KR20110117286A

KR20110117286A - Economical Cloning Method for Multiple Pregnancy Pets

Info

Publication number: KR20110117286A
Application number: KR1020100036659A
Authority: KR
Inventors: 김경희
Original assignee: 김경희
Priority date: 2010-04-21
Filing date: 2010-04-21
Publication date: 2011-10-27

Abstract

The present invention provides a method of preparing a plurality of nuclear transfer eggs for different orderers by transplanting and fusing each somatic cell of a homologous multiple pregnancy pet of different orderers into donor nuclei, respectively, into denuclearized eggs of the same species. Activating each of the different orderer's nuclear replacement eggs to produce a plurality of different orderer's nuclear replacement embryos, and mixing the different orderer's nuclear replacement embryos in the body of a surrogate animal of the same species. Transplanted and implanted together so that, after a period of time, pets of multiple orderers with different genetic traits are simultaneously reproduced in a surrogate mother as cloned animals, and each DNA used as a donor nuclear source Cloned animals born by judging their match by comparing DNA genetic information of somatic cells It is an economical cloning method of a multi-pregnancy pet, characterized in that it comprises a step of determining which ordered animal for this order.

By doing so, in cloning an animal with multiple pregnancies, at the time of one successful birth of a surrogate animal, multiple-order cloned animals (eg dogs, cats) are born at the same time, thereby reducing the cost of replication assigned to each orderer. There are features and effects that make it possible.

Description

Economical cloning of multiple pregnant pets {omitted}

The present invention relates to an economical method of cloning animals. In particular, it relates to the reproduction of inexpensive multi-pregnancy pets (eg dogs, cats) at low cost.

Cloning of animals, particularly mammalian animals, has been documented as the first successful case of Walmut's sheep cloning. Since then, many mammals have reported success in cloning. Examples of cloned animals reported so far have been reported production of cloned litter by somatic cell nuclear transfer methods in cattle, mice, goats, pigs and rabbits (WO9937143A2, EP930009A1, WO9934669A1, WO9901164A1 and US5,945,577). Wolf (Korean Patent 10-0824218), Dog (Korean Patent 10-0733012, 10-0829426), Transgenic Dog (Korean Patent Publication 10-2009-0075637), Transgenic Cat (Korean Patent Publication 10-2009-0060191) Has been reported, and the success of cloning horses and camels, which are horseshoe animals, has been reported.

The techniques used in these mammalian cloning examples are all based essentially on the original techniques used to clone sheep on Walmute.

Known basic techniques of replication that apply to the replication of all animals to date are as follows.

(a) implanting and nucleating the nuclei of differentiated diploid donor cells into denuclearized eggs (second oocytes, hereinafter referred to as eggs) to produce nuclear substituted eggs;

(b) reprogramming the nuclear replacement column of step (a) for a period of time;

(c) activating the nuclear replacement egg to produce a nuclear substituted embryo;

(d) transplanting the nuclear-substituted embryo prepared in step (c) into a surrogate mother animal of the same kind so that a cloned living body is born after a certain time; Consists of To describe them in more detail, the steps of harvesting the mature egg (oval with the 2nd chromosome in which the first polar body of the secondary meiosis is formed) and somatic cells (chromosome: 2n) of the animal to be cloned are prepared. Denuclearizing the nucleus of the mature egg, implanting and fusing the nucleus (or somatic cell) of the somatic cell of the animal to be cloned into the denuclearized mature egg to produce a nuclear substituted egg, Reprogramming for a period of time, and activating the reprogrammed nuclear transfer eggs by electrical or chemical methods to generate nuclear transfer embryos. Known cloning techniques include implanting the nucleated embryo into the surrogate mother's body (uterus or fallopian tube) to implant it into the uterus of the surrogate mother and causing the surrogate animal to give birth to the cloned animal after a period of pregnancy. .

In a conventional conventionally known method of animal cloning, activation is carried out after a chemical treatment that suppresses the occurrence of the second polar body in order for the embryo to maintain the 2n chromosome when activating the nuclear substituted eggs. For example, the nuclear replacement eggs are activated after treatment with 6-DMAP. Known methods for activating nuclear substituted eggs are as follows. That is, the nuclear transfer embryos were placed in mSOF (Table 4) containing 10 μM ionophores (Sigma), incubated at 39 ° C. for 4 minutes, the nuclear transfer embryos were washed, and 1.9 mM 6-dimethylaminopurine (6-DMAP) was added. It was chemically activated by further incubating for 4 hours in mSOF (Korean Patent 10-0733012). Activation of nuclear transfer eggs can also be activated by electrical methods. For example, the conventional cat cloning technique successfully produces cloned cats by an electric activation method. Electrical activation of the nuclear replacement eggs of the cat was as follows. That is, the fused embryos were equilibrated in 0.3 M mannitol containing 0.1 mM Ca 2+ and 0.1 mM Mg 2+, and then placed in a fusion chamber containing the same culture, and the time of 20 μsec at a DC voltage of 1.0 kV / cm and Each was energized with a time of 0.1 sec to activate. The activated feline somatic clone embryos were then washed, and then cultured in TCM199 culture with 0.3% BSA and 2 mM 6-DMAP in a 38 ° C., 5% CO 2 incubator (Korean Patent Publication 10-2009-0060191).

In addition, the method of denuclearization of an egg in a conventional animal cloning technique removes the nucleus of an egg by using a method of inhaling a small amount of egg cytoplasm around the first pole body, or by gently pressing the egg cytoplasm around the first pole body. The method is well known in the art. In the prior art, when denuclearizing the nucleus of an egg, the first polar body was also removed so that the first polar body could not be fused again.

In addition, when the nucleus of the somatic cell is transplanted into the denuclearized egg, the nucleus of the somatic cell is removed and transplanted into the denuclearized egg (the second oocyte, hereinafter referred to as an egg). Alternatively, the somatic cell itself is denuclearized. After transplantation into the cytoplasm or gastric cavity, methods for cell fusion of somatic cells and eggs by electrical stimulation have been used in this field. The electrical stimulation at the time of cell fusion can be AC voltage, but a method of allowing DC pulse voltage to be applied for a short time is commonly used. For example, in the case of dog replication, the electrical stimulation during cell fusion is proposed to be DC 3.0-3.5KV / cm, 10-30 us, 1-3 times (Korean Patent Publication 10-2007-0013432). Other voltages during electrical stimulation have been proposed to 3.8-5.0 KV / cm (Korean Patent Registration No. 0829426).

Cell fusion voltage conditions in cat replication resulted in the insertion of donor nucleated cells into the gastric cavity of each denuclearized egg by micromanipulation. Cytoplast / cell couplets were equilibrated in 0.3 M mannitol containing 0.1 mM Mg2 + and transferred to an electrofusion chamber containing the same culture, followed by an electrogenerator (Nepagene, Ichikawa, Chiba, Japan). Cell fusion was carried out by energizing twice at a time of 20 μsec at a DC voltage of 2.0 kV / cm. The fused embryos were removed from the chamber, washed several times, placed in a TCM199 culture medium containing 0.3% BSA, and then cultured in a 38 ° C., 5% CO 2 incubator to complete cell fusion (Korean Patent Publication No. 10-2009-A). 0060191).

The electrical stimulation voltage at the time of cell fusion was changed little by little depending on the type of the species and the electrode type of the animal providing the egg and the somatic cell, but the basic principles were the same.

On the other hand, it is well known that the oocytes to be used for nuclear replacement should collect the suspended oocytes in the middle stage of the second meiosis, in which the first polar body is released. In normal animal cloning, such mature eggs can be obtained by collecting eggs after ovulation is confirmed. However, in the case of dogs, mature eggs are collected in dogs due to the immature ovulation of eggs at the time of ovulation. The oocytes can be harvested from fallopian tubes by harvesting eggs from 48 to 72 hours after ovulation is observed. More preferably, 72 hours after ovulation is known to increase the harvesting rate of mature eggs.

The method of determining the ovulation time is as follows when cited from a known technique (Korean Patent 10-0733012). In other words, "Determination date is determined by measuring vaginal smear and serum progesterone levels daily in dogs with estrous periods, and serum progesterone concentrations are collected by centrifuging 3-5 ml of blood daily and centrifuged. Obtained and analyzed using DSL-3900 ACTIVE progesterone coated tube radioimmunoassay kit (Diagnostic Systems Laboratories, Inc., TX), and when progesterone concentrations range from 4.0 to 7.5 ng / ml are considered ovulation days (Hase et al. , J. Vet. Med. Sci., 62: 243-248, 2000).

In addition, the "vaginal cell smear test for determination of ovulation time is performed by obtaining a sample daily from the day of the initial symptoms of estrus, and the vaginal cell sample is collected by inserting a cotton swab into the vulva, and smearing it on a slide glass, Next, Diff-Quik staining (International chemical co., Japan) and staining under a microscope to determine the epidermal cells (cornified index, Evans JM et al., Vet. Rec, 7: 598 -599, 1970) over 80% of cases are considered ovulation. " It was.

In the prior art, somatic cells used as donor nuclei are known to increase the success rate of embryonic development by keeping the cell cycle stationary at the G0 phase or G0 or G1 phase.

For this purpose, a method of culturing serum starvation has been proposed. As another method for keeping the cell cycle stationary at the G0 phase or G0 or G1 phase, a method of culturing by adding a cell cycle synchronization material at the time of culturing somatic cells as donor nuclei has been proposed. Examples include Roscovitine, Cyclohesimide, DMSO, Butyrolactone I, Aphidicolin, Demecolcine, Mimosine, Mimosine, A method of culturing somatic cells, a donor nucleus, has been proposed by adding one cell cycle synchronization inducer selected from the group consisting of colchicine and Hoechst 33342 (Korean Patent Publication No. 2009-0051716).

In addition, in the prior art, methods for transplanting into a surrogate mother body of a nuclear-substituted embryo have been proposed to be directly implanted in the uterus and to a fallopian tube. In general, implantation into the fallopian tube is commonly used, and in the case of dog cloning, implantation in the uterus is known to be difficult to implant (Korean Patent 10-0733012).

Also, when cloning multiple pregnant animals, the number of enough embryos to be implanted into the surrogate mother's body is sufficient to account for implantation success and childbirth success rate (e.g. 40-60 dogs for multiple pregnancies of 10 or more at a time). The method of transplanting the nuclear transfer embryos into the surrogate mother's body (for example, fallopian tubes) at a time is commonly used. Of course, if future implantation success rates and pregnancy birth success rates are higher than they are now, fewer nuclear replacement embryos will be implanted.

In addition, considering that dogs and cats are multi-pregnant animals, which usually give birth to about 5-10 animals at a time, nuclear-transferred embryos are also multi-pregnant animals. Is implanted in the surrogate mother's body. Especially in the case of multiple pregnant animals, if only a small number of embryos are implanted (pregnant), there is a risk that the body may be mistaken for miscarriage and cause miscarriage. Nuclear transfer embryos are transplanted into the surrogate's body (eg fallopian tubes). For this reason, cloned births of multiple gestational animals are usually transplanted into homologous gestational surrogate animals with sufficient numbers (approximately 5-10) of nuclear transgenic embryos capable of conceiving about 10 (approximately 5-10). Done.

However, the animals most loved as pets are dogs and cats, and they are multi-pregnant animals. As a result, about 10 successful cloned individuals (off-springs) are usually delivered when successful reproduction is given.

This excess fertility in multi-pregnancy pet cloning results in unnecessary surplus cloned animal populations, which in turn contributes to unnecessary expense. This is because too many unnecessary surplus cloned animals will be delivered together than the number of individuals required by the orderer who commercially requested the reproduction of the pet.

Typically, a person who wants to restore a pet, a pet with deep memories, usually needs only 1-2 clones of the same shape as his pet. Therefore, if 10 were given birth, 8 cloned pets would remain as surplus clones if 2 were delivered to the clone orderer. This excess quantity of surplus clones, which remain after being delivered to the orderer, cannot be discarded or euthanized, making the treatment very difficult.

Until now, since the cloning was conducted mainly for research purposes, the above-mentioned surplus clones were kept for research observation or given to the people around them, but the problem of disposing of the surplus clones was solved. Cats) In the case of commercially large-scale replication, the above-described redundant clones have become a very difficult problem in the prior art. In addition, as described above, although only one or two cloned animals (such as dogs or cats) are required to be delivered to the orderer, a large number of cloned embryos may be transplanted to prevent abortion when cloning multiple pregnant pets. This resulted in the production of a large number of surplus cloned animals, which resulted in an increase in the cost of cloning, which had the prior art. Due to the problems of the prior art described above, the prior art has hindered the industrialization of multiple pregnancy pets and had problems.

In addition to the above-mentioned reasons for increasing the replication cost, another factor for increasing the replication cost of the prior art will be described below.

In the case of normal dog cloning, eggs were taken from the ovary of female dogs in estrous state to harvest eggs. These oocyte-gathering dogs must be in the estrus state to harvest eggs.

In addition to the oocyte-providing dogs, dogs used as surrogate mothers must also be estroused so that they can be implanted when nuclear-transplanted embryos are transplanted.

However, in order to perform dog cloning in a large-scale industry, it is necessary to always make it possible to collect dog eggs at any time desired. In addition, the nuclear transfer embryos must be able to be transplanted into surrogate mothers at any time desired. To do this, a dog must be in heat each day. However, the estrous cycle of dogs is much longer than other livestock, which varies slightly depending on the breed of the dog, but averagely every eight months. In large dogs, there are some species that heat up once a year. In other words, on average, three times every two years. Therefore, if necessary, eggs can be harvested at any time, and in order to be used as surrogate mothers, a large number of female dogs must be kept on a daily basis so that there is always a certain number of dogs that are statistically present every day. Of course, depending on the quantity and the urgency of the replication order, there may be cases where a certain number of estrous dogs are needed every 2-3 days, or once a week, instead of being found every day. However, in the case of industrial cloning of dogs, a certain increase in order volume requires a certain number of dogs to be in estrus almost every day, and to meet these conditions, depending on the success rate and the quantity of the order, there are several hundred statistically. Only a few thousand or tens of thousands of dogs are kept at all times to meet the industrial demand for continuous cloning. For reference, when dog cloning was first successful, the success rate was about 0.2%. This situation, ie, having to keep a large number of female dogs at all times, was another major factor that led to excessive expenses in cloning dogs. As described above, the oocyte collecting dog and the surrogate mother dog can be used only if the dog is in a estrous state. Therefore, in order to digest a certain quantity of orders every day, the problem is that the females of a sufficiently large number of statistically calculated populations that can always show a certain number of estrous dogs must be kept at all times. there was. This is another important factor that increases the cost of cloning dogs.

For reference, in the case of dog cloning, it is known that an orderer pays about 50 to 200 million won per order. This amount of conventional dog cloning is realistically a problem that is too expensive for the public to use it commercially.

The present invention to solve the above-mentioned problems in the prior art is to propose a cloning technique of multiple pregnancy pets that can realize the reduction of the replication cost for large-scale industrialization of multiple pregnancy pets (dogs, cats) replication .

Animal cloning techniques can be used to nullify the populations of rare, endangered, or animals with special genetic traits.Another purpose is to use ovaries as somatic cells of sterile mini pigs genetically engineered to carry human genes. It can also be used to replace diseased human organs by replicating sterile mini-pigs by replacing the nucleus of. In addition to these purposes, another special purpose of cloning animals is the tender sympathy between humans and pets as pets, when their pets, who have interacted with them and lived together, have died. It can also be used for the purpose of resolving a hurting heart for a human pet (companion) that wants to restore it again.

At the present time, the industrial market size of the pet cloning industry is much larger than that of other purposes, and the industrialization is very high. However, animal cloning techniques to date have been so expensive for the above-mentioned reasons that it is preventing the activation of the pet industry (eg, dogs, cats) cloning industry as described above.

In the field of cloning of animals, in particular, we propose an economical method of cloning pets that can reduce the cost of cloning multiple pregnant pets (eg dogs, cats).

1. Producing a plurality of nuclear transfer eggs for different orderers by transplanting and fusing somatic cells of the same multiple gestational pet individuals of different orderers into denuclearized eggs of the same species;

2. activating the respective nuclear replacement eggs for different orderers to produce a plurality of nuclear replacement embryos for different orderers;

3. Transplanting and implanting the nuclear transfer embryos for different orderers together in the body of a surrogate animal of the same species so that, after a period of time, pets of multiple orderers with different genotypes are simultaneously born; It is configured to include.

When any one surrogate animal gives birth to a cloned animal, the cloned pets for multiple orderers are given birth at the same time, thereby reducing the cost of cloning a pet assigned to one orderer. In addition, it is possible to minimize surplus cloned animals that remain difficult to process after being delivered to the orderer generated during the reproduction of multiple pregnant pets (eg, dogs and cats), thereby reducing costs and difficulty in handling surplus cloned animals. There is an effect that can be solved. In addition, this has the effect of activating the pet cloning industry.

[Configuration]

1. A large number of somatic cells of multiple gestational pets to be cloned of the same order from different orderers are transplanted and fused to denucleared eggs of the same species, respectively, and replaced with different donor nuclei (somatic cell nuclei). Preparing four nuclear substituted eggs;

2. activating each of the first nuclear replacement eggs in a solution containing a solution for inhibiting the generation of a second polar body, thereby generating a plurality of nuclear replacement embryos substituted with different donor nuclei;

3. Mixing and implanting the nuclear-substituted embryos according to step 2 into the bodies of surrogate mothers of the same species so that, after a period of time, a plurality of breeds having different genotypes are born together from one surrogate mother;

4. Animals born by comparing the genetic information (DNA fingerprint analysis information) of cloned animals born in accordance with step 3 with the genetic information (DNA fingerprint analysis information) of residual somatic cells used as donor nuclei of the given cloned animals. And determining which orderer is a cloned animal.

In the configuration of the present invention, donor nucleus-causing somatic cells are somatic cells of an animal to be replicated for different orderers. Thus, while somatic cells of the same species (eg dogs), breeds (eg greyhounds, Jindo dogs, Pung San dogs, lion dogs) may be different somatic cells. Alternatively, the varieties requested by a plurality of orderers may be the same varieties, and in this case, donor nuclei of the same varieties may be mixed among the nuclear transfer embryos transplanted together in one surrogate mother.

In the constitution of the present invention, a method for determining egg harvest time, a method for collecting eggs, a method for denuclearizing eggs (e.g., a method for squeezing the cytoplasm of the first polar region, or a method for denuclearizing by inhalation), and a donor nuclear source Methods for preparing somatic cells (e.g., starvation serum culture to synchronize cell cycles), and replacing the nuclei of eggs with donor nuclei (somatic cell nuclei) by implanting and fusion of somatic nuclei into denuclearized eggs (e.g., denucleated) A method of implanting somatic cells of donor nucleus into the gastric cavity of oocytes and then cell fusion by electrical stimulation An example of electrical stimulation: the electrical pulse stimulation method uses a DC pulse voltage of 3.0 kv / cm to 3.5kv / cm, 1 to 3 times for 30 mA, or 2 times for 15 to 25 mA at 3.8 kv / cm to 5.0 kv / cm using a needle type electrode, or 1.75 kv / cm DC Pulse, or 2.0 kv / cm to 1 to 3 times for 6 kv / cm DC Pulse 10 to 3.0 등, etc.), Method of activating nuclear substituted eggs (e.g., 5-10 μM of calcium ionophores at 37-39 ° C for 3-5 minutes, then 1.5-2.5 mM 6-dimethylaminopurine at 37-39 ° C for 4-5 hours) Treatment), the method of carrying out the specific technique required for each step for animal cloning, such as a method of transplanting the nuclear-substituted embryo into the body of a surrogate animal (eg, uterus or fallopian tube) to give birth, may use known prior art. have. As an example, techniques known in WO9937143A2, EP930009A1, WO9934669A1, WO9901164A1, US5,945,577, Korean Patent Publication No. 10-1999-0044149 can be used.

If the present invention is applied to cloning a dog which is a pet of multiple pregnancy, known techniques of Korean Patent Registration No. 10-0733012, Korean Patent Registration No. 10-0829426, and Korean Patent Publication No. 10-2009-0075637 may be used. . In addition, new techniques for animal cloning, which will be invented in the future, may be utilized in the practice of the invention. Even in this case, the originality of the present invention and the gist of the present invention are not changed. If the present invention is applied to cat cloning, the specific technical process for each stage for animal cloning may use the technique of Korean Patent Publication No. 10-2009-0060191.

Example 1.

First, the present embodiment will be described in detail by taking an example of the number of orderers requesting dog cloning each day (365 cases per year).

First of all, the number of surrogate mother dogs necessary for carrying out the conventional method and the method of the present invention, the number of surplus cloned dogs, and the number of individual dogs that should be kept normally for use as surrogate dogs and egg donor dogs are first calculated and compared. I want to see.

Accordingly, the cost of cloning a dog by the conventional method and the cost of cloning a dog by the method of the present invention will be compared with each other.

A. Cloning of Dogs by Conventional Methods

Dogs that are largely pregnant are usually given about 6-10 dogs per birth. For convenience of calculation, suppose that on average eight pregnant women are giving birth. Therefore, the number of nuclear transfer embryos implanted in surrogate mother's fallopian tube is considered to be 20% considering implantation success rate and 20% success rate after successful implantation. In order to implant in%, five times nuclear transfer embryos should be transplanted. Therefore, in order to implant and implant 8 individuals, 8 individuals x 5 times = 40 nuclear transfer embryos must be implanted so that 8 individuals will be implanted at a 20% success rate.

In addition, the embryonated success rate for implanted embryos was set at 20%, which is the average value of conventional current technology. Thus, in order to achieve 100% success in birth, at least five surrogate mothers must be transplanted with each of the nuclear transfer embryos. Thus, 40 cloned embryos should be implanted in each of the 5 surrogate dogs for a single order dog clone. Thus, total transplanted nucleated embryos must be transplanted into 5 surrogate dogs with 5 x 40 nuclear substituted embryos = 200 nuclear substituted embryos. That is, five surrogate mother dogs and a total of 200 nuclear transfer embryos are needed to produce only one or two cloned dogs to be delivered to an orderer. The surrogate success rate was assumed to be the average of 20%, so one out of five surrogate mothers gave birth successfully. As a result, surrogate mothers give birth to eight cloned dogs. As a result, when one dog is delivered to the orderer, there are seven surplus cloned dogs left after delivery.

That is, the number of surrogate dogs required for the request of a single orderer is 5, and the number of surrogate dogs is 8, and when the ordering quantity is 1, the surplus cloned dogs remaining after delivery are 7 dogs. If, for example, the manufacturing cost for the copy requested by an orderer was P1 (for reference, the current cost M is from about 50 million to about 200 million won), 1 The production cost for the cloned dogs will be P. In reality, however, the P cost includes a manufacturing cost for producing 7 surplus cloned dogs, but these costs are all transferred to the one delivered to the orderer, resulting in the cost of one clone being delivered. .

The expenses that make up these replication costs, P, will vary. For example, the cost of technology development will include depreciation costs, technician labor costs, financial costs, reagent costs, surrogate and egg collection costs, and the usual costs of operating them.

Among the above costs, the costs of oocyte-gathering dogs and surrogate mothers and their usual operating expenses are as follows.

Ovulation in dogs ovulates several dozens of immature eggs in one ovulation. Immature eggs become mature eggs 48-72 hours after ovulation in the body. Small dogs have fewer ovulations, and large dogs generally have higher ovulation levels. The number of eggs in ovulation depends on the age at which they grow in the same dog, and differ slightly for each individual. On average, one egg can ovulate up to 10 eggs.

In addition, the estrous cycle of dogs is very long, unlike cattle, horses, and pigs that heat up almost every month. That is, a large dog, such as a greyhound, has a estrous cycle about once a year, a small dog about once every 6-7 months, and usually once every eight months.

Therefore, in order to perform one orderer's request every day, 200 nuclear transfer embryos were required for a single orderer's order as described in the conventional dog cloning method. However, in order to produce 200 nuclear replacement embryos, at least 20 dogs must be in a estrous state every day in order to produce 200 nuclear-substituted eggs every day when it is assumed that 10 eggs can be used in one ovulation. In practice, however, the rate of cell fusion success should also be taken into account. If the cell fusion rate is considered to be 80%, then 100 dogs must be present in estrous state to be used as oocyte harvesting dogs. I can make it. In addition, as described above, since 40 nuclear-substituted embryos are transplanted into one surrogate dog, 200 dogs must be in estrous state every day in order for 200 nuclear-substituted embryos to be transplanted into the surrogate. Therefore, if one order comes in every day, 25 estrous oocytes with estrus + 5 estrous surrogate dogs = a total of 30 estrous dogs are needed every day. This suggests that statistically large numbers of dogs should be kept on a regular basis, given that the estrous cycle averages once every eight months. In other words, in order to be statistically estrous every day, a single estrous dog exists every day when the average estrus cycle is 8 months x 30 days / month = 240 dogs. Therefore, if 30 estrous dogs exist every day, it means that 240 x 30 (horned dogs) = 7,200 dogs should be kept as usual. In other words, to have one clone order per day, 30 dogs must be in heat each day, and at least 7,200 dogs must be kept, managed and monitored at all times to discover 30 heat-established dogs each day. Also, if a few years later become sick or grow old and cannot be used as an oocyte or surrogate dog, you should normally raise at least 7,200 enough dogs (e.g., approximately 10,000) to make one clone order each day. I can digest it. The number of dogs that must be kept in these daily lives is the number required to continuously carry out one replication order per day. Of course, the results of this calculation may be somewhat reduced if the yield increases with the development of dog cloning in the future, but it is now an important factor to increase the cost of dog cloning.

In other words, in the case of commercial cloning of dogs in a conventional manner, if one clone per day and 365 cases per year, there will be some fluctuations depending on the success rate of each stage technology in the cloning process. As a rule, approximately 7,200 to about 10,000 female dogs should be raised as usual, which was a major factor in the increase in manufacturing costs in conventional dog cloning techniques.

Considering the above-mentioned example in which a factor of another manufacturing cost in the conventional dog cloning technique is regarded as giving birth to 8 cloned dogs at one birth is as follows. If you deliver one cloned dog to an orderer who has cloned a pet, there will be seven surplus cloned dogs each day. As a result, in the event of a successful reproduction of eight cloned dogs, the remaining number of cloned dogs left and delivered to the orderer is 365 days x 7 per day = 2,555, i.e. 1 About 2,600 untreated surplus cloned dogs remain in the year. In other words, digesting one cloned dog order daily leaves approximately 2,600 surplus surplus dogs a year. Breeding these surplus dogs not only raises another manufacturing cost, but also raises a problem that the dog is a companion animal and cannot be thrown away.

In addition, in order to continuously digest one order each day, five surrogate mothers need to have a nuclear transfer embryo implanted in the surrogate mother's dog every day, and at the same time, five births a day. In order to continue digestion of the spell, approximately 25 surgeries for oocyte retrieval should be performed each day. That is, a total of 35 surgical expenses are used every day to continuously digest one replication order every day. This cost of surgery is also a major factor that increases the manufacturing cost of one cloned dog delivered to the orderer.

B. When carried out by the method of the present invention

The present invention is a method of simultaneously implanting a nuclear replacement egg of multiple orderers in one surrogate mother. Taking the example of digesting 365 orders per year (average 1 daily) by the method of the present invention, the present invention will be described as follows.

1. Simultaneously transplant nuclear-substituted embryos for multiple orderers (eg 8 orderers) and transplant them into one surrogate dog simultaneously.

That is, in order to compare with the above-described example in the prior art, a total of 40 nuclear-substituted embryos having the same conditions are transplanted into one surrogate mother, and 40 nuclear-substituted embryos of 40 surrogate embryos each of 5 surrogate mothers are considered in consideration of the fertility success rate. Implant each. However, in the above-described conventional method, the nuclear substituted embryos transplanted into five surrogate dogs are all nuclear substituted embryos having the same genetic traits (same somatic cell nuclei) for the same orderer, whereas in the present invention, each of the five surrogate mothers Each of the 40 nuclear transfer embryos to be transplanted, for example, is a mixture of 8 replacement nuclear transfer embryos that are simultaneously implanted in one surrogate dog. That is, 8 types of nuclear replacement embryos replaced with somatic cell nucleus of pet dog to be cloned in order 1, order 2, order 3, order 4 ...... At the same time, a surrogate dog's fallopian tube is implanted. Simultaneous mixed transplantation of these multi-order nuclear replacement embryos into the same surrogate mothers is performed on each of the five surrogate mothers under the same conditions as the example of the prior art in consideration of the success rate of birth.

Therefore, one of the five surrogate mothers (if the birth success rate is 25) has given birth to a surrogate mother, and if eight (usually 6-10) births, then several out of eight cloned dogs are born. Of cloned animals are mixed at the same time in a probable proportion to give birth. Thus, one successful copy completes one or more orders for eight orders. Therefore, the reproduction cost allocated to one orderer is reduced to 1/8 if the reproduction dogs for eight orderers, which is the case of giving birth at the ideal ratio, are mixed and delivered one by one. Of course, depending on the odds, only two orderers are born, or only three orderers are born, or only four orderers are born, or only five orderers are born, or only six orderers are born, or The cost of reproduction costs assigned to each orderer will vary according to the rate at which only seven orderers are born, or only eight orderers are born or the birth is successful, but in any case the present invention compares to the conventional method. The manufacturing cost can be reduced up to 1/8 (1/8 of the conventional manufacturing cost P).

In another aspect, the present invention provides the same number of oocyte-providing dogs, the same number of surrogate mothers, the same number of nuclear substituted embryos, the same number of dogs that must be in constant breeding, compared to the prior art example described above. With the same costs, such as the number of procedures and the like, the method of the present invention means that multiple (for example, eight times) orderer copies can be processed at one time at a conventional cost. That is, the present invention has a feature that the cost of solving the duplication for one orderer can be significantly reduced (for example, to 1/8) than the prior art example described above. In the ideal case, where the number of individual breeding dogs is 1 for each breed, not only will the costs of cloning allocated to each orderer be minimized, but there will be little or no unpleasant surplus cloned dogs delivered to and left by the orderer. There are features that can be.

In the surrogate mother transplantation method of the present invention, in the transfer and delivery of the nuclear replacement embryos for a number of different orders to five surrogate mothers, another method for reducing the replication cost of the present invention is each surrogate dog Of course, transplantation and delivery of nuclear-substituted embryos may be performed sequentially at regular time intervals. In this case, as shown in Fig. 1, the first surrogate mother is a nuclear surrogate embryo for a large number of orderers (e.g., for eight orderers A, B, C, D, E, F, GH). Transplant to (1) to give birth. If the birth of a cloned animal for orderer H fails from the first surrogate animal (1), new orderers (eg, H, I, J, K, L, Nuclear substituted embryos for M, N, O) are implanted in a second surrogate mother animal as shown in FIG. 2 to give birth. After confirming the birth status of the first surrogate mother (1), the two surrogate embryos for the new orderer and the nuclear replacement embryos for the new orderer are excluded. The first surrogate mother (2) may be transplanted into an animal. Also, if there is an orderer who failed to give birth after watching the birth of the second surrogate mother, a nuclear transfer embryo for the orderer who failed to give birth and a nuclear replacement embryo for another new orderer are mixed and transplanted into the third surrogate mother. And childbirth. In this way, a 4th surrogate mother can be transplanted and given birth. If the cloned animals for A, B, C, D, E, F, and G orderers succeeded in giving birth in the first surrogate mother, the second surrogate had nuclear replacement for failed orderer H in the first surrogate mother as shown in FIG. Embryos are mixed with nuclear substituted embryos for another majority orderer (eg, I, J, K, L, M, N, O orderer) and implanted in the second surrogate mother (2). In other words, in transplanting and giving birth to nuclear replacement embryos for different orderers in each surrogate mother, the next order of surrogate mothers may be sequentially performed while observing the success of the birth.

Even when the sequential surrogate mother is transplanted, when comparing the prior art and the present invention under the same conditions, the cost of the present invention is as follows based on the case of transplanting 8 replacement nuclear transfer embryos at a time. That is, in the prior art, if the first surrogate mother succeeds in giving birth, only eight cloned animals for one orderer are born (when multiple births are eight). On the other hand, in the case of sequential surrogate mother transplantation, under the same conditions as in the above-described conventional method, if the first surrogate mother succeeds in giving birth, a cloned animal for up to 8 orders (nuclear transfer embryo for 8 orders) Are mixed and transplanted at the same time) to give birth (when multiple pregnancies are eight), resulting in one-eighth the manufacturing cost of a cloned animal assigned to one orderer. Also, if only one cloned animal is delivered to the orderer, in the prior art, there are 7 surplus cloned animals remaining after delivery, but in the present invention, if the birth rate is the same, one animal is delivered to the orderer one by one. There is no surplus cloned animal remaining.

As described above, when comparing the cell fusion success rate, surrogate mother implantation success rate, and fertility success rate under the same conditions in the case of nuclear replacement, the present invention is compared to the pet cloning according to the prior art. Since the number of cloned dogs for multiple orderers can be born at the same time in the delivery of surrogate mothers, the cost of cloning allocated to each orderer for cloning is reduced. In addition, the present invention is due to the birth of a clone pet for a large number of orders in a single birth, while breeding dogs of surrogate mothers and oocytes for the same number of dogs in normal breeding, as an example, to digest up to about 8 times the order amount more than conventional There may also be features. This feature is characterized by the fact that it is possible to reduce the quantity of the usual breeding dog population to digest the same order quantity by one eighth as compared to the prior art as an example. In addition, the number of operations (eg, oocyte extraction surgery, surrogate mother transplant surgery, and childbirth surgery) for digesting the same order amount may be reduced to about 1/8, for example. In addition, the present invention has a feature that can significantly reduce the number of surplus cloned pets left and delivered to the orderer compared to the prior art. For these reasons, the present invention is characterized in that it is possible to reproduce a dog or cat, which is a multi-pregnancy pet, very economically than before.

In the practice of the present invention, if the pet to be cloned has a very long estrous period, the estrus should be judged for determination at the time of oocyte collection and for the determination of whether the dog is a surrogate mother. Reference is made to conventional methods known in the art. In other words, it is also possible to determine whether or not estrus by visually seeing the color of the pubic area and the color and concentration of the secretion, and another method is to detect the hormone (e.g. It can also be determined by checking whether or not.

As an example, quoting a known method is as follows. That is, the method of determining the ovulation time can be cited from a known technique (Korean Patent 10-0733012). That is, "Determination date is determined daily by measuring vaginal smear and serum progesterone concentration in dogs with estrous period, and serum progesterone concentration is collected by centrifuging 3-5 ml of blood every day and centrifuged. Obtained and analyzed using DSL-3900 ACTIVE progesterone coated tube radioimmunoassay kit (Diagnostic Systems Laboratories, Inc., TX), and when progesterone concentrations range from 4.0 to 7.5 ng / ml are considered ovulation days (Hase et al. , J. Vet. Med. Sci., 62: 243-248, 2000).

The method of determining which ordered cloned animals are cloned animals for the order is genetic information obtained by performing DN fingerprint analysis of somatic cells of cloned animals delivered from surrogate mothers and donor nuclear sources to nuclear-substituted embryos implanted in the surrogate animals. The DNA fingerprint analysis genetic information of the somatic cell nuclei provided by each orderer is compared with each other to find a match to determine that the cloned animal is for a specific orderer.

1 is a conceptual diagram of the present invention showing that nuclear replacement embryos for multiple orderers are mixed and implanted in a first surrogate mother animal at the same time.

FIG. 2 shows that multiple replacement order embryos containing nuclear replacement embryos for the orderer H who failed to give birth when the reproduction of the cloned animal for the orderer H in the first surrogate animal are mixed are simultaneously mixed in the second surrogate animal. A conceptual diagram of the present invention showing the implantation state.

※ Description of main code in drawing

1. the first surrogate animal 2. the second surrogate animal

A. Nuclear Transfer Embryos for Orderer A B. Nuclear Transfer Embryos for Orderer B

C. Nuclear Transfer Embryos for Orderer C D. Nuclear Transfer Embryos for Orderer D

E. Nuclear Transfer Embryos for Orderer E F. Nuclear Transfer Embryos for Orderer F

G. Nuclear Transfer Embryos for Orderer G H. Nuclear Transfer Embryos for Orderer H

I. Nuclear Transfer Embryos for Orderer I J. Nuclear Transfer Embryos for Orderer J

K. Nuclear Transfer Embryos for Orderer K L. Nuclear Transfer Embryos for Orderer L

M. Nuclear Transfer Embryos for Orderer M N. Nuclear Transfer Embryos for Orderer N

O. Nuclear Transfer Embryos for Orderer O

Claims

In the conventional method of cloning a multi-pregnancy pet,

1. Producing a plurality of nuclear transfer eggs for different orderers by transplanting and cell fusion into denucleated eggs of the same species, respectively, using somatic cells of the same multiple gestational pets of different orderers as donor nuclei;

2. each of the different ordered nuclear replacement eggs is individually activated after treatment to inhibit the generation of a second polar body, resulting in a plurality of different ordered nuclear replacement embryos;

3. The nuclear transfer embryos for different orderers are mixed in the body of surrogate mothers of the same species and transplanted and implanted so that, after a period of time, the pets of multiple orderers having different genotypes are reproduced together as cloned animals. ;

4. The genetic information of animals born from surrogate animals and the genetic information of donor nuclear sources (somatic cells) for each ordered transplanted to the surrogate animals are compared to determine whether the birthed animal is a cloned animal for which ordered animals. Economical cloning method of a multi-pregnancy pet, characterized in that comprises a step.

The method according to claim 1,

Economical cloning method of a multiple pregnancy pet, characterized in that the multiple pregnancy pet is an individual.

The method according to claim 1,

The multi-pregnancy pet is an economical replication method of the multi-pregnancy pet, characterized in that the cat.

The method according to claim 1,

Transplantation to the surrogate animal causes transplantation and delivery of mixed multi-order nuclear replacement embryos, which in turn include subsequent nuclear replacement embryos that failed to deliver in surrogate mothers. And a method of economically replicating a multi-pregnancy pet, characterized in that the sequential transplantation and delivery of such surrogate mother animals are performed sequentially.

The method according to claim 1,

The nuclear replacement eggs are prepared by denuclearizing the nuclei of oocytes (ovals) of the second stage of meiosis (MII), and transplanting and cell fusion of the somatic cell nucleus of the animal to be cloned by the orderer to the denucleated oocytes (ovals). Economical cloning method of multiple pregnancy pets, characterized in that.

The method according to claim 1,

The cell fusion is an economical replication method of a multi-pregnancy pet, characterized in that by applying a DC pulse voltage to the oocytes in the state in which the somatic nucleus is transplanted into the denucleated oocytes.

The method according to claim 1,

The nuclear-substituted embryos are economical replication method of a multi-pregnancy pet, characterized in that the activation by treatment with calcium ionophore and 6-dimethylaminopurine.

The method according to claim 1,

The cell fusion is an economical replication method for a multi-pregnancy pet, characterized in that it is performed 1 to 3 times for 10-30 Hz at a DC pulse voltage of 3.0 to 3.5 kv / cm.

The method according to claim 1,

The cell fusion is an economical replication method of a multi-pregnancy pet, characterized in that it is carried out twice for 15 to 25 Hz at a DC pulse voltage of 1.75 to 6.0 kv / cm.