KR20200060836A - Mouse embryonic stem cell line derived from outbred mouse and preparing method thereof - Google Patents

Abstract

The present application relates to an embryonic stem cell line derived from an outbred mouse. More specifically, the present invention relates to an embryonic stem cell line derived from an ICR mouse, a manufacturing method thereof, and a culture method thereof. The embryonic stem cell line derived from the outbred mouse according to the present invention can significantly reduce the time and cost required for research and development.

Description

Mouse embryonic stem cell line derived from outbred mouse and preparing method thereof

The present application relates to an embryonic stem cell line derived from an outbred mouse, and more particularly, to an embryonic stem cell line derived from an ICR mouse, a manufacturing method thereof, and a culture method thereof.

To date, embryonic stem cells (ESCs), which have excellent abilities in self-renewal and pluripotency, are used for cell therapy in organogenesis models and regenerative medicine during embryonic development in developmental biology. It is receiving great attention as a basic resource cell. In addition, embryonic stem cells were used to test for drugs without generating genetically modified animals, developing custom drugs, and conducting clinical trials. Therefore, embryonic stem cells are actively being applied not only to basic research in regenerative medicine, transgenic animal production, and pharmaceutical fields, but also to clinical research in industrial fields.

Early in stem cell research, embryonic stem cells derived from blastocysts of mice of various genetic backgrounds were widely used to develop many stem cell-related technologies. Beginning with the generation of the first mouse embryonic stem cells derived from the 129SvE strain in 1981, attempts have been made to establish mouse embryonic stem cells in various strains. However, the successful establishment of mouse embryonic stem cells has been limited to several subspecies such as 129 sub-strains and C57BL/6, producing embryonic stem cells such as ICR, CBA, NOD, DBA and Balb/c strains. The efficiency of mouse embryonic stem cell production from these demanding non-permissive varieties was extremely low. Induction efficiency of mouse embryonic stem cells in these non-acceptable varieties was very low, but establishment of mouse embryonic stem cells has been reported from other non-acceptable varieties except ICR. However, the establishment of embryonic stem cells derived from non-subsidiary ICR mouse blastocysts has not been reported.

Based on the fact that the genetic identity between mouse and human is about 99%, a variety of laboratory mouse strains, including inbred, hybrid and outbred mice, are widely used for individual research purposes. . Researchers can get consistent results from mice in the vicinity of mice that have features such as genetic and phenotypic stability, but recovery is difficult when the regulatory genes that maintain homeostasis are damaged because of genetic homology in the chromosome. Hybrid mice produced by intentionally crossing two different mice are maintained with the same genetic characteristics and phenotype as those of the mice in the vicinity, but it may be difficult to accurately collect data related to the genetic background. On the other hand, non-sub-mice mice have genetic characteristics, such as human-like phenotypic variations and high heterogeneity, which are not genetically defined. At the same time, they demonstrated a very successful case as the primary population for selection when producing new or improved humanized mouse models. Therefore, when the results are applied to humans, the results obtained in non-subclinical mice are considered to be more valuable than those obtained from sub-clinical or hybrid mice. For this reason, non-subsidiary mice are actively applied in toxicology, pharmacology and basic biomedical research.

U.S. Patent Publication No. 2018-0155680

Because of the high genetic similarity between mice and humans, mice and their stem cells are widely used in biomedical research, new drug development, and cell therapy research, but they are often not applied to humans even if the developed technology shows effects in mice. For this reason, technologies developed through mice are being re-verified for their effects through large animals that have physiological phenomena similar to humans before being applied to clinical trials. This complex revalidation process can be solved by using a non-cross-linked mouse with a genetic variation similar to humans and very high heterogeneity, but embryonic stem cells derived from a non-cross-linked mouse have not been established. Accordingly, the present application is intended to provide embryonic stem cells derived from non-subsidiary mice that have not been reported worldwide and methods for manufacturing the same.

However, the problems to be solved by the present application are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

A first aspect of the present application includes: (a) crossing an outbred mouse to obtain a fertilized egg in a zygote state; (b) developing a fertilized egg in a zygote state to a 4-cell state; (c) developing a four-cell fertilized egg into a blastocyst; (d) removing the transparent zone of the blastocyst; And (e) obtaining embryonic stem cell colonies by culturing a blastocyst with a clear band removed on fibroblast feeder cells derived from a non-subsidiary mouse fetus, to a method for generating non-subsidiary mouse embryonic stem cells. .

The second aspect of the present application relates to non-subsidiary mouse embryonic stem cells produced by the method of the first aspect of the present application.

The third aspect of the present application relates to non-subsidiary mouse embryonic stem cells deposited with accession number KCTC13658BP.

The fourth aspect of the present application relates to mouse germ cells differentiated from non-subsidiary mouse embryonic stem cells of the third aspect of the present application.

The fifth aspect of the present application comprises the step of subcultured on the fibroblast feeder cells derived from the hybrid mouse (hybrid) mouse, the non-subsidiary mouse embryonic stem cell of the third aspect of the present application, It is about.

A sixth aspect of the present application relates to a method for subculture of non-mice mouse embryonic stem cells, comprising culturing the non-mice mouse embryonic stem cells of the third aspect of the present application on fibroblast feeder cells derived from the non-muscle mouse. .

The above-described problem solving means are merely exemplary and should not be interpreted as an intention to limit the present invention. In addition to the exemplary embodiments described above, there may be additional embodiments and embodiments described in the drawings and detailed description of the invention.

The present invention provides an embryonic stem cell line first established from a non-subsidiary mouse and a method for manufacturing the same. Specifically, embryonic stem cells were separated and cultured in vitro from the inner cell mass of the blastocyst derived from a non-subcutaneous ICR mouse, and the root muscles were confirmed by confirming characteristics such as self-replication and differentiation potentials of the cultured embryonic stem cells. The successful establishment of mating mouse embryonic stem cell lines was verified.

Non-sub-mice mouse embryonic stem cells according to the present invention have many genetic mutations and very high heterogeneity similar to humans, so if they are used, it is possible to omit the preclinical research process using large animals similar to humans, thus enabling clinically applicable stems. It can greatly reduce the time and cost of research and development of cell therapy products and cell therapy technologies.

Figure 1 shows the colony morphology of embryonic stem cells ( _ICR ESCs) established from non-subsidiary ICR mouse blastocyst according to an embodiment of the present application (n = 3). _ICR ESCs colonies confirmed that they maintained a perfect dome shape during the incubation period, similar to those detected in _E14 ESCs colonies (top right) (Scale bar = 200 μm).
Figure 2 confirms the expression (B) and the activity (C) of the alkaline phosphatase (AP) protein of embryonic stem cells ( _ICR ESCs) established from the blastocysts of non-systemic ICR mice according to an embodiment of the present application (n = 3). _Although AP protein expression (B) and activity (C) were partially confirmed in colonies of _ICR ESCs, colonies of _E14 ESCs showed complete strong AP protein expression (top right of B) and activity (top right of C). (Scale bar = 200 μm).
Figure 3 shows the self-renewal-related genes Oct4 , Sox2, Nanog , Tert, and AP transcriptional expression _E14 for embryonic stem cells ( _ICR ESCs) established from non-subsidiary ICR mouse blastocysts according to an embodiment of the present application. Compared to ESCs. As detected in _E14 ESCs _{It was} confirmed that transcripts of Oct4 , Sox2 , Nanog , Tert and AP , self-replicating genes were detected in _ICR ESCs.
Figure 4 compares the expression expression of the autologous replication-related genes Oct4 , Sox2 , and Nanog against _E14 ESCs with respect to embryonic stem cells ( _ICR ESCs) established from non-subsidiary ICR mouse blastocysts according to an embodiment of the present application ( _E14 ESCs) n = 3). It was confirmed that _ICR ESCs, like _E14 ESCs (top right of E to G), also positively express Oct4, Sox2, and Nanog (Scale bar = 200 μm).
Figure 5 compares the expression of Tra-1-60 and Tra-1-81 genes with _E14 ESCs for embryonic stem cells ( _ICR ESCs) established from a non-subsidiary ICR mouse blastocyst according to an embodiment of the present application. (n = 3). Like _E14 ESCs (top right of H~I), _ICR ESCs also confirmed that Tra-1-60 and Tra-1-81 were negatively expressed (Scale bar = 200 μm).
Figure 6 shows the formation of embryonic bodies (Embryonic bodies; EBs) of embryonic stem cells ( _ICR ESCs) established from blastocysts of non-systemic ICR mice according to an embodiment of the present application. _ICR ESCs were cultured for 3 days in an environment free of feeder cells and LIF to differentiate into embryoid bodies, and spherical embryoid bodies were observed (Scale bar = 200 μm).
Figure 7 confirms the spontaneous in vitro differentiation into embryonic bodies of embryos differentiated from _ICR ESCs according to an embodiment of the present application (n = 3), the embryos cultured for 7 days in an environment without feeder cells and LIF The cells derived from the differentiated embryoid body were further differentiated, and the cells were derived from neurofilament (exoderm; K), alpha-smooth muscle actin (mesoderm; L) and cytokeratin 18 (cytokeratin 18) ( It showed reactivity to endoderm; M) (Scale bar = 200 μm).
FIG. 8 is a study of transplantation of _ICR ESCs into nude mice according to an embodiment of the present application to confirm whether trilobal differentiation is in vivo, and confirmed that teratoma was formed 5 weeks after transplantation [simple columnar epithelial cells (endoderm) Lineage cells, N1), blood vessels (endodermal lineage cells, N2), simple cubic cells (endodermal lineage cells, N3), chondrocytes (mesoderm lineage cells, N4), adipocytes (mesodermal lineage cells, N5) muscle cells (mesoderm lineage Cells, N6), neural tube (ectoderm lineage cells, N7), germinal hair bulb like structure with pigmented cells on core region (ectoderm lineage cells, N8) and nerve tissue (ectoderm lineage cells, N9)] (Scale bar = 50 μm ).
Figure 9 shows the differentiation of _ICR ESCs into oocytes according to an embodiment of the present application (Scale bar = 50 μm).
10 is an analysis of the karyotype of _ICR ESCs according to an embodiment of the present application, 40 normal diploid karyotypes were identified.
Figure 11 confirms the sex determination of _ICR ESCs according to an embodiment of the present application, the sex was confirmed in the genome for the presence of X chromosome specific Xist or Y chromosome specific Zfy1. It was confirmed that Xist was present in the genome and Zfy1 was absent, so that the sex of _ICR ESCs was female.
FIG. 12 shows colony formation of pluripotent stem cells and embryonic germ cells after 14 days of incubation of feeder cells ( _ICR MEFs) prepared by harvesting mouse embryonic fibroblasts (MEFs) from fetuses of non-subcutaneous ICR mice. It was confirmed whether or not (day 0, 6, 12, and 14) (Scale bar = 200 μm).
FIG. 13 is a flow cytometry analysis of the presence of pluripotent stem cells and embryonic germ cells after 14 days of incubation of feeder cells ( _ICR MEFs) prepared by collecting mouse embryonic fibroblasts (MEFs) from fetuses of non-cross-linked ICR mice in standard ESC medium. (FACS) (Oct4, Sox2, and Nanog: pluripotent stem cell specific protein; VASA: embryonic germ cell specific protein). All data are presented as the mean±SD of three independent experiments.
FIG. 14 shows the doubling time after culturing _ICR ESCs on MECR feeder cells derived from ICR, C57BL/6 and B6CBAF1 mice for 4 days, respectively. All data are presented as the mean±SD of three independent experiments (* p <0.05).
Figure 15 shows the colony size after culturing _ICR ESCs for 4 days on MEF feeder cells derived from ICR, C57BL/6 and B6CBAF1 mice, respectively. All data are presented as the mean±SD of three independent experiments (* p <0.05).
Figure 16 shows the number of colonies (# of _ICR ESC colonies) after culturing _ICR ESCs in MEF feeder cells derived from ICR, C57BL/6 and B6CBAF1 mice for 4 days, respectively. All data are presented as the mean±SD of three independent experiments (* p <0.05).
Figure 17 shows the degree of expression of Oct4, Sox2 and Nanog after culturing _ICR ESCs in MEF feeder cells derived from ICR, C57BL/6 and B6CBAF1 mice for 4 days, respectively. All data are presented as the mean±SD of three independent experiments (* p <0.05).
Figure 18 compares colony morphology of _ICR ESCs and _E14 ESCs cultured for a long time on _B6CBAF1 MEFs (n = 3). _ICR ESCs colonies were dome-shaped during the incubation period (Scale bar = 200 μm), similar to those detected in _E14 ESCs colonies (top right).
Figure 19 compares the expression (B) and activity (C) of alkaline phosphatase (AP) protein of _ICR ESCs and _E14 ESCs cultured for a long time on _B6CBAF1 MEFs (n = 3). _{Although the} weak AP protein expression (B) and activity (C) were partially confirmed in colonies of _ICR ESCs, the colonies of _E14 ESCs showed complete strong AP protein expression (top right of B) and activity (top right of C). Will (Scale bar = 200 μm).
FIG. 20 shows the transcriptional expression of Oct4 , Sox2, Nanog , Tert, and AP transcriptions related to self-replicating genes for long-term cultured _ICR ESCs (below 35 passage cultures) and short-term cultured _ICR ESCs (20 passages or less) on _B6CBAF1 MEFs. As a comparison, it was confirmed that there was no significant difference in the level of transcription for each gene.
Figure 21 is a comparison of the long term culture of ESCs _ICR and whether the self Oct4, translation expression of Sox2, and Nanog replication-related gene with respect to _E14 on ESCs and MEFs _{B6CBAF1 E14} ESCs (n = 3). It was confirmed that _ICR ESCs, like _E14 ESCs (top right of E to G), also positively express Oct4, Sox2, and Nanog (Scale bar = 200 μm).
22 is a long period of time whether Tra-1-60 and Tra-1-81 expression of translation with respect to the gene in cultured _ICR ESCs and the ESCs _E14 compares and _E14 ESCs (n = 3) on _B6CBAF1 MEFs. Like _E14 ESCs (top right of H~I), _ICR ESCs also confirmed that Tra-1-60 and Tra-1-81 were negatively expressed (Scale bar = 200 μm).
23 shows embryonic formation of _ICR ESCs cultured for a long time on _B6CBAF1 MEFs. _ICR ESCs cultured for a long time were cultured for 3 days in an environment free of feeder cells and LIF to differentiate into embryoid bodies, and spherical embryoid bodies were observed (Scale bar = 200 μm).
Figure 24 _confirms spontaneous extracorporeal differentiation into trilobites for EBs differentiated from _ICR ESCs cultured for a long time on _B6CBAF1 MEFs (n = 3), the embryoid body cultured for 7 days in an environment without feeder cells and LIF, Differentiated, cells derived from differentiated embryoid bodies showed responsiveness to neurofilaments (ectoderm; K), alpha-smooth muscle actin (mesoderm; L) and saosatokeratin 18 (endoderm; M) (Scale bar = 200 μm) ).
FIG. 25 confirms whether teratoma differentiation was _observed in vivo by transplanting _ICR ESCs cultured on _B6CBAF1 MEFs for a long period of time into nude mice. Bilayered apocrine tubes (endodermal lineage cells, N2), tubes composed of simple cubic cells (endodermal lineage cells, N3), adipocytes (mesodermal lineage cells, N4), striped muscle (mesodermal lineage cells, N5), smooth muscle cells ( Mesodermal lineage cells, N6), epithelial cells with keratinization (endodermal lineage cells, N7), neural bundles (ectoderm lineage cells, N8) and neural epithelium (ectoderm lineage cells, N9)] (Scale bar = 50 μm).
Figure 26 shows the differentiation of long-term cultured _ICR ESCs on _B6CBAF1 MEFs into oocytes (Scale bar = 50 μm).
FIG. 27 is an analysis of the karyotype of _ICR ESCs cultured for a long time on _B6CBAF1 MEFs, and 40 normal diploid karyotypes were identified.
FIG. 28 _confirms the sex determination of _ICR ESCs cultured over a long period of time on _B6CBAF1 MEFs, and gender confirms the presence of X chromosome specific Xist or Y chromosome specific Zfy1 in the genome. It was confirmed that Xist was present in the genome and Zfy1 was absent, so that the sex of _ICR ESCs was female.
29 shows _{microsatellite} DNA analysis for _{C57BL / 6} MEFs, _B6CBAF1 MEFs, _ICR MEFs, and long-term cultured _ICR ESCs, using D1Mit16 and D17Mit124 markers. The alleles between _ICR MEFs and _ICR ESCs detected by the D1Mit16 and D17Mit124 markers were identical, and in contrast, it was confirmed that the alleles of _ICR ESC were different from _C57BL/6 MEFs and _B6CBAF1 MEFs.

Since the present application can apply various transformations and have various embodiments, specific embodiments will be illustrated in the drawings and described in detail in the detailed description. However, it is not intended to limit the present application to the specific embodiments, it should be understood to include all conversions, equivalents, and substitutes included in the spirit and scope of the present application. In the description of the present application, when it is determined that the detailed description of related known technologies may obscure the subject matter of the present application, the detailed description will be omitted.

 The terms used herein are only used to describe specific embodiments and are not intended to limit the present invention. Singular expressions include plural expressions unless the context clearly indicates otherwise. As used herein, the terms “include” or “have” are intended to indicate that a feature, number, step, action, component, part, or combination thereof described in the specification exists, or that one or more other features or It should be understood that the presence or addition possibilities of numbers, steps, actions, components, parts or combinations thereof are not excluded in advance.

Terms such as first and second may be used to describe various components, but the components should not be limited by the terms. The terms are used only for the purpose of distinguishing one component from other components.

Throughout the present specification, when a step is said to be “on” or “before” another step, this is not only the case where one step is in direct time series relationship with the other step, but also the mixing step after each step. The sequence of steps may include the same rights as those in an indirect time-series relationship where the time-series sequence can change.

The terms “about”, “substantially”, etc., used to the extent of the present specification are used in or near the numerical values when manufacturing and material tolerances unique to the stated meanings are presented, and understanding the present invention In order to help, accurate or absolute figures are used to prevent unscrupulous abuse of unconscionable infringements of the disclosed disclosure. As used throughout this specification, the term “step (to)” or “step of” does not mean “step for”.

Hereinafter, the method for generating non-sub-mice mouse embryonic stem cells of the present application and non-sub-mouse mouse embryonic stem cells, mouse germ cells differentiated therefrom, and the subculture method thereof will be described in detail with reference to embodiments and examples and drawings. Explain. However, the present application is not limited to these embodiments and examples and drawings.

A first aspect of the present application includes: (a) crossing an outbred mouse to obtain a fertilized egg in a zygote state; (b) developing a fertilized egg in a zygote state to a 4-cell state; (c) developing a four-cell fertilized egg into a blastocyst; (d) removing the transparent zone of the blastocyst; And (e) obtaining embryonic stem cell colonies by culturing a blastocyst with a clear band removed on fibroblast feeder cells derived from a non-subsidiary mouse fetus.

The non-submuscular mouse of the present invention is a concept distinguished from an inbrde mouse and a hybrid mouse. Inbred mice are inbred mice, mice that have continued sibling or paternity for more than 20 generations, and have a kinetic coefficient (R) of 99.6%, which makes them nearly identical to identical twins, so that individuals are genetically homogeneous. There are sub-clinical mice such as C57BL/6, C58, 129, DBA and CBA, and each line has a specific disease. A hybrid (hybrid) mouse is a mouse produced by cross-breeding of a suburban mouse and has hybrid stress. The first generation of the two suburban mice are genetically identical. (Example: D2B6F1 hybrid mice are offspring (F1) of female DBA2 and male C57BL/6J)

Non-inbred mice are mice that have not been inbred, such as siblings, in order to maintain the greatest genetic diversity. Advantages include low cost, long lifespan, low disease incidence and fertility, and genetic heterogeneity is established by crossing as many sub-systems as possible to obtain heterogeneity when genetic heterogeneity is required. Due to various genetic variations and high heterogeneity, non-sub-mice mice have not been reported to establish embryonic stem cells, unlike sub-mice and hybrid mice. Therefore, the non-subsidiary mouse embryonic stem cells produced by the method for generating non-subsidiary mouse embryonic stem cells according to the present invention are the first embryonic stem cells derived from non-subsidiary mice.

The non-subsidiary mouse embryonic stem cells thus generated were deposited with the KCTC Biological Resource Center.

Depository Name: Korean collection for type cultures

Accession number: KCTC13658BP

Date of Deposit: 20181005

According to one embodiment of the present application, the non-subsidiary mouse may be an ICR (Institute of Cancer Research) mouse, but may not be limited thereto, and is usually not obtained from non-sub-mouse subspecies prepared without inbred breeding such as sibling breeding. The technician can choose accordingly.

According to one embodiment of the present application, the fertilized egg in the zygote state of step (a) may be obtained by sequentially ovulating pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) into female non-subcutaneous mice, , It may not be limited. For example, female ICR mice are over-ovulated by injection of PMSG, and after about 48 hours, hCG is injected and mated with male ICR mice at the same time, and after about 18 hours of hCG injection, mice are sacrificed and cultured by harvesting fertilized eggs and hCG injection. After about 66 hours, the fertilized egg of the 4-cell stage is transferred to a medium containing bovine serum albumin, and then cultured. After about 138 hours of hCG injection, embryonic stem cells can be obtained from the fertilized egg of the blastocyst stage.

The fertilized egg, freshly harvested from the mouse, contains ovary cells around it, and the hyaluronidase can be used to remove the egg cells. In this case, the removal of the ovarian cells may be performed using M2 medium containing hyaluronidase, specifically, M2 medium containing 0.2% (w/v) hyaluronidase, but may not be limited thereto. .

According to one embodiment of the present application, step (b) may include culturing a fertilized egg in a zygote state in M2 medium, but may not be limited thereto. The M2 medium may not contain bovine serum albumin.

According to one embodiment of the present application, step (c) may include culturing a fertilized egg of a 4-cell state in M2 medium containing bovine serum albumin (BSA), wherein the bovine serum albumin is about 1 to It may be included in a concentration of 5 mg/ml, about 2 to 4 mg/ml, or about 3 mg/ml, but may not be limited thereto.

According to one embodiment of the present application, the transparent zone of the blastocyst may be mechanically removed in step (d), for example, only the transparent zone may be mechanically removed from the blastocyst using an insulin syringe, but is not limited thereto. Can be. The blastocyst from which the transparent band is removed may be cultured under conditions to help cultivation and proliferation of embryonic stem cells in order to cultivate the embryonic stem cells present in the inner cell mass. At this time, conditions that help the culture and proliferation of embryonic stem cells may include the presence of feeder cells, the presence of fetal bovine serum, and the presence of mLIF, but may not be limited thereto.

According to one embodiment of the present application, the culture of the blastocyst with the clear band removed in step (e) may be performed in DMEM medium containing fetal bovine serum (FBS) and mLIF (mouse leukemia inhibitory factor), wherein Fetal serum may be heat-inactivated, may be qualified for culturing embryonic stem cells, and may be about 5-20% (v/v), about 10-20% (v/v) in the medium. , Or about 15% (v/v) may be included, but may not be limited thereto. In addition, the mLIF may be used in an amount of about 200 to 2,000 units/ml, about 500 to 1,500 units/ml, or about 1,000 units/ml, and further includes, for example, antibiotics, non-essential amino acids, L-glutamine and beta mercaptoethanol It may, but may not be limited to this.

According to one embodiment of the present application, non-subsidiary mouse embryonic stem cells may express characteristic markers of embryonic stem cells, but may not be limited thereto. For example, the characteristic markers of the embryonic stem cells may be those selected from Oct4, Sox2, Nanog, Tert, and AP, and the non-subsidiary mouse embryonic stem cells according to the present invention are at least one of the markers, at least 2 Dogs, at least 3, at least 4, or all 5 can be expressed. In addition, the non-subsidiary mouse embryonic stem cells may not express TRA-1-60 and TRA-1-81, which are known not to be expressed in normal mouse embryonic stem cells.

According to one embodiment of the present application, the non-subsidiary mouse embryonic stem cells may be capable of differentiation into endoderm, mesoderm, and ectoderm, and may also be capable of differentiation into germ cells. This differentiation can be confirmed by embryoid formation, teratoma formation and immunocytochemistry. The fact that the embryonic stem cells of the non-subsidiary mouse of the present invention can differentiate into germ cells has great significance in that it is possible to form a complete adult mouse that can finally be reproduced.

According to one embodiment of the present application, the method may further include sub-culturing the embryonic stem cell colonies obtained in step (e) on fibroblast feeder cells (MEF) derived from a non-subsidiary mouse fetus. It may not be limited. Here, the passage may be performed 5 times, 10 times, 20 times, 30 times, 50 times, or 100 times or more, but may not be limited thereto.

The use of the non-subsidiary mouse MEF may be advantageous for the establishment of non-subsidiary mouse embryonic stem cells compared to MEFs derived from other mouse subspecies. For example, the MEF derived from the non-subsidiary mouse fetus may be a MEF derived from an ICR mouse.

According to one embodiment of the present application, the passage may include, but is not limited to, mechanically separating the colonies and moving them onto new feeder cells, for example, after separating the colonies using a glass capillary pipette. It may be passaged over the feeder cells.

The second aspect of the present application relates to non-subsidiary mouse embryonic stem cells produced by the method of the first aspect of the present application. Non-subsidiary mouse embryonic stem cells produced by the method of the present invention can exhibit all of the gene and protein expression characteristics of commonly known mouse embryonic stem cells, self-replicating is possible, differentiation into three germ cells and into germ cells. It may be a complete mouse embryonic stem cell capable of differentiation.

The third aspect of the present application relates to non-subsidiary mouse embryonic stem cells deposited with accession number KCTC13658BP.

The fourth aspect of the present application relates to mouse germ cells differentiated from non-subsidiary mouse embryonic stem cells of the third aspect of the present application.

The fifth aspect of the present application comprises passage of the non-subsidiary mouse embryonic stem cells according to the third aspect of the present application on fibroblast feeder cells derived from hybrid mice. It's about how.

According to one embodiment of the present application, the hybrid mouse may be a female C57BL/6 mouse and a B6CBAF1 mouse, a descendant of a male CBA mouse, but may not be limited thereto.

A sixth aspect of the present application relates to a method for subculture of non-subsidiary mouse embryonic stem cells, comprising culturing the non-subsidiary mouse embryonic stem cells according to the third aspect of the application on fibroblast feeder cells derived from non-subsidiary mice. will be.

According to one embodiment of the present application, the non-subsidiary mouse may be an ICR mouse, but may not be limited thereto.

When MEF derived from a hybrid or non-muscle mouse, not a murine mouse, is used for long-term culture of embryonic stem cells of the present invention, characteristics such as pluripotency marker expression, autoreplication and proliferation are better maintained. Can be. The medium used for the culture of non-subsidiary mouse embryonic stem cells includes a universal medium used to obtain colonies of mammalian embryonic stem cells. For example, the medium is Dulbecco's modified Eagle's medium (DMEM), knock-out DMEM, DMEM with fetal calf serum, serum substitute, DMEM with Chatot, Ziomek and Bavister (CZB) medium, children Ham's F-10 with serum (FCS), Tyrodes-albumin-latate-pyruvate (TALP), Dulbecco's phosphate buffered saline (PBS), Eagle's and Whitten's medium. Preferably, the culture medium is DMEM medium comprising beta-mercaptoethanol, non-essential amino acids, FBS, antibiotics and/or LIF (leukemia inhibitory factor). The detailed description of the medium is described in R Ian Freshney, Culture of Animal Cells, A Manual of Basic Technique, Alan R Liss, Inc, New York, WO 97/47734 and WO 98/30679, and the document is herein Is inserted as a reference.

Hereinafter, the present invention will be described in detail by the following examples.

However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

< Example >

The experimental contents for the production and culture of embryonic stem cells ( _ICR ESCs) derived from non-subsidiary ICR mice are described below.

Experimental Example  1: Animal preparation

Female ICR mice were purchased from Daehan Biolink to obtain embryos, and 6-week-old male NCr nude mice were purchased from Nara Biotech and used to generate teratomas in the experimental examples described below. In addition, to isolate mouse embryo fibroblasts (MEFs) used for the purpose of culturing embryonic stem cells, ICR (Daehan Biolink), C57BL/6 (Nara Biotech), and male CBA mice (Coartec) ) And female mice of C57BL/6 (Coretec) naturally mated.

All animal experiments according to this example were performed according to the guidelines with the approval of Kangwon National University Animal Experiment Ethics Committee (No. KW-170131-1).

Experimental Example  2: mouse fetal fibroblasts ( MEFs ) Feeder  Cell preparation

Feeder cells for culturing embryonic stem cells of non-cross-linked ICR mice were prepared as follows.

Female mice at 13.5 days of pregnancy (ICR, C57BL/6 and C57BL/6 Х CBA) were sacrificed by cervical dislocation method, each uterine petri containing DPBS (Dulbecco's phosphate-buffered saline; Welgene Inc., Daegu, Korea) I moved to Dish. Fetal heads, legs, tails, and tissues within the embryo were removed from the uterus, and only the skin of the fetus was recovered. The recovered tissue was then digested with 0.25% trypsin-EDTA (Welgene) for 10 minutes at 37° C., 5% CO ₂ , and the undigested MEF was removed with a 70-μm nylon mesh (SPL, Pocheon, Korea). DMEM with 10% (v/v) heat-inactivated fetal bovine serum (FBS; Welgene) and 1% (v/v) antibiotic-antimycotic (Welgene) After washing the MEFs with MEF culture medium consisting of (Dulbecco's modified Eagle's medium; Welgene), 37°C, 5% CO ₂ incubator until MEF reaches 90% of the culture dish area while supplying fresh medium every 2 days after culture. Cultured in MEFs reaching 90% of the dish area in the primary passage were inactivated by 10 μg/ml mitomycin C (mitomycin C, Sigma-Aldrich, St. Louis, MO) for 3 hours at 37° C., 5% CO ₂ After washing with DPBS, it was separated from the culture dish by 0.05% trypsin-EDTA (Welgene). The inactivated MEF suspension was prepared in 4-well culture dishes (SPL) coated with 0.1% (w/v) gelatin (Sigma-Aldrich), and later used as feeder cells of embryonic stem cells derived from non-subsequent ICR mice.

Experimental Example  3: Embryo collection and culture

Female ICR mice were inoculated with 5 IU of gestational mare serum gonad stimulating hormone (PMSG; Sigma-Aldrich) and injected with 5 IU of human chorionic gonadotropin (hCG, LG Chem, Seoul, Korea) 48 hours after ovulation. And mating between over-ovulated female and male ICR mice was initiated with hCG injection. Female mice were sacrificed by cervical dislocation 18 h after hCG injection and the fallopian tubes transferred to a Petri dish containing DPBS with 0.5% (w/v) bovine serum albumin (BSA; Sigma-Aldrich) added. Subsequently, harvesting of the conjugate surrounded by the oocytes in the fallopian tube was mechanically performed from the fallopian tube bulge in M2 medium (Sigma-Aldrich), and the oocytes were 0.2% (w/v) hyaluronidase (Sigma-Aldrich). ) Was removed from the recovered conjugate by treatment at 37° C., 5% CO ₂ for 1 minute in added M2 medium. After washing with fresh M2 medium, the zygote with the second pole and pronucleus was incubated in M16 medium (Sigma-Aldrich) at 37°C and 5% CO ₂ conditions. Was transferred to M16 medium supplemented with 3 mg/ml BSA and incubated at 37° C., 5% CO ₂ . The blastocysts were then collected and used for the production of embryonic stem cells 138 hours after hCG injection.

Experimental Example  4: Outbred ICR  mouse In blastocyst Derived  Establishment and culture of embryonic stem cells

The clear stand of the collected blastocyst was mechanically removed with an insulin syringe under a stereo microscope (Olympus, Tokyo, Japan). The cleared blastocysts are 15% (v/v) heat-inactivated ES-qualified FBS (Welgene), 1% in DMEM on MEF feeder ( _ICR MEFs) cells derived from the fetus of non-subclinical ICR mice. (v/v) Antibiotic-antimycotic, 1% (v/v) non-essential amino acid (Invitrogen, Carlsbad, CA), 2 mM L-glutamine (Invitrogen), 0.1 mM beta-mercaptoethanol (Invitrogen ) And 1,000 units/ml mouse leukemia inhibitory factor (LIF), Chemicon International, Inc., Temecula, CA) in standardized embryonic stem cell culture medium at 37° C., 5% CO ₂ under conditions of 7 Incubated for 1 day and culture medium was changed daily. Subsequently, the grown colonies were mechanically cut with a glass capillary pipette, transferred onto fresh _ICR MEF feeder cells, and incubated with standard ESC culture medium at 37°C and 5% CO ₂ for 4 days. Proliferated colonies were dissociated with 0.05% trypsin-EDTA and subcultured on _ICR MEF feeder cells at 37° C., 5% CO ₂ and standard embryonic stem cell media for 4 days. Embryonic stem cells ( _ICR ESCs) derived from ICR mouse blastocysts exchanged the medium every 2 days, and continued to proliferate through the passage process every 4 days. In addition, self-replicating embryonic stem cells ( _E14 ESCs) (American Type Culture Collection, Manassas, VA) derived from 129/Ola mouse blastocysts exchanged medium every 2 days and standard ESC culture on _ICR MEF feeder cells at 4 day intervals The medium was maintained by passage under 37° C. and 5% CO ₂ conditions and was used as a positive control.

Experimental Example  5: gene expression analysis

Dynabeads® mRNA Direct ^TM Kit (Ambion, Austin, TX) according to the manufacturer's instructions for using the total mRNA extraction of embryonic stem cells and the synthesis of cDNA from the extracted mRNA is ReverTra Ace qPCR RT Master Mix with gDNA remover kit used (Toyobo , Osaka, Japan). Thereafter, a polymerase chain reaction (PCR) was performed using Premix Ex Taq ^TM Hot Start Version (TaKaRa Bio Inc., Oshu, Japan) under the following conditions: 5 min at 94° C. for initial denaturation Subsequently, for 30 seconds at 94° C., for annealing of each primer, the process was repeated 35 times at 60° C. for 1 minute and at 72° C. for 30 seconds. The final extension was performed at 72° C. for 10 minutes. PCR products were size fractionated by electrophoresis using a 1.5% (wt/v) agarose gel (Sigma-Aldrich), using a Gel Doc ^TM XR+ imaging system (Bio-Rad Laboratories, Hercules, CA). It was visualized.

For quantitative real-time PCR (qPCR), amplification of each gene was performed using THUNDERBIRD SYBR qPCR Mix (Toyobo) under a 7500 real-time PCR system (Applied Biosystems, Foster City, CA). PCR reactions were checked by confirming the melting curve data and specific gene expression was compared to Gapdh transcription level. Relative mRNA levels were calculated as 2 ^-ΔΔCt . (Ct=critical cycle for target amplification, ΔCt = Ct _target Gene (specific gene for each sample)-Ct _{international reference} (Gapdh for each sample). ΔΔCt = ΔCt _sample (processed sample in each experiment)-value in ΔCt _calibrator (control sample in each experiment). All primers used for PCR and qPCR were constructed based on cDNA sequences obtained from GenBank and Primer3 software for mice (Whitehead Institute/MIT Genome Research Center). The general information and sequences of the primers are shown in Table 1.

Experimental Example  6: Alkaline Phosphatase  (AP) dyeing

The cultured embryonic stem cells were fixed with 4% (v/v) formaldehyde (Junsei Chemical Co., Ltd, Ogu, Japan) for 15 minutes at room temperature and washed twice with DPBS. Immobilized cells in 0.1 M pH 8.2 Tris buffer (Duchefa Biochemie, Haarlem, The Netherlands) 0.2 mg/ml naphthol AS-MX phosphate (Sigma-Aldrich), 2% (v/v) N,N-dimethyl formamide (Sigma -Aldrich) (pH 7.4) and 1 mg/ml Fast Red TR salt (Sigma-Aldrich) added AP staining solution for 30 minutes at room temperature. Then, the stained cells were washed twice with DPBS and observed under an inverted microscope (CKX41; Olympus).

Experimental Example  7: immune cell chemistry

Embryonic stem cells fixed with 4% (v/v) formaldehyde were washed twice with DPBS. Next, endogenous peroxidase activity was blocked with Dako REAL ^™ Peroxidase-Blocking Solution (Dako, Glosrtup, Denmark) for 10 minutes at room temperature, and washed twice with PBS. After that, the embryonic stem cells were primary anti-Oct4, anti-Sox2, anti-Nanog and anti-AP (positive markers related to autoreplication in mouse pluripotent stem cells) diluted in 0.1% Triton X-100 (Sigma-Aldrich). Anti-TRA-1-60 and anti-TRA-1-81 (negative markers for autoreplication in mouse pluripotent stem cells) diluted in antibody and DPBS were stained for 30 minutes at room temperature with primary antibodies. After being washed twice with DPBS, the primary antibody was added to Dako REAL ^TM Envision ^TM /HRP, Rabbit/Mouse (ENV) (Dako) from Dako REAL ^TM EnVision ^TM Detection system, Peroxidase/DAB, Rabbit/Mouse (Dako) kit. Was detected at room temperature for 30 minutes. The stained ESCs were then washed twice with DPBS and visualized by treatment with Dako REAL ^™ DAB + Chromogen (Dako) for 10 minutes. The detailed information and dilution ratio of the primary antibody used are shown in Table 2.

Experimental Example  8: Embryoid body  ( EB ) Create and Ex vivo Trilobite  differentiation

Embryonic bodies (EBs) were formed using the'hanging drop' method. A 20 μL drop containing 500 _ICR ESCs was transferred to the lid of a 60 mm plastic culture dish (SPL), and the lid was turned over and placed on a culture dish filled with DPBS. Then, the cells were cultured at 37°C and 5% CO ₂ for 3 days. The resulting EB was then transferred to a 4-well culture dish and incubated for 7 days in basic MEF culture medium. EBs that spontaneously differentiated were stained with markers for neurofilaments (related to ectoderm), alpha-smooth muscle actin (related to mesoderm) and cytokeratin 18 (related to endoderm). Primary antibodies were detected using the Dako REAL ^TM EnVision ^TM Detection system, Peroxidase/DAB, Rabbit/Mouse according to the supplier's instructions. Detailed information and dilution ratio of the primary antibody used are described in Table 2 above.

Experimental Example  9: Teratoma ( teratoma ) Formation and Analysis

Cultured 1×10 ⁶ _ICR ESCs were injected subcutaneously into male 6 week old NCr nude mice. After 5 weeks, the teratoma formed at the subcutaneous site was recovered and fixed with 4% (v/v) paraformaldehyde (Sigma-Aldrich), treated with paraffin sections and stained with hematoxylin and eosin (H&E). Then, the stained tissue was analyzed histologically under a phase contrast microscope (BX53, Olympus).

Experimental Example  10: In vitro differentiation into eggs

_The previously described protocol (Hubner et al., 2003) was used to modify _ICR ESCs into oocytes in vitro. _ICR ESCs were cultured in 0.1% (w/v) gelatin coated plastic culture dishes in standard ESC media without MEF feeder cells and LIF. After 3 or 4 days, floating cells were removed and replaced with standard ESC medium without LIF. Subsequently, incubation of _ICR ESCs attached to the bottom of the gelatin coated culture dish in standard ESC medium without LIF was performed for an additional 3 days to form small aggregates in suspension. Aggregates were collected in 15 ml conical tubes (Hyundai micro Co., Seoul, Korea) and cultured using standard ESC medium without 400 µL of LIF and medium exchange was performed every 2 days. One month later, the presence of oocyte-like cells with a clear zone was monitored under an inverted microscope (CKX-41).

Experimental Example  11: doubling time and Colony  Measurement of size and number

_ICR ESCs were cultured for 4 days in a 37° C., 5% CO ₂ incubator using standard ESC medium on MEF feeder cells derived from fetuses of ICR, C57BL6 or B6CBAF1 strains. Then, the doubling time was calculated by t log ₂ /(log N _t -log N ₀ ), where t is the time until _ICR ESCs reach confluency, and N _t is confluence. The number of _ICR ESCs reached at any time, and N ₀ is the initial number of _ICR ESCs before incubation. To determine the size and number of _ICR ESCs colonies, cultured _ICR ESCs were fixed with 4% (v/v) formaldehyde for 15 minutes at room temperature and washed twice with DPBS. Thereafter, the size and number of _ICR ESCs were analyzed under an inverted microscope (CKX-41) using Image and Microscope Technology (IMT) solution software (IMT i-solution Inc., Vancouver, Canada).

Experimental Example  12: Flow cell  analysis

Cells were fixed in 4% (v/v) paraformaldehyde fixative. Cells were washed with DPBS, then anti-Oct4, Sox2 and Nanog (abnormal self-replication related markers) and Vasa (embryonic germ cell markers) diluted with DPBS added with 0.1% (v/v) Triton X-100 The antibody was stained at 4° C. for 45 minutes. Then, each primary antibody was detected by a secondary antibody conjugated with Alexa Fluor 488. Detailed information and dilution ratio of the primary or secondary antibody used are shown in Table 2 above. The stained cells were then washed with DPBS and analyzed by FACSCalibur (Becton, Dickinson and Co., Franklin Lakes, NJ). In addition, BD CellQuest Pro software (Becton, Dickinson and Co.,) was used for data analysis.

Experimental Example  13: Karyotype analysis

_ICR ESCs were incubated in a 0.1% (w/v) gelatin coated dish with standard ESC medium at 37°C. After incubation for 3 days, _ICR ESCs were treated for 3 hours in DMEM (high glucose) with 0.4 μg/ml demecolcine (Sigma-Aldrich) at 37° C. and treated with 0.05% (v/v) trypsin-EDTA. Was harvested. The harvested _ICR ESCs were then treated in 0.075 M KCl (Sigma-Aldrich) for 15 minutes and fixed with a 3:1 (v:v) methanol acetic acid solution. Immobilized _ICR ESCs were dropped on a heated slide and dried on a hot plate and stained with KaryoMAX Giemsa staining solution (Gibco Invitrogen, Grand Island, NY). Then, the stained slide was washed with distilled water, dried at room temperature, and observed under an inverted microscope (CKX-41).

Experimental Example  14: sex determination

Genomic DNA of _ICR ESCs was extracted using Genomic DNA Extraction Blood DNA Mini Kit (Favorgen Biotech Co., Pingtung County, Taiwan) according to the manufacturer's instructions. The extracted genomic DNA was amplified with primers for Zfy1 (Y-chromosome specific) and Xist (X-chromosome specific) using Premix Ex Taq ^TM Hot Start Version, and the PCR product was 1.5% (w/v) agar. Fractionated into Los Gel and visualized using Gel Doc ^™ XR+ imaging system. Detailed information on the X and Y chromosome primers is as described in Table 1 above.

Experimental Example  15: Microsatellite DNA analysis

The genomic DNA of MEFs and _ICR ESCs derived from non-suburban ICR, sub-system C57BL/6 and hybrid B6CBAF1 was extracted using the Genomic DNA Extraction Blood DNA Mini Kit, and the microsatellite marker was D1Mit16 (5'-AGAGTTAGCTGCCTAGCTTGAGTG-3', Primers and Premix Ex Taq ^{TM of} 5'- TGGAAAGATCTAGGGTTGTCAAAA-3'; Dietrich et al., 1992) and D17Mit124 (5'- TGTTGATGAGATCTTAAATCAGCC-3', 5'-TTTAACTAGTTGTTATTGCATGTGTG-3'; Rocha et al., 2004) It was detected using Hot Start Version. Next, PCR products were fractionated by 4% (w/v) agarose gel and visualized using Gel Doc ^™ XR+ imaging system.

Experimental Example  16: Statistical analysis

Statistical analysis of all numerical data was performed using Statistical Analysis System (SAS) software (SAS Institute Inc., Cary, NY). The significance test for the experiment was performed using the general linear model (PROC-GLM) included in the SAS package. Statistical significance was defined as the case where the p value value was less than 0.05.

Example  One: Non-suburban ICR  Mouse-derived embryonic stem cells ( _ICR ESCs ) Of all-round analysis

Of the 218 blastocysts produced in non-subclinical ICR mice, 115 blastocysts were attached to _ICR MEF feeder cells, and 106 colonies grew from the inner cell mass of blastocysts attached to 115 _ICR MEFs. However, only one embryonic stem cell from 106 grown colonies was successfully maintained until passage 14 times. Then, the characteristics of the established _ICR ESCs were analyzed between 15 and 20 passages.

Colonies of _ICR ESCs showed neat borders and dome-like morphology, similar to _E14 ESC colonies (top right in Figure 1) (Figure 1). Unlike the strong AP protein expression of _E14 ESC colonies (top right of B in Fig. 2) and activity (top right of C in Fig. 2), AP protein expression (B in Fig. 2) and activity in some of the _ICR ESC colonies (Fig. 2C) ) Was partially weakly observed.

In addition, the established _ICR ESCs showed the same expression pattern as _E14 ESC in transcription and translation of self-replicating genes. Positive expression and Tra-1 of Oct4 (E in Figure 4), Sox2 (F in Figure 4) and Nanog (G in Figure 4), with successful transcriptional expression of Oct4 , Sox2 , Nanog , Tert and AP (Figure 3) Negative expression of -60 (H in Figure 5) and Tra-1-81 (I in Figure 5) was detected in both _ICR ESCs (big picture in Figures 4 and 5) and _E14 ESCs (small picture in Figures 4 and 5) Became.

In addition, embryoid bodies formed in _ICR ESCs (FIG. 6) showed lineage-specific differentiation of endoderm, mesoderm, and ectoderm into trilobites. The spontaneously differentiated embryoid bodies are neurofilaments used as ectodermal markers (K in FIG. 7 ), alpha-smooth muscle actin used as mesodermal marker (L in FIG. 7) and cytokeratin 18 used as endoderm marker (M in FIG. 7 ). ). _ICR ESCs implanted in nude mice include simple columnar epithelial cells (endodermal lineage cells, N1 in FIG. 8), blood vessels (endodermal lineage cells, N2 in FIG. 8), simple cubic cells (endodermal lineage cells, N3 in FIG. 8), cartilage Cells (mesodermal lineage cells, N4 in FIG. 8), adipocytes (mesodermal lineage cells, N5 in FIG. 8) muscle cells (mesodermal lineage cells, N6 in FIG. 8), neural tube (ectoderm lineage cells, N7 in FIG. 8), Teratomas including germinal hair bulb like structure with pigmented cells on core region (ectoderm lineage cells, N8 in FIG. 8) and neural tissue (ectoderm lineage cells, N9 in FIG. 8) were formed.

Through the differentiation of _ICR ESCs into germ cells, oocyte-like cells with clear bands were successfully generated (FIG. 9, arrow head). The established _ICR ESCs were 40 normal diploid karyotypes (FIG. 10), and their sex was confirmed as females by confirming the presence of X chromosome specific Xist and absence of Y chromosome specific Zfy1 (FIG. 11).

The _ICR MEF feeder cells used in the ESC establishment process were then cultured for 14 days in standard ESC medium to confirm that the _ICR ESCs were derived from embryonic germ cells or pluripotent stem cells that may be present in _ICR MEF feeder cells. During the culture period, the formation of domed colonies in cultured _ICR MEF feeder cells (FIG. 12) was not detected, and for pluripotent stem cell specific proteins (Oct4, Sox2 and Nanog) and embryonic germ cell specific proteins (VASA). Positive cells were detected at a very low level of less than 1% (FIG. 13).

Through the above results, it was confirmed that the established _ICR ESCs were not derived from pluripotent stem cells or embryonic germ cells in the _ICR MEF feeder cell population. Furthermore, it was confirmed that self-replicating and pluripotent _ICR ESCs can be successfully established from the inner cell mass of blastocysts derived from non-subcutaneous ICR mice.

Example  2: _ICR ESCs  Customized MEF Feeder  Establish cell-based culture system

Next, in order to investigate the strain of MEF feeder cells that can efficiently support the in vitro maintenance of self-replication of _ICR ESCs, in MEF feeder cells derived from non-subsidiary ICR, sub-clinical C57BL/6 and hybrid B6CBAF1 The doubling time and colony size and number of cultured _ICR ESCs and the expression of self-replicating proteins were analyzed. As a result, _ICR ESCs cultured in _C57BL/6 MEFs were significantly longer doubling times (Figure 14), smaller colony size (Figure 15) and fewer colonies (Figure 16) than _ICR ESCs cultured in _ICR MEFs and _B6CBAF1 MEFs. ) And _no significant difference was detected between _ICR MEFs and _B6CBAF1 MEFs for each parameter. Moreover, there was no significant difference in the expression of _{autoreplication-} related proteins (Oct4, Sox2 and Nanog) between _ICR ESCs cultured in _ICR MEFs, _C57BL/6 MEFs, and _B6CBAF1 MEFs (Figure 17).

These results demonstrate that MEF feeder cells derived from ICR and B6CBAF1 mice are useful for maintaining autoreplication of embryonic stem cells derived from ICR mice. In addition, considering that the genetic background of the feeder cells used for in vitro culture is different from the embryonic stem cells to be cultured to avoid cell contamination, in vitro culture of _ICR ESCs of MEF feeder cells derived from B6CBAF1 mice It has been found that the use is suitable for maintenance of embryonic stem cell autoreplication. Subsequently, in Example 3 to be described later, cultivation of _ICR ESCs was performed on hybrid _B6CBAF1 MEFs from passage 21.

Example  3: _ICR ESCs  optimization MEF Feeder  Long-term culture in a cell-based culture system _ICR Characterization of ESCs

For in the long-term maintenance of the _ICR ESCs to check availability of the MEF feeder cell-based culture systems optimized for the _ICR ESCs, subculture 21st _ICR ESCs were cultured in hybrids _B6CBAF1 MEFs until the 34th subculture of long-term cultured _ICR ESCs Characteristics were analyzed between passages 35 to 40.

All colonies derived from _E14 ESCs (top right in Figure 18) and _ICR ESCs (Figure 18) exhibited neat borders and dome-like morphology. For AP protein expression and activity, strong AP protein expression (upper right B in FIG. 19) and activity (upper right C in FIG. 19) were detected in all colonies derived from _E14 ESCs, whereas partial in colonies derived from _ICR ESCs. And weak AP protein expression (FIG. 19B) and activity (FIG. 19C) were detected. Moreover, _no significant difference in expression at the transcription level of Oct4 , Sox2 , Nanog , Tert and AP successfully expressed in long-term cultured _ICR ESCs in _B6CBAF1 MEFs was observed compared to short-term cultured _ICR ESCs (Figure 20). . Positive expression and Tra-1-60 (H in Fig. 22) and Tra-1 for Oct4 (E in Fig. 21), Sox2 (F in Fig. 21) and Nanog (G in Fig. 21) in long-term cultured _ICR ESCs The negative expression pattern for -81 (I in FIG. 22) was identically detected in _E14 ESCs (small pictures in FIGS.

In spontaneous differentiation of embryonic bodies (FIG. 23) derived from long-term cultured _ICR ESCs, the endodermal marker neurofilament (K in FIG. 24), the mesodermal marker alpha-smooth muscle actin (L in FIG. 24), the endoderm marker cytokeratin 18 (M in Fig. 24) was detected.

In addition, _ICR ESCs cultured for a long time after transplantation into nude mice were composed of intestinal epithelial cells (endodermal lineage cells, N1 in FIG. 25), bilayered apocrine tubes (endodermal lineage cells, N2 in FIG. 25), and tubes consisting of simple cubic cells. (Endodermal lineage cells, N3 in FIG. 25), adipocytes (mesodermal lineage cells, N4 in FIG. 25), striated muscle (mesodermal lineage cells, N5 in FIG. 25), smooth muscle cells (mesodermal lineage cells, N6 in FIG. 25), Teratoma with keratinocytes (endodermal lineage cells, N7 in FIG. 25), neural bundles (ectoderm lineage cells, N8 in FIG. 25) and neural epithelium (ectoderm lineage cells, N9 in FIG. 25) were formed. .

Through the differentiation of long-term cultured _ICR ESCs into germ cells, oocyte-like cells with clear bands were successfully generated (FIG. 26, arrow head). 40 normal diploid karyotypes (FIG. 27) were observed in long-term cultured _ICR ESCs, and the presence and absence of X chromosome-specific Xist and Y chromosome-specific Zfy1 were observed in the genome, respectively, and consequently, females (FIG. 28). Moreover, the alleles detected by the microsatellite markers D1Mit16 and D17Mit124 in _ICR ESCs were equally observed in _ICR MEFs, whereas they _were different from those of _C57BL/6 MEFs and _B6CBAF1 MEFs (Figure 29).

The results clearly show that _ICR ESCs are derived from blastocysts of non-subsidiary ICR mice. Despite prolonged incubation of _ICR ESCs in _B6CBAF1 MEFs, _{autoreplication} , pluripotency and chromosomal normality were successfully maintained without any modification. Therefore, it was _{confirmed that} in vitro culture of _ICR ESCs in _B6CBAF1 MEFs is a MEF feeder cell based culture system optimized for established _ICR ESCs.

In the above, although the present invention has been illustrated and described in connection with specific preferred embodiments, the invention can be variously modified and changed within the limits that do not depart from the technical features or fields of the present invention provided by the following claims. Being present is obvious to those of ordinary skill in the art.

Claims (20)

(a) crossing outbred mice to obtain a fertilized egg in a zygote state;
(b) developing a fertilized egg in a zygote state to a 4-cell state;
(c) developing a four-cell fertilized egg into a blastocyst;
(d) removing the transparent zone of the blastocyst; And
(e) obtaining embryonic stem cell colonies by culturing a blastocyst with a clear band removed on fibroblast feeder cells derived from a non-subsidiary mouse fetus.
Including, non-sub-mouse mouse embryonic stem cells (embroynic stem cell) production method.
The method of claim 1, wherein the non-subsidiary mouse is an Institute of Cancer Research (ICR) mouse.
The method according to claim 1, wherein the fertilized egg in the zygote state of step (a) is obtained by sequential injection of pregnant mare's serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) into female non-subcutaneous mice.
The method of claim 1, wherein step (b) comprises culturing the fertilized egg in a zygote state in M2 medium.
The method of claim 1, wherein the step (c) comprises culturing a fertilized egg of a 4-cell state in M2 medium containing bovine serum albumin (BSA).
The method according to claim 1, wherein the transparent zone of the blastocyst is mechanically removed in the step (d).
The method of claim 1, wherein the culture of the blastocyst with the clear band removed in the step (e) is performed in DMEM medium containing fetal bovine serum (FBS) and mouse leukemia inhibitory factor (mLIF).
The method of claim 1, wherein the generated non-subsidiary mouse embryonic stem cells express characteristic markers of the embryonic stem cells.
The method of claim 8, wherein the characteristic marker of the embryonic stem cell is selected from Oct4 , Sox2 , Nanog, Tert and AP .
The method according to claim 1, wherein the generated non-subsidiary mouse embryonic stem cells are capable of differentiation into endoderm, mesoderm and ectoderm.
The method of claim 1, wherein the generated non-subsidiary mouse embryonic stem cells are capable of differentiation into germ cells.
The method of claim 1, further comprising sub-culturing the embryonic stem cell colonies obtained in step (e) on fibroblast feeder cells (MEF) derived from a non-subsidiary mouse fetus.
13. The method of claim 12, wherein the subculture comprises mechanically separating the colonies and transferring them onto new feeder cells.
A non-subsidiary mouse embryonic stem cell produced by the method of any one of claims 1 to 13.
Non-subsidiary mouse embryonic stem cells deposited with accession number KCTC13658BP.
A mouse germ cell differentiated from the non-subsidiary mouse embryonic stem cell of claim 15.
A method for sub-culture of a non-sub-mouse mouse embryonic stem cell, comprising the step of sub-culturing the non-sub-mouse mouse embryonic stem cell of claim 15 on fibroblast feeder cells derived from a hybrid mouse.
18. The method of claim 17, wherein the hybrid mouse is a B6CBAF1 mouse that is a progeny of a female C57BL/6 mouse and a male CBA mouse.
A method for subculture of non-sub-mouse mouse embryonic stem cells, comprising culturing the non-sub-mouse mouse embryonic stem cells of claim 15 on fibroblast feeder cells derived from non-sub-mouse mice.
20. The method of claim 19, wherein the non-subsidiary mouse is an ICR mouse.

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