout, and gene knockin. The production of genetically modified animals through genome editing is typically performed by microinjecting artificial restriction enzyme genes into pronuclear embryos. The introduction of Cas9 mRNA and gRNA into fertilized rat eggs by electroporation can be described, for example, in Non-Patent Document 7 (which is incorporated herein by reference). If the cryopreserved rat eggs are unfertilized, the thawed unfertilized rat eggs can be fertilized in vitro with sperm, and then the above-mentioned genetic manipulation can be performed using the fertilized eggs. In vitro fertilization can be achieved by in vitro fertilization of unfertilized eggs with sperm collected from a male rat. The sperm collected from male rats may be fresh, refrigerated, or cryopreserved. In vitro fertilization can be performed by appropriately referring to known methods, for example, Nakagata et al. (Non-Patent Document 8, which is incorporated herein by reference).

[0028] 4. Embryo Transfer of Fertilized Eggs Embryo transfer of rat fertilized eggs can be performed by appropriate reference to known methods. For example, thawed fertilized eggs or genomically edited fertilized eggs, or fertilized eggs obtained by in vitro fertilization of thawed unfertilized eggs or genomically edited fertilized eggs thereof, are cultured immediately or in a culture medium until they develop into two cells, and the two-cell embryos are then transplanted into the oviduct of a recipient female rat on the day a vaginal plug is observed (day 1 of pseudopregnancy). Culture media that can be used to culture the fertilized eggs include any culture medium used in in vitro fertilization or embryo culture, and examples include HTF and mHTF. The embryos are then transferred, the female rats are raised, and offspring are confirmed.

[0029] 5. Preparation of Rat Fertilized Eggs (5-1) Obtaining Unfertilized Rat Eggs Fertilized rat eggs can be obtained by in vitro fertilization of unfertilized eggs and sperm. Unfertilized rat eggs can be obtained by inducing superovulation in 3- to 7-week-old female rats. Superovulation induction can be preferably performed by administering inhibin antiserum and equine chorionic gonadotropin (eCG) to 3- to 5-week-old female rats, followed by administering human chorionic gonadotropin (hCG), or by administering LH-RH, inhibin antiserum, and equine chorionic gonadotropin (eCG) to 6- to 7-week-old female rats, followed by administering human chorionic gonadotropin (hCG). The method of superovulating female rats to obtain unfertilized eggs can be carried out by referring to the description in the international patent application (PCT / JP2024 / 015563: Patent Document 1) filed by the present inventors, for example. When unfertilized rat eggs are cryopreserved, the unfertilized rat eggs obtained as described above are cryopreserved.

[0030] (5-2) Obtaining Fertilized Rat Eggs In the present invention, fertilized rat eggs can be obtained by in vitro fertilization of unfertilized eggs collected from 3- to 7-week-old, preferably 4- to 7-week-old, and more preferably 6- to 7-week-old female rats with sperm collected from male rats. The sperm collected from male rats may be fresh, refrigerated, or cryopreserved, but fresh sperm is preferred. When using fresh sperm, for example, a male rat is euthanized by cervical dislocation, and the cauda epididymis is collected and transferred to a sperm pre-culture dish covered with paraffin oil. A sperm mass is then recovered from the cauda epididymis using a glass rod, transferred to, for example, a drop of mHTF culture medium, diluted to the desired final sperm concentration, and used for in vitro fertilization with eggs. When using refrigerated sperm, the refrigerated sperm stored at 4°C for several days, e.g., 2-5 days, are transferred to a culture medium, e.g., mHTF culture medium, and motile sperm are collected and used for in vitro fertilization with eggs. When using cryopreserved sperm, the cryopreserved sperm are thawed according to standard procedures, transferred to a culture medium, e.g., mHTF culture medium, and motile sperm are collected and used for in vitro fertilization with eggs. The fertilized eggs to be cryopreserved may be fertilized eggs at any stage between 5 hours after insemination (fertilized eggs in which sperm have penetrated the egg cytoplasm) and 22 hours after insemination (fertilized eggs immediately prior to becoming two-cell embryos), but preferably are pronuclear stage fertilized eggs approximately 7-8 hours after insemination. In vitro fertilization can be performed by appropriately referring to known methods, and rat fertilized eggs can be obtained. For example, the method described in Nakagata et al. (Non-Patent Document 8) can be used as a reference. The fertilized eggs thus obtained can be cryopreserved, thawed using the method of the present invention, and used to develop offspring, or genetically manipulated to produce genetically modified rats.

[0031] The anti-inhibin antibody used in the superovulation induction method for preparing rat eggs may be any antibody that has the activity of neutralizing endogenous inhibin in rats. For example, an inhibin antiserum containing anti-inhibin antibodies can be obtained by immunizing a castrated goat with purified 32 kDa pig, mouse, or rat inhibin as an antigen. Anti-inhibin activity can be measured by conventional methods. For example, immunopotency can be measured by immunoassay. In the present invention, the anti-inhibin antibody may be in the form of an inhibin antiserum or a purified (including crudely purified) antibody. When an inhibin antiserum is used, the prepared antiserum is administered, for example, at 0.05 mL to 0.3 mL, preferably 0.05 mL to 0.2 mL, per rat, although this is not a limitation.

[0032] The amount of equine chorionic gonadotropin (eCG) administered to female rats in the method for inducing superovulation to prepare rat eggs is the same as that typically used in superovulation treatments for laboratory rats. For example, the amount is 2 to 50 IU, preferably 5 to 40 IU, per rat. By using this amount of eCG in combination with inhibin antiserum, excellent induction of superovulation can be achieved.

[0033] The amount of human chorionic gonadotropin (hCG) administered to female rats in the method for inducing superovulation to prepare rat eggs is the same as that usually used in superovulation treatment of laboratory rats, for example, 2 to 50 IU, preferably 5 to 40 IU per rat.

[0034] The amount of LH-RH administered to female rats in the method for inducing superovulation to prepare rat eggs is the same as that usually used in superovulation treatment of laboratory rats, for example, 0.005 to 0.2 mg, preferably 0.01 to 0.1 mg per rat.

[0035] The present invention will be described in detail below using examples, but the present invention is not limited to the following examples. 1. Materials and Methods (1-1) Animals Female and male rats of each strain were purchased from Jackson Laboratory Japan (formerly Charles River Japan). Female rats of the indicated ages were used as egg donors, and male rats at 12 to 14 weeks of age were used as sperm donors. All animals were provided with food and water ad libitum and housed at 22°C ± 1°C under a 12-hour dark-light cycle (light hours: 7:00-19:00). Animal experiments were performed according to a protocol approved by the Kumamoto University Animal Care and Use Committee.

[0036] (1-2) Culture Medium Modified human tubal fluid (mHTF) was used as the preculture medium for sperm, the in vitro fertilization culture medium, and the culture medium for thawed fertilized eggs.

[0037] (1-3) Ovulation and Egg Collection Female rats aged 3 to 5 weeks were administered a combination of IASE (inhibin antiserum and eCG administered simultaneously) and hCG (human chorionic gonadotropin), while female rats aged 6 to 7 weeks were administered a combination of LH-RH (purchased from Peptide Institute, Inc.), IASE, and hCG. Inhibin antiserum was prepared using mouse inhibin peptide according to the method described in Kishi et al.'s paper (Non-Patent Document 9, which is incorporated herein by reference), and the prepared antiserum was used as is. The antibody activity of the prepared inhibin antiserum was measured using ELISA and standardized as necessary before use. Specifically, mouse inhibin peptide at a concentration of 5 μg / mL was adsorbed onto an ELISA plate, followed by washing and blocking. Inhibin antiserum diluted 1,000-128,000 times was treated, and ELISA was performed using horseradish peroxidase-labeled anti-goat IgG antibody as the secondary antibody. It was confirmed that inhibin antibody could be sufficiently detected even at the maximum dilution, and this was used in the following experiments.

[0038] Superovulation induction was performed as follows. For female rats aged 3 to 5 weeks, IASE and hCG were administered 53 to 57 hours after IASE (0.1 mL of inhibin antiserum + 20 IU of eCG / rat). For female rats aged 6 to 7 weeks, IASE and hCG were administered 52 to 53 hours after LH-RH (0.06 mg, dissolved in 300 μL of saline), IASE (0.15 mL of inhibin antiserum + 30 IU of eCG / rat) was administered, and then 53 to 57 hours after that, hCG (30 IU / rat) was administered.

[0039] (1-4) In vitro fertilization sperm were collected from Sprague-Dawley (SD) or Long-Evans (LE) strain rats as follows: Male rats were euthanized by cervical dislocation, and the cauda epididymis was collected and transferred to a sperm preculture dish covered with paraffin oil. Sperm clumps were collected from the cauda epididymis using a glass rod and transferred to a 400 μL drop of mHTF medium. The clumps were then further diluted with a 200 μL drop of mHTF medium to a final sperm concentration of 500 sperm / μL. One day after administration of hCG (Aska Pharmaceutical Co., Ltd.), rats were euthanized by cervical dislocation, and their oviducts were quickly removed. Cumulus-oocyte complexes (COCs) were collected from the oviducts under microscopic observation and introduced into a drop of 200 μL of mHTF culture medium containing sperm (insemination).

[0040] (1-5) Cryopreservation of Fertilized Eggs Seven hours after insemination, the fertilized eggs were transferred to a drop (100 μL) of 1 M dimethyl sulfoxide (DMSO)-containing PB1 at room temperature and left for 5 minutes. The fertilized eggs were then removed from the drop and placed in a cryotube with 5 μL of 1 M DMSO-containing PB1 and left at 0°C for 5 minutes. 45 μL of a simple vitrification solution (DAP213; a phosphate buffer containing 2 M dimethyl sulfoxide, 1 M acetamide, and 3 M propylene glycol) was then added, and the eggs were left at 0°C for 5 minutes. After that, the rat fertilized eggs (pronuclear stage fertilized eggs) were cryopreserved by immersion in liquid nitrogen.

[0041] (1-6) Thawing of Fertilized Eggs: Cryotubes containing 50 μL of storage solution containing frozen preserved rat fertilized eggs were removed from liquid nitrogen and placed at room temperature for 60 seconds. Next, 0.9 mL of PB1 solution containing various concentrations of sucrose was added at 37°C to thaw the frozen fertilized eggs. The thawed fertilized eggs were then washed in four drops (60 μL) of mHTF.

[0042] (1-7) Genome editing of fertilized eggs After washing the thawed fertilized eggs, morphologically normal fertilized eggs were selected and genome editing was performed as follows. The selected fertilized eggs were electroporated to introduce an sgRNA / Cas9 protein complex (ribonucleoprotein: RNP) and disrupt the tyrosinase gene (Tyr) involved in determining rat coat color. Mutations in this gene cause an albino phenotype, a white coat and red eye phenotype. Specifically, sgRNA targeting exon 1 was used, and fertilized eggs into which RNP had been introduced were immediately used for embryo transfer. In addition, frozen fertilized eggs from SD rats were thawed, and RNP was introduced into the fertilized eggs by electroporation using sgRNA designed for exon 3 of the solute carrier family 6 member 19 (Slc6a19) gene, followed by embryo transfer.

[0043] (1-8) Embryo Transfer Embryo transfer was performed according to the procedure previously reported by the present inventor (Non-Patent Document 10, which is incorporated herein by reference). Thawed fertilized eggs or thawed, genome-edited fertilized eggs were immediately transferred into the oviducts of CD (SD) female rats on the day a vaginal plug was observed (day 1 of pseudopregnancy) (10 embryos / oviduct). The number of offspring was recorded 22 days later.

[0044] (1-9) Statistical Analysis: Statistical analysis was performed using Prism version 5.0 (GraphPad). Results are presented as mean ± standard deviation (SD). Group results were compared using analysis of variance after arcsine transformation of percentages. p < 0.05 was considered statistically significant.

[0045] Example 1: Study of sucrose concentration in thawing solution. Four-week-old Crl:CD(SD) female rats were administered IASE and hCG to induce superovulation, resulting in the production of oocytes. The collected oocytes were then fertilized in vitro with fresh sperm from mature Crl:CD(SD) male rats, and the resulting fertilized oocytes were cryopreserved. The thawing of the fertilized oocytes was performed by removing 50 μL of cryopreserved rat fertilized oocytes from liquid nitrogen, leaving them at room temperature for 60 seconds, and then adding 0.9 mL of PB1 solution containing various concentrations of sucrose (0.05 M, 0.1 M, 0.2 M, and 0.3 M). The sucrose concentrations of the thawing solution were 0.47 M, 0.95 M, 0.19 M, and 0.28 M, respectively. One hundred frozen oocytes were used for each condition. After thawing and washing, the eggs were cultured for 60 minutes and the number of morphologically normal fertilized eggs was counted. All morphologically normal fertilized eggs were further cultured and developed to the two-cell stage. The results are shown in the table below.

[0046] By adding 0.05M to 0.2M sucrose solutions, morphologically normal fertilized eggs were recovered after thawing, and sufficient development to the 2-cell stage was confirmed, with the addition of 0.1M sucrose solution being particularly effective.

[0047] Example 2: Study of Female Rats' Ages Since Example 1 showed favorable results with a 0.1 M sucrose concentration, the following study was performed using a 0.1 M sucrose solution. Fertilized eggs were prepared from 3- to 7-week-old female rats, and cryopreserved rat fertilized eggs were produced. Three- to 5-week-old Crl:CD(SD) female rats were administered IASE and hCG to induce superovulation, resulting in the production of oocytes (unfertilized eggs). Six- to 7-week-old Crl:CD(SD) female rats were administered LH-RH, IASE, and hCG to induce superovulation, resulting in the production of oocytes (unfertilized eggs). In vitro fertilization was performed using each oocyte with fresh sperm from mature Crl:CD(SD) male rats, and the resulting fertilized eggs were cryopreserved. The fertilized eggs were thawed by removing a cryotube containing 50 μL of storage solution containing frozen preserved rat fertilized eggs from liquid nitrogen, leaving it at room temperature for 60 seconds, and then adding 0.9 mL of PB1 solution containing 0.1 M sucrose. After thawing, the eggs were transferred to a drop of 0.2 mL of PB1 solution, and the number of morphologically normal fertilized eggs was counted. Morphologically normal fertilized eggs were immediately transplanted, and offspring were developed. The results are shown in the table below.

[0048] After thawing, good recovery of morphologically normal fertilized eggs was confirmed in female rats of all ages, but the production of offspring after transplantation was remarkable when eggs from female rats aged 6 and 7 weeks were used. At 6 and 7 weeks of age, almost all of the thawed fertilized eggs were morphologically normal, and the postnatal development rate from the thawed fertilized eggs was remarkable at approximately 50%.

[0049] (Example 3) Genome editing of fertilized eggs Based on the results of Example 2, fertilized eggs were produced using eggs (unfertilized eggs: oocytes) from 7-week-old female rats. The fertilized eggs obtained by in vitro fertilization between eggs collected from 7-week-old female rats (SD: albino) and sperm collected from LE rats (black and white spotted) were frozen and thawed, and then an sgRNA / Cas9 protein complex (ribonucleoprotein: RNP) targeting the tyrosinase gene (a gene determining coat color) was introduced. The fertilized eggs were then transplanted into the oviducts of recipient females to generate offspring. The results are shown in Figure 2. As a result, albino individuals (individuals with white coats and red eyes) in which the tyrosinase gene was disrupted were successfully produced (Figure 2B). On the other hand, offspring from the control group in which RNP was not introduced exhibited coat color with black and white spots derived from LE (Figure 2A).

[0050] Example 4: Confirmation of Gene Disruption Fertilized eggs were produced using oocytes (unfertilized eggs) from 7-week-old female rats. In vitro fertilization was performed between eggs collected from 7-week-old female SD rats and sperm collected from male SD rats. The fertilized eggs obtained were frozen and thawed, and then RNPs targeting the Slc6a19 gene were introduced. These fertilized eggs were then transplanted into the oviducts of recipient females to generate offspring. A portion of the tail was collected from the offspring, and genomic DNA was extracted. The extracted genomic DNA was used to amplify the target region of the Slc6a19 gene by PCR, followed by agarose gel electrophoresis. The target band was then excised, DNA extracted, and sequenced by Sanger sequencing to confirm base deletions and insertions. As a result, the production of individuals with a disrupted Slc6a19 gene was confirmed.

[0051] Even when using rat fertilized eggs cryopreserved by the method of the present invention, the proportion of morphologically normal eggs after thawing is high, and the development of offspring after embryo transfer is also good. Therefore, by using the method of the present invention, if a large number of fertilized eggs are produced and cryopreserved in advance, genetically modified rats can be produced at any time, facilitating the production, preservation, and propagation of genetically modified rats.

[0052] The above detailed description is merely illustrative of the objects and scope of the present invention and is not intended to limit the scope of the appended claims. Various modifications and substitutions to the described embodiments will be apparent to those skilled in the art from the teachings set forth herein, without departing from the scope of the appended claims.

[0053] The present invention provides a method for thawing frozen-preserved rat eggs, and the rat eggs prepared using the thawing method of the present invention have a high rate of morphologically normal eggs and produce good offspring after embryo transfer. The present invention is useful because it enables the advance production and cryopreservation of large quantities of rat eggs for use.

Claims

1. A method for thawing frozen stored rat eggs, characterized in that the thawing is carried out in a thawing solution containing about 0.05M to about 0.2M sucrose.

2. The method of claim 1, wherein the rat eggs are in vitro fertilized eggs.

3. The method according to claim 2, wherein the in vitro fertilized eggs are in vitro fertilized eggs obtained by induced superovulation of unfertilized eggs and sperm collected from female rats aged 6 to 7 weeks.

4. The method according to claim 1, wherein the rat eggs are oocytes collected from superovulated female rats aged 6 to 7 weeks.

5. The method according to claim 3 or 4, wherein the superovulation induction is carried out by simultaneously administering anti-inhibin antibody and equine chorionic gonadotropin (eCG) to female rats aged 6 to 7 weeks, followed by administering human chorionic gonadotropin (hCG).

6. A method for producing offspring from cryopreserved rat eggs, comprising the following steps: (1) thawing and culturing cryopreserved rat eggs in a melting solution containing about 0.05M to about 0.2M sucrose; (2) selecting and culturing morphologically normal eggs as necessary; (3) if the cryopreserved rat eggs are unfertilized, fertilizing the unfertilized eggs with sperm in vitro and culturing the fertilized eggs; and (4) implanting the cultured fertilized rat eggs into recipient female rats to generate offspring.

7. The method according to claim 6, comprising, before step (4), the following step: (A) subjecting a fertilized egg to genetic manipulation to produce and culture a genetically modified fertilized egg.

8. The method according to claim 7, wherein the genetic manipulation is selected from the group consisting of CRISPR-Cas9, TALEN, ZFN, RNAi, gene knockout, and gene knockin.

9. The method according to any one of claims 6 to 8, wherein the rat eggs are in vitro fertilized eggs.

10. The method according to claim 9, wherein the in vitro fertilized eggs are in vitro fertilized eggs obtained by combining unfertilized eggs and sperm collected from superovulated female rats aged 6 to 7 weeks.

11. The method according to any one of claims 6 to 8, wherein the rat eggs are oocytes collected from superovulated female rats aged 6 to 7 weeks.

12. The method according to claim 10, wherein the superovulation induction is carried out by simultaneously administering anti-inhibin antibody and equine chorionic gonadotropin (eCG) to female rats aged 6 to 7 weeks, followed by administering human chorionic gonadotropin (hCG).

13. The method according to claim 11, wherein the superovulation induction is carried out by simultaneously administering anti-inhibin antibody and equine chorionic gonadotropin (eCG) to female rats aged 6 to 7 weeks, followed by administering human chorionic gonadotropin (hCG).

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