KR20170005203A - Method for distinguishing pluripotent stem cells using double transgenic mice with dual fluorescent reporter system - Google Patents

Abstract

The present invention relates to a method for distinguishing pluripotent stem cell transgenic mice using a double fluorescent marker transgenic mouse. More particularly, the present invention relates to a method for producing a double fluorescent marker transgenic mouse by crossing an O4-DE-GFP mouse and an O4- And a double fluorescent marker transgenic mouse produced by the above production method. The present invention also provides a method for distinguishing pluripotent stem cells using the double fluorescent marker transgenic mouse. The double fluorescent marker mice developed in the present invention can differentiate cells from the existing Oct4-GFP mice. In other words, it can be used to differentiate GFP ⁺ , GFP ⁺ RFP ⁺ , and RFP ⁺ 3 pluripotent stem cells, and to develop culture conditions and factors necessary for complete and semi-pluripotency.

Description

[0001] The present invention relates to a method for differentiating pluripotent stem cells using double fluorescent marker transgenic mice with dual fluorescent reporter system,

The present invention relates to a method for discriminating pluripotent stem cells using a double fluorescent marker transgenic mouse.

Pluripotent stem cells (PSCs) are able to regenerate infinitely and have the ability to differentiate into all somatic and germ cells. PSCs can be derived from the inner cell mass (ICM) of the blastocyst or from the implanted epiblast cells. In vitro ( in In vitro , when ICM and epiblast cells are cultured in stem cell maintenance medium, they can grow into PSCs. Interestingly, the ICMs of blastocysts form "naive" embryonic stem cells (ESCs) and epiblast cells form "primed" epiblast stem cells cells; EpiSCs). In the cell ( in In vivo , semi-pluripotent stem cells (PSCs) have limited ability to differentiate, and they contribute little to chimeric formation by blastocyst injection analysis. Semi-pluripotent stem cells maintain stem cell function through basic fibroblast growth factor (bFGF) and Activin / Nodal signaling pathways, but not the STAT3 and BMP4 pathways. The two kinds of PSCs exhibit different molecular characteristics, but still share many important markers. One of the genes frequently expressed in these cells is Oct4 , which is a PSC and a reproductive cell marker. In addition to maintaining PSCs, Oct4 can convert differentiated cells to PSCs, also known as induced PSCs (iPSCs). The Oct4 gene contains three different cis-regulatory elements, the proximal promoter (PP), the distal enhancer (DE) and the proximal enhancer (PE) Have. The respective cis-regulatory elements (cis-regulatory elements) is in the regulation of Oct4 expression depending on the cell type, DE adjusts the Oct4 expression of a complete all-round cells, PE is known to control the Oct4 expression in a semi-round stem cells have.

Korean Patent Laid-Open No. 10-2012-0034184 (published Apr. 10, 2012)

It is an object of the present invention to provide a method for producing a dual fluorescent marker transgenic mouse by crossing an O4-DE-GFP mouse and an O4-PE-RFP mouse to produce a double fluorescent marker transfected mouse, Pluripotent stem cells).

In order to achieve the above object, the present invention provides a method for producing a double fluorescent marker transgenic mouse by crossing an O4-DE-GFP mouse and an O4-PE-RFP mouse.

The present invention also provides a double fluorescent marker transgenic mouse produced by the above method.

The present invention also provides a method for distinguishing pluripotent stem cells using the double fluorescent marker transgenic mouse.

The present invention relates to a method for distinguishing pluripotent stem cell transgenic mice from pluripotent stem cell transgenic mice. The double fluorescent marker mouse developed in the present invention can discriminate cells more diverse than those using conventional Oct4-GFP mice. In other words, use in the development of GFP ^+, GFP ⁺ RFP ^+, RFP ⁺ 3 kinds of by distinguishing pluripotent stem cells, fully pluripotent stem cells and semi-round culture conditions and factors necessary to differentiate the stem cells only fully functional and gender semi-round .

Figure 1 shows the production and dual Oct4 regulator of the Oct4 regulatory factor in a dual transgenic mouse (O4-DE-GFP / O4-PE-RFP). (A) Genetic map of wild-type endogenous Oct4 , Oct4- ΔPE-GFP (O4-DE-GFP) and and Oct4- ΔDE-RFP (O4-PE-RFP). (B) Expression patterns of O4-DE-GFP and O4-PE-RFP in pre-implantation embryos (2C ~ blastocyst). O4-DE-GFP / O4-PE-RFP pre-implantation embryo expressing only O4-DE-GFP. Scale bar = 20 μm. 0.0 > O4-DE-GFP < / RTI > and O4-PE-RFP in the embryo after (CH) (C) 6.5 dpc embryo, Scale bar = 100 μm; (D) 7.25 dpc embryo, Scale bar = 100 [mu] m; (E) 8.5 dpc embryo, Scale bar = 100 [mu] m; (F) 9.5 dpc embryonic and mobile PGCs, Scale bar = 100 [mu] m; (G) 10.5 dpc embryos and PGCs, Scale bar = 100 [mu] m; And (H) 13.5 dpc embryos and gonads; scale bar = 100 μm. Lt; RTI ID = 0.0 > O4-DE-GFP < / RTI > and O4-PE-RFP in testes (I and J) (I) 7 dpp testis and seminiferous tubules and (J) 4 week old adult mouse testes and seminiferous tubules, scale bar = 100 μm. (K) neurospheres did not express O4-DE-GFP or O4-PE-RFP. scale bar = 100 μm. Thus, the expression of O4-DE-GFP and O4-PE-RFP proved to be restricted to pluripotent cells and germ cells.
Figure 2 shows that the ESC culture conditions were Oct4 It affects the activity of the enhancer. (AB) O4-DE-GFP / O4-PE-RFP Induction of ESCs from blastocysts. O4-PE-RFP was first expressed in the establishment of ESCs. scale bar = 50 μm. (C) Fluorescence image of O4-DE-GFP / O4-PE-RFP ESCs in serum + LIF medium. scale bar = 100 μm. (D) Flow cytometric analysis of O4-DE-GFP ⁺ single cells or O4-DE-GFP ⁺ and O4-PE-RFP ⁺ cell ratios in serum + LIF medium. (E) O4-DE-GFP + single cell or O4-DE-GFP ⁺ and O4-PE-RFP ⁺ cells were collected, the serum + about 10 based on the LIF medium O4-DE-GFP ⁺ single cell or O4-DE Flow cytometric analysis of the ratio of GFP ⁺ and O4-PE-RFP ⁺ cells. (F) Fluorescence image of O4-DE-GFP / O4-PE-RFP ESCs in serum + LIF + 2i medium. scale bar = 100 μm. (G) O4-DE-GFP + single cell or O4-DE-GFP ⁺ and O4-PE-RFP ⁺ cells were collected, the serum + LIF + 2i medium at 10 based on O4-DE-GFP ⁺ single cell or O4 Flow cytometric analysis of the ratio of DE-GFP ⁺ and O4-PE-RFP ⁺ cells. (I) Fluorescence image of O4-DE-GFP / O4-PE-RFP ESCs in serum + LIF + 2i medium. scale bar = 100 μm. (J and K) Flow cytometry analysis on O4-DE-GFP ⁺ single cells or O4-DE-GFP ⁺ and O4-PE-RFP ⁺ cell ratios for 2 days cultured in N2B27 + LIF + . DE-GFP ⁺ single cells or O4-DE-GFP ⁺ and O4-PE-RFP ⁺ cell ratios derived from (L) N2B27 + LIF + 2i medium and cultured for 2 days in serum + LIF medium Analysis. Therefore, in order to maintain O4-DE-GFP ⁺ fully pluripotent stem cells, LIF + 2i supplemented with no serum should be cultured.
Fig. 3 shows that 2i-GFP positive cells cultured in N2B27 + LIF + 2i medium and GFP or GR positive cells cultured in serum + LIF medium exhibit different gene expression patterns. (A) Topographic map of the entire gene expression pattern for 2i-GFP ⁺ , GFP + RFP ^- (GFP) and GFP ⁺ RFP ⁺ (GR) cells. (B and C) Hierarchical clustering and MDS plot analysis showed that 2i-GFP positive cells were different from GFP or GR positive cells. (D) 2I-GFP and GFP positive cells, (E) 2i-GFP and GR positive cells, and (F) GFP and GR positive cells. (G) Results of thermal maps showing that GFP-positive cells express germ cell-associated genes including Wnt3a , Rhox5 , Rhox6 , Rhox9 , Nanos3 and Tcfap2c at a higher level than GR-positive cells. (H) 2i-GFP-positive cells express high expression of naive pluripotency-related genes, and GFP and GR positive cells express high expression markers. (I) universal markers (Oct4, Nanog, Sox2, Esrrb ), full (naive) universal markers (Rex1, Klf2, Klf4, Tcl1 , Tbx3, Prdm14) and differentiation markers (Dnmt1, Dnmt3a, Dnmt3b, T , Fgf5) Quantitative gene expression analysis of. Data were repeated three times ( n = 3) and expressed as mean ± SD.
FIG. 4 shows that Oct4- .DELTA.DE-RFP positive cells constitute primed pluripotent stem cells through gene expression patterns and womens genetic status. (A) Fluorescence image of EpiSC-like cells (EpiLCs) derived from O4-DE-GFP / O4-PE-RFP ESCs. EpiLCs express Oct4- DELTA DE-RFP but not Oct4- DELTA PE-GFP. cale bar = 100 [mu] m. ^{(B) 2i-GFP +,} GFP + RFP - (GFP +), GFP + RFP + (GR), GFP - RFP + (RFP) cells, ESCs, also open the entire gene expression in EpiSCs and MEF. (C and D) Hierarchical clustering and MDS plot analysis showed that RFP-positive cells were more similar to EpiSCs than ESCs, 2i-GFP, GFP or GR positive cells. (E) EpiSCs and GFP positive cells, (F) EpiSCs and GR positive cells, and (G) EpiSCs and RFP-positive cells. (H and I) RFP-positive cells did not express naive pluripotency-related genes, but expressed primed universal-related genes. Data were repeated three times ( n = 3) and expressed as mean ± SD. (JN) Each Nanog , Stella , Dppa5 , LINE and IAP in 2i-GFP, GFP, GR and RFP- Results of bisulfate genomic sequencing of the region.
FIG. 5 is a graph showing changes in the intracellular content of Oct4- DE-RFP-positive cells ( In vivo developmental ability. (A) Aggregation of GFP, GR and RFP-positive cells with normal embryos. RFP-positive cells were not injected into the embryo. Scale bar = 50μm. (B) Coagulation efficiency of GFP, GR, and RFP-positive cells.
Figure 6 shows the womb genetic status of Oct4 regulators in naive and primed pluripotent stem cells. (A) ChIP-qPCR analysis to determine H3K27ac, H3K27me3 and H3K9me3 concentrations on Oct4 distal and proximal enhancers. Data were repeated three times ( n = 3) and expressed as mean ± SD. (B) Results of bisulfate genomic sequencing of the progenitor, proenzyme, proenzyme, and adjacent promoter region, which are located at each Oct4 position in 2i-GFP, GFP, GR, RFP positive cells and MEF. (C) Schematic representation of the regulatory factor of Oct4 in the process of pre-differentiation. (D) welfare genetic status of Oct4 enhancers in naive and primed universality.

The present invention provides a method for producing a double fluorescent marker transgenic mouse by crossing an O4-DE-GFP mouse and an O4-PE-RFP mouse.

Specifically, the O4-DE-GFP mouse lacks the proximal enhancer (PE) site of the Oct4 gene and under the control of the distal enhancer (DE), the green fluorescent protein (GFP) as a reporter protein.

In detail, the O4-PE-RFP mouse lacks the distal enhancer (DE) site of the Oct4 gene and under the control of the proximal enhancer (PE), the red fluorescent protein protein (RFP) as a reporter protein.

The present invention also relates to a method for expressing a green fluorescent protein (GFP) under the control of the distal enhancer (DE), which is a distal end of the Oct4 gene, or expressing the proximal enhancer (PE) Lt; / RTI > to provide a dual fluorescent marker transgenic mouse expressing red fluorescent protein (RFP).

The present invention also provides a method for discriminating pluripotent stem cells (PSCs) comprising measuring the degree of luminescence of RFP or GFP expressing stem cells of a double fluorescent marker transgenic mouse.

Specifically, the pluripotent stem cells may be naive pluripotent stem cells or primed pluripotent stem cells.

Specifically, the pluripotent stem cell differentiation method can be considered as a naive pluripotent stem cell when embryonic stem cells (ESCs) derived from the double fluorescent marker transgenic mouse are GFP ⁺ RFP ^- cells.

In detail, the pluripotent stem cell differentiation method is characterized in that EpiSC-like cells differentiated from epidermal stem cells (EpiSCs) or embryonic stem cells (ESCs) derived from the double fluorescent marker transgenic mouse express GFP ^- RFP ⁺ cells can be judged as primed pluripotent stem cells.

In the present invention, "GFP ⁺ RFP ^- cell" means a cell in which GFP expression is positive and RFP expression is negative, "GFP ^- RFP ⁺ cell" means a cell in which GFP expression is negative and RFP expression is positive do.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

The following experimental examples are intended to provide experimental examples that are commonly applied to the respective embodiments according to the present invention.

< Experimental Example >

One. O4 - DE - GFP  / O4 - PE - RFP  Mouse production

(Oct4-ΔDE-RFP) containing the red fluorescent protein (tdTomato) in the Oct4 high base sequence deficient in the distal enhancer (DE) was constructed to confirm the activity of the proximal enhancer (PE). The Oct4-ΔDE-RFP vector regulates the expression of the red fluorescent protein by the activity of proximal enhancers. These vectors were used to produce transgenic mice (O4-PE-RFP) using microinjection experiments. Existing O4-DE-GFP mice and O4-PE-RFP mice were crossed to produce double fluorescent marker mice. Through the fluorescence microscope, the activity of the distal and proximal enhancers during development in the body through the embryo obtained by crossing with the double fluorescent marker mouse and the general mouse was confirmed.

2. Double Fluorescent marker  Establish embryonic stem cells from embryos

The blastocysts obtained by crossing between the double fluorescent marker mice and the normal mice were induced to develop the blastocysts in vitro. These blastocysts were cultured on embryonic stem cell culture medium after culturing on a culture dish with supporting cells. Embryonic stem cell medium contains DMEM containing 15% calf serum, 1x penicillin / streptomycin / glutamine (Gibco), 1X Beta-mercaptoethanol and 1000 U LIF. Cells grown in support cells were separated and subcultured using trypsin / EDTA. Established embryonic stem cells were cultured in the presence of two inhibitors (CHIR99021 (3 μM) PD0325901 (1 μM)) in normal embryonic stem cell culture medium or with LIF and two inhibitors in N2B27 medium. N2B27 medium was mixed with DMEM and Neural basal medium 1: 1, N2 and B27 additives were added, and 1x penicillin / streptomycin / glutamine (Gibco) and 0.005% BSA were added.

3. From embryonic stem cells Embryonic stem cell  differentiation

Double fluorescent marker embryonic stem cells were cultured in N2B27 medium without or supplemented with supporting cells in the cervical stem cell differentiation medium containing activin A (20 ng / ml), bFGF (12 ng / ml) and KSR (1% Lt; / RTI &gt; The cervical stem cell differentiation medium was replaced every day. Embryonic stem cells expressing differentiated red fluorescent proteins were isolated and subcultured using collagenase (Collaganase).

4. RNA  Separation and quantitative RT - PCR  analysis

RNA was isolated using RNeasy Mini Kit (QIAGEN) and treated with DNA degrading enzyme to remove genomic DNA contamination. 1 μg of RNA was amplified using SuperScript III Reverse Transcriptase Kit (Invitrogen) and Oligo (dT) primer (Invitrogen) according to the manufacturer's instructions. Quantitative polymerase chain reaction (PCR) reactions were performed in duplicate with Power SYBR Green Master Mix (Dakara) and analyzed with Roche LightCycler 5480 (Roche). The base sequence used was as follows. Oct4 sense 5'-GATGCTGTGAGCCAAGGCAAG-3 ', Oct4 antisense 5'- GGCTCCTGATCAACAGCATCAC-3'; Nanog sense 5'-CTTTCACCTATTAAGGTGCTTGC-3 ', Nanog antisense 5'-TGGCATCGGTTCATCATGGTAC-3'; Sox2 sense 5'-CATGAGAGCAAGTACTGGCAAG-3 ', Sox2 antisense 5'- CCAACGATATCAACCTGCATGG-3'; Rex1 sense 5'-TCCATGGCATAGTTCCAACAG-3 ', Rex1 antisense 5'-TAACTGATTTTCTGCCGTATGC-3'; Esrrb sense 5'-CAGGCAAGGATGACAGACG-3 ', Esrrb antisense 5'-GAGACAGCACGAAGGACTGC-3'; Klf2 sense 5'-TCGAGGCTAGATGCCTTGTGA-3 ', Klf2 antisense 5'-AAACGAAGCAGGCGGCAGA-3'; Klf4 sense 5'-AGGAGCCCAAGCCAAAGAGG-3 ' , Klf4 antisense 5'-CGCAGGTGTGCCTTGAGATG-3'; Tcl1 sense 5'-TGGCCTCACTAGAACAAGAGG-3 ', Tcl1 antisense 5'-CTCGGTCAAGGATGGAAGC-3'; Tbx3 sense 5'-TTATTTCCAGGTCAGGAGATGGC-3 ', Tbx3 antisense 5'-GGTCGTTTGAACCAAGTCCCTC-3'; Prdm14 sense 5'-ACAGCCAAGCAATTTGCACTAC-3 ', Prdm14 antisense 5'-TTACCTGGCATTTTCATTGCTC-3'; Dnmt3a sense 5'-GACTCGCGTGCAATAACCTTAG -3 ', Dnmt3a antisense 5'-GGTCACTTTCCCTCACTCTGG -3', Dnmt3b sense 5'-CTCGCAAGGTGTGGGCTTTTGTAAC-3 ', and Dnmt3b antisense 5'-CTGGGCATCTGTCATCTTTGCACC-3', Dnmt3l sense 5'-CCAGGGCAGATTTCTTCCTAAGGTC-3 ' , and Dnmt3l antisense 5'-TGAGCTGCACAGAGGCATCC-3 ', T / Brachyury sense 5'-ATCAGAGTCCTTTGCTAGGTAG-3', and T / Brachyury antisense 5'-GTTACAATCTTCTGGCTATGC-3 ', Fgf5 sense 5'- AAAACCTGGTGCACCCTAGAAG- 5'-GCTAAACCGTCTGTGGTTTCTG-3 ', Fgfr1 sense 5'-CTACCAACCCTGTCCCCAGT-3', and Fgfr1 antisense 5'-CACAGGAAGGCCTCAGTCAG-3 ', Fgfr2 sense 5'-CAAGGAGCTCTTGTTCTTCAGG-3', and Fgfr2 antisense 5'-TAACACTGCCGTTTATGTGTGG-3 '.

5. DNA  Methylation analysis

DNA was isolated from each cell, and the bisulfite reaction was carried out using the EZ methylation kit bisulfite kit according to the manufacturer's instructions. And amplified by PCR using a primer set of a specific site. The base sequence used was as follows. Oct4 -DE sense 5'- TTTAGGTTTTAGAGGTTGGTTTTG-3 ', Oct4 -DE antisense 5'-CCAATTTCTATACATTCATTATAAAACAAT-3'; Oct4 -PE first sense 5'- GGTTTTTTGAGGTTGTGTGATTTAT-3 ', Oct4 -PE first antisense 5'- CTCCCCTAAAAACAACTTCCTACTC-3'; Oct4 -PE second sense 5'- GGGATTTTTATATGGGTTTAGAAAA-3 ', Oct4 -PE second antisense 5'- CTCCTCAAAAACAAAACCTCAAATA-3', Oct4 -PP first sense 5'-TTTGTTTTTTTATTTATTTAGGGGG-3 ', Oct4- PP first antisense 5'- ATCCCCAATACCTCTAAACCTAATC-3 '; Oct4 -PP second sense 5'-GGGTTAGAGGTTAAGGTTAGAGGG-3 ', Oct4 -PP second antisense 5'- CCCCCACCTAATAAAAATAAAAAAA-3'; Nanog first sense 5'- TTTGTAGGTGGGATTAATTGTGAA-3 ', Nanog first antisense 5'- AAAAAATTTTAAACAACAACCAAAAA-3 ', Nanog second sense 5'- TTTGTAGGTTGGGATTAATTGTGAA-3', Nanog second antisense 5'- AAAAAAACAAAACACCAACCAAAT-3 '; Stella first sense 5'- TTTTTTTATTTGTGATTAGGGTTG-3 ', Stella first antisense 5'- CTTCACCTAAACTACACCTTTAAAC-3'; Stella second sense 5'- TTTGTTTTAGTTTTTTTTGGAATTGG-3 ', Stella second antisense 5'- CTTCACCTAAACTACACTTTAAAC-3', Dppa5 first sense 5'- GGTTTGTTTTAGTTTTTTTAGGGGTATA-3 ', Dppa5 first antisense 5'- CCACAACTCCAAATTCAAAAAAT-3'; Dppa5 second sense 5'-TTTAGTTTTTTTAGGGGTATAGTTTG-3 ', Dppa5 second antisense 5'- CACAACTCCAAATTCAAAAAATTTTA-3 ', LINE sense 5'- TCAAACACTATATACTTTAACAATTCCCA-3', LINE antisense 5'-CCCCCACCTAATAAAAATAAAAAAA-3 '; IAP first sense 5'- TTGATAGTTGTGTTTTAAGTGGTAAATAAA-3 ', IAP first antisense 5'- AAAACACCACAAACCAAAATCTTCTAC-3 ', IAP second sense 5'- TTGTGTTTTAAGTGGTAAATAAATAATTTG-3', and IAP second antisense 5'- CAAAAAAACACACAAACCAAAAT -3 '.

The base sequence amplified by PCR was separated using agarose gel (1%) and cloned into T vector. The methylation of the replicated T vector was determined by sequencing.

6. Luminescent enzyme analysis

To determine the activity of the Oct4 enhancer, two kilobates of the Oct4 topoisomerase, each lacking a distal enhancer or lacking a proximal promoter, were cloned into the pGL3 base vector. The pGL3-Oct4Δ distal enhancer and the pGL3-Oct4Δ progenitor progenitor were prepared in two steps. First, the distal enhancer 5 ', the proximal hamster 5' fragment was PCR amplified from the pOct4-GFP plasmid via specific primers, cut with KpnI and MluI restriction enzymes, and replicated into the pGL3 basic vector. The proximal hamster 3 'and proximal hamster 3' fragments were PCR amplified from the pOct4-GFP plasmid via a specific primer, cleaved with MluI and BglII restriction enzymes, and amplified with pGL3-distant hamster 5 'or pGL3- 'Plasmid. Luminescent enzyme assays were performed using the Dual-Luciferase Reporter Assay System (Promega, USA). For the indicator of Oct4 enhancer activity, the pGL3-Oct4 delta enhancer, pGL3-Oct4-delta pro-nuclear factor, was transfected into each cell. After 48 hours of transfection, the growth medium was removed and the cells were rinsed with 1x phosphate buffered saline. Then, the cells were dissolved in 1x lysis solution and incubated with shaking at room temperature for 10 minutes. The cell lysate was transferred to a 1.5 milliliter tube and centrifuged at 10,000 revolutions per minute for 5 minutes. After transferring 10 microliters of the supernatant to a 96-well culture dish, luminescent enzyme expression was analyzed by light emission. Each experiment was repeated three times and the values were recorded in relative light emitting units.

7. Chromatin Immune sedimentation reaction

The cultured cells were cross-linked with 1% formaldehyde and washed with phosphate-buffered saline containing protease inhibitor. Genomic DNA extraction and chromatin immunoprecipitation were performed using the SimpleChIP Plus Enzymatic Chromatin IP Kit (Cell Signaling), a chromatin immunoprecipitation kit for simple chromatin immune response. The antibodies used were H3K27ac (Abcam), H3K27me3 (Cell signaling) and H3K9me3 (Abcam). Chromatin Immunopathogenesis - The primers used in the quantitative PCR were Oct4-DE sense 5'-GGCTGCAGGCATACTTGAAC-3 ', Oct4-DE antisense 5'-AGGGCAGAGCTATCATGCAC-3'; Oct4-PE sense 5'-TCCTCCTAATCCCGTCTCCT-3 ', and Oct4-PE antisense 5'-GGACTCCGGTGTTCATCCT-3'.

8. Microarray  analysis

Labeled cRNA samples (750 ng) were mixed with the MouseRef-8 v2 Expression BeadChip, respectively. Signal detection was performed with Amersham Fluorolink Streptavidin-Cy3 (GE Healthcare Bio-Science).

The original data was extracted using Illumina GenomeStudio v2011.1, Gene Expression Module v1.9.0 software. The array data was filtered with a p-value of 0.05 or less in at least 50% of the samples.

9. Agglutination with normal embryo

Embryonic stem cells or cyst epithelial-like stem cells aggregated with embryos of 8-year-old aphasia. 8-cell embryos were obtained from 2.5 dpc B6D2F1 female mice and cultured in mineral oil-covered embryo culture medium. Selected embryonic stem cells or cadaveric cortex-like stem cell masses (4-10 cells) were transferred to a microdrop containing 8-cell embryos free of zona pellucida. Embryonic stem cells or cadaveric embryonic stem cells were co-cultured at 37 ℃ and 5% CO ₂ .

< Example  1> In the process of mouse embryo development Oct4 Lt; RTI ID = 0.0 &gt; expression-specific &lt; / RTI &gt;

The present inventors produced double transgenic mice expressing GFP and RFP under the control of either DE or PE of Oct4 . The O4-DE-GFP mouse originally has the Oct4- DELTA PE-GFP transgene named OG2 and the O4-PE-RFP mouse has the Oct4- DELTA DE-RFP transgene (Figure 1A). O4-PE-RFP mice were crossed with homozygous O4-DE-GFP mice and then O4-DE-GFP ^+/- / O4-PE-RFP ^{+ / +} double transgenic mice were obtained (Figure 1A). ^{O4-DE-GFP +/- / O4} -PE-RFP + / + O4-DE-GFP through the mating of male mice and wild-type female mice ^+/- / O4-PE-RFP ^+/- gained embryos. 2-cell stage embryos did not express both GFP or RFP (Fig. 1B), consistent with the fact that the junctional genome is not activated at this stage. GFP was first detected in 8-cell embryos and was strongly expressed in the inner cell mass (ICM) of the blastocyst stage, whereas RFP was not detected in the blastocyst stage (FIG. 1B) Indicating that PE is unnecessary in the expression of Oct4 in the embryo.

Next, the present inventors confirmed the expression of O4-DE-GFP and O4-PE-RFP at the post-implantation stage (6.5 dpc to 13.5 dpc). 6.5 dpc epidermal cells (epiblast) expressed both GFP and RFP (Fig. 1C). At 7.25 dpc, the intensity of the GFP signal was reduced, but the RFP signal remained intact in the cyst epithelial cells (Fig. 1D). Primordial germ cells (PGCs) can not be distinguished at this stage. However, at 8.5 dpc, GFP-positive cells were located behind the embryos in which PGCs clustered and began to migrate to the genital ridge (Fig. 1E). At 9.5 dpc, GFP-positive cells were detected in the hindgut region (Fig. 1F). RFP-positive cells disappeared from somatic cells, but some of the cells in the retroperitoneal area expressed both RFP and GFP (about 34.7%), suggesting that migrating PGCs at 9.5 dpc were expressed in two populations, GFP ⁺ and GFP ⁺ / RFP ⁺ Indicating that it can be divided. At the 10.5 dpc stage, most of the PGCs expressed Oct4 -GFP and RFP as they got closer to the genital ridge (Fig. 1G). This was also the case when PGCs reached the gonads and proliferated (13.5 dpc; Fig. 1H).

Oct4 is expressed in prospermatogonia and type A spermatogonia where mitosis is stopped, but downregulated in type B spermatogonia and spermatocytes of adult testis Respectively. Expression of GFP and RFP was detected in seminiferous tubules of male transgenic testes at 7 dpp (Fig. 1I). Interestingly, GFP ⁺ and RFP ⁺ cells were detected in 4-week-old adult male mouse testes, whereas GFP ⁺ cells were located only around the seminiferous tubules (near basement membrane) and RFP ⁺ cells were seminiferous tubules ) (Fig. 1J). Immunohistochemical analysis revealed that GFP ⁺ cells were present around the tubules (type A garden niche) and RFP ⁺ cells were detected near the lumen of the tubule (differentiated germ cell site). These results are ex-works meiosis stage DE, and in, but the PE can use all four weeks old mouse type A spermatogonia of the adult testis, only DE Oct4 expression to express a type A spermatogonial cells Oct4 in (7 dpp) . Committed germ cells located near the lumen also expressed PE-regulated Oct4 .

The present inventors could not detect either GFP or RFP in neural stem cells (NSCs) (Fig. 1K), demonstrating that the transformation system of the present invention is specific to PSCs and germ cells.

< Example  2> Double fluorescence  Transformation A blastocyst  Used full ( naive Induction of pure populations of pluripotent embryonic stem cells

The present inventors have tried to demonstrate the heterogeneity of embryonic stem cells (ESCs) using the dual transfection system of the present invention and to derive a pure population of naive pluripotent embryonic stem cells. The blastocysts were inoculated on a feeder-layer vessel containing conventional mouse ESC medium (serum + LIF) (FIG. 2A and FIG. 2B). Initially, only GFP was expressed in ICM of blastocysts, but RFP was also expressed after 3 days. GFP ⁺ RFP ^- cells became GFP ⁺ RFP ⁺ cells when the ICM outgrowth expanded (FIG. 2B). These results support previous reports that bladder specificity is required for induction of universal ESC. Two other populations were cultured in serum ESC medium + LIF (GFP ⁺ RFP ^- and GFP ⁺ RFP ^+; Fig. 2C). GFP ⁺ RFP putative full (naive) PSCs ^- cells were composed only of only 45% of the population, GFP ⁺ RFP ⁺ cells were composed of 55% of the population (Fig. 2D), GFP ^- RFP ⁺ cells was observed . When GFP ⁺ RFP ^- cells were collected and re-grown in serum + LIF medium, about 82.9% of the cells returned to GFP ⁺ RFP ⁺ cells after 10 passages (FIG. 2E). In contrast, GFP ⁺ RFP ⁺ cells collected serum + LIF when cultured in a culture medium, after passage 10 about 3.7% of the GFP ⁺ cells of ^RFP-was converted to cells (Fig. 2E). These results demonstrate that ESCs are heterogeneous in serum + LIF medium, indicating that they contain populations with two compatible cell types. The GFP ⁺ RFP ⁺ and GFP ⁺ RFP ^- cell populations are indistinguishable using the Oct4 -ΔPE-GFP single-transduction system.

Next, we modified the culture conditions by adding two inhibitors (2i), ERK1 / 2 (PD184352) and GSK3 (CHIR99021) to serum + LIF medium. Under serum + LIF + 2i conditions, only 49.5% of the cells were GFP ⁺ RFP ^- and 50.5% were GFP ⁺ RFP ⁺ (FIGS. 2F and 2G). When GFP ⁺ RFP ^- cells were collected and cultured for 10 times under serum + LIF + 2i conditions, 12.5% became GFP ⁺ RFP ⁺ (Fig. 2H, left panel). Conversely, when GFP ⁺ RFP ⁺ cells were collected and cultured, they were converted to GFP ⁺ RFP ⁺ cells (58.6%), which was higher than that observed in serum + LIF culture conditions (FIG. 2H, right panel). These results indicate that serum + LIF + 2i culture conditions are insufficient to maintain GFP ⁺ RFP ^- cells. That is, ESCs are metastable in medium containing serum and are compatible between GFP ⁺ RFP ^- and GFP ⁺ RFP ⁺ cells. Next, mouse ESCs were shown to have elevated ERK1 / 2 phosphorylation levels, so we removed serum from the serum containing medium. Serum free ESC culture medium (N2B27 medium) supplemented with 2i with LIF20-22 was used for ESC culture. ESCs (from serum + LIF culture) were transferred to N2B27 + LIF + 2i medium. N2B27 + LIF + 2i move it to the culture medium on the second day (FIG. 2I), most of the GFP ⁺ RFP ⁺ GFP ⁺ cells RFP ^- was converted to cells (96.5%; Fig. 2J). When cultured further, almost all cells were maintained as GFP ⁺ RFP ^- cells (99%; Fig. 2K). Next, ESCs (derived from N2B27 + LIF + 2i medium) were transferred to serum + LIF medium. On day 2 of culture, almost all GFP ⁺ RFP ^- cells were converted to GFP ⁺ RFP ⁺ cells (90.7%; Fig. 2L). That is, serum-free culture conditions are essential for culturing a pure population of naive PSCs. In order to confirm that the expression of Oct4- DELTA PD-GFP and Oct4- DELTA DE-RFP appears correctly in the naive and primed PSC states, we used GFP ⁺ RFP ^- and GFP ⁺ RFP ⁺ The total gene expression patterns of the cells were compared. N2B27 + LIF + 2i (2i- GFP + RFP -) of GFP ⁺ RFP cultured ^in-gene expression profile of the cells it is cultured in serum-GFP ⁺ RFP + ^LIF-differed from the cells (Fig. 3A). Hierarchical clustering (hierarchical clustering), multi-dimensional scaling (multidimensional scaling; MDS) plot and ssangbyeol scatter analysis (pairwise scatter plot analyses) are GFP ^{+ RFP-GFP +} RFP ⁺ than ^cell-gene expression patterns 2i-GFP ⁺ RFP of cells Lt; / RTI &gt; cells (Figures 3B-3F). Differentially expressed genes in GFP ⁺ RFP ^- and GFP ⁺ RFP ⁺ cells are strongly associated with gonadal development. GFP ⁺ RFP ^- cells expressed Wnt3a , Rhox5 , Rhox6 , Rhox9 , Nanos3 , and Tcfap2c (Fig. 3G). The present inventors have found that GFP ⁺ RFP ^- it was confirmed that 249 genes were up-regulated in the cells and 446 genes that were down-regulated ^{cell-2i-GFP +} RFP on. Pou5f1 ( Oct4 ), Nanog , Zfp42 (Rex1), Esrrb, Sall4, and the expression level of all-purpose property such factors as the Lin28 is ^{2i-GFP + RFP -, GFP} + RFP - and GFP ⁺ RFP ⁺ (Fig. 3H) were almost same in the cell. However, complete (naive) is a universal markers, Klf4, Tcl1 and Tbx3 the GFP ⁺ RFP ^- was expressed in more cells ^- and GFP ⁺ RFP ⁺ cells than in the 2i-GFP ⁺ RFP. Conversely, Dnmt3b , brachyury (T), differentiation associated gene, such as Otx2 and Fgf5 is 2i-GFP ⁺ RFP ^- cells than GFP ⁺ RFP ^- and in GFP ⁺ RFP ⁺ cells was more significantly upregulate. The gene expression pattern was further confirmed by quantitative RT-PCR (qRT-PCR) analysis (Fig. 3I). These results indicate that GFP ⁺ RFP ^- cells under the serum + LIF culture conditions exhibit more naive properties (such as high expression of reproductive cell markers), but they are metastable and readily convert to GFP ⁺ RFP ⁺ cells . 2i-GFP ^{+ RFP-cells} are GFP ⁺ RFP cultured in containing a serum-free medium ^- different and is a cell, jyeoteumeuro found to express a larger number of full (naive) all-round markers, the stable full (naive) PSCs are But only in serum-free N2B27 + LIF + 2i medium.

< Example  3> Zune primed ) PSCs Representative of GFP ^- RFP ⁺  cell

The present inventors have attempted to identify pure populations of primed PSCs using a dual transfection system. During EpiSC induction from O4-DE-GFP ^+/- / O4-PE-RFP ^+/- cervical epithelial cells, early expression of GFP decreased and only RFP ⁺ cells expanded. Next, we sought to differentiate GFP ⁺ RFP ⁺ ESCs into GFP ^- RFP ⁺ EpiSC-like cells (EpiLCs). GFP ⁺ RFP ⁺ ESCs morphologically changed to form flat colonies of GFP ^- RFP ⁺ cells (Fig. 4A). Thermal mapping and hierarchical clustering analysis showed that gene expression patterns of GFP ^- RFP ⁺ cells were more similar to EpiSCs than ESCs (FIGS. 4B and 4C). MDS and scatter plot analysis, GFP ^- RFP ⁺ cells ^{2i-GFP +, GFP + RFP} - ⁺ RFP or GFP ⁺ cells were more similar than EpiSCs (Fig. 4D through 4G). These results indicate that the transfer of amplification activity from DE to PE is similar to the transition from the naive to the primed universal state. Next, we confirmed the expression of core universal, naive, universal and differentiation related genes in GFP ^- RFP ⁺ EpiLCs (FIG. 4H). Oct4 And Nanog were highly expressed in EpiLCs, EpiSCs and all ESC cell lines (Fig. 4H). However, GFP ^- RFP ⁺ EpiLCs were the expression of a very low level of perfection (naive) universal sex-related genes Klf2, Klf4, Klf5, Dppa3, Dppa4, Zfp42, Tbx3 and Tcl1, which was expressed at low EpiSCs. However, the differentiation related genes Krt18 , Fgf5 , Fgf8 , Otx2 , T ( brachyury ) and Nestin were highly expressed in GFP ^- RFP ⁺ EpiLCs and EpiSCs. qRT-PCR analysis revealed that the core universal gene ( Oct4) in GFP ^- RFP ⁺ EpiLCs and EpiSCs And Nanog) and EpiSC markers (high expression, and low expression of Sox2, Klf4 and Klf2 of T and Fgf5) was observed (Fig. 4I). These results indicate that GFP ^- RFP ⁺ EpiLCs are similar to EpiSCs in morphology and gene expression profiles.

Next, the present inventors have found that ^{GFP + RFP -, GFP + RFP} +, 2i-GFP + RFP - and GFP ^- in RFP ⁺ EpiLCs, Nanog, Stella ( Dppa3 ) and the Dppa5 promoter region were examined (Fig. 4J to Fig. 4L). Nanog The promoter region was hypomethylated in all samples (Figure 4J). However, Stella And the promoter region of the GFP Dppa5 ^- was methylated as in RFP ⁺ EpiLCs (Fig. 4K and 4L). LINE1 Region GFP ⁺ cells (GFP ⁺ RFP ^-, GFP ⁺ RFP ⁺ and GFP ⁺ 2i-RFP ^-) than GFP ^- were more methylated in the RFP ⁺ EpiLCs (FIG. 4M). IAP Region GFP ⁺ RFP ^- and 2i-GFP ⁺ RFP ^- cells than RFP ⁺ cells (GFP ⁺ RFP ⁺ and GFP ^- RFP ⁺ EpiLCs) were more methylated (Fig. 4N). Taken together, these results indicate that the O4-PE-RFP ⁺ reporter is a applicable primed PSC marker.

Next, GFP ^- RFP ⁺ EpiLCs as well as GFP ⁺ RFP ^- sikyeotgo the aggregation, GFP ⁺ RFP ⁺ cells with the wild-type 8-cell embryos were cultured to the blastocyst stage. GFP ⁺ RFP ^- RFP ⁺ and GFP ⁺ cells were effectively introduced into the ICM of a blastocyst (Fig. 6A and Fig. 6B). Interestingly, after aggregation day, it injected GFP ^{+ RFP-RFP} cells are simultaneously analogy GFP ⁺ RFP ⁺ cells was expressed (Fig. 5A). The aggregation efficiencies of GFP ⁺ RFP ^- and GFP ⁺ RFP ⁺ cells were 91.1% (41/45) and 93.3% (42/45), respectively (FIG. However, all GFP ^- RFP ⁺ EpiLCs failed to inject into the blastocyst ICM and the coagulation efficiency was 0% (0/27). Single agglutinated embryos of RFP ⁺ cells were observed, but GFP ^- RFP ⁺ cells were not identified in ICM (FIG. 5A and FIG. 5B). These results show that GFP ⁺ cells exhibit the characteristics of naive universality and that GFP ^- RFP ⁺ EpiLCs characterize primed universal-like EpiSCs.

< Example  4> complete naive ) And semi- primed ) PSCs in DE  And PE  Argumentative Welfare genetic status

Recent studies have shown that histone modification is closely related to the activity of enhancers. Acetylation of histone H3 lysine 27 (histone H3 lysine 27; H3K27) is a marker of the active inhibitor, and deacetylation of H3K27 is associated with reduced gene expression or a poised enhancer. In addition, H3K27me3 and H3K9me3 have been proposed as an indication of a prepared enhancer. The present inventors have found that by using a Chip-qPCR ^{analysis, 2i-GFP +, GFP +} RFP - examined the chromatin concentrated state of H3K27ac, H3K27me3 and H3K9me3 from RFP ⁺ cells (FIG. 6A) ^-, GFP ⁺ RFP ⁺ and GFP . H3K27ac all Oct4 - expressing cells, full ^{(naive) (2i-GFP +} ), metastability (GFP ⁺ RFP ^- and GFP ⁺ RFP ⁺⁾ and gave (primed) PSCs ^- of Oct4 in (GFP RFP ⁺⁾ DE and PE (Fig. 6A). The prepared poised enhancer markers, H3K27me3 and H3K9me3, were further enriched on PE rather than DE in 2i-GFP ⁺ cells. The levels of H3K27me3 on PE in metastable cells (GFP ⁺ RFP ^- and GFP ⁺ RFP ⁺ ) were slightly higher than DE, but the levels of H3K9me3 on DE and PE were not significantly different. Thus, the welfare genetic markers are likely to be highly variable in the metastable state, and the activity of PE and DE can not be distinguished by the histone mark. In primed universal GFP ^- RFP ⁺ cells, H3K9me3 was more enriched on DE than on PE, while levels of H3K27me3 were not different. These results show that the PE on H3K9me3 and H3K27me3 show a naive all-around state when enriched and a primed all-around state on enrichment of DE on H3K9me3. Since DNA methylation and histone modification both regulate gene expression and influence each other, the inventors Oct4 DNA methylation patterns of regulators were examined. The DNA methylation status of DE, PE and PP of Oct4 was analyzed by bisulfate DNA sequencing (Fig. 6B). Of Oct4 DE, PE and PP is complete (naive) PSCs (2i-GFP +), GFP + RFP - the RFP ⁺ and GFP ⁺ cells was completely non methylated. However, gave ^{(primed) PSCs (GFP - RFP} +) is naetneunde receive a relatively Methylation patterns at the Oct4 DE, which indicates that gave (primed) PSCs of Oct4 DE, as well as the H3K9me3 in that controlled by the DNA methylation (Figs. 6C and 6D).

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (7)

 O4-DE-GFP mouse and an O4-PE-RFP mouse to prepare a double fluorescent marker transgenic mouse. The O4-PE-RFP mouse according to claim 1, wherein the O4-PE-RFP mouse lacks the distal enhancer (DE) site of the Oct4 gene and is regulated by the proximal enhancer (PE) red fluorescent protein (RFP) as a reporter protein. The expression of the green fluorescent protein (GFP) under the control of the distal enhancer (DE) at the distal end of the Oct4 gene or the regulation of the proximal enhancer (PE) Dual fluorescent marker transgenic mice expressing red fluorescent protein (RFP). A method for differentiating pluripotent stem cells (PSCs) comprising measuring the degree of luminescence of RFP or GFP expressing stem cells of a double fluorescent marker transfected mouse according to claim 3. 5. The method according to claim 4, wherein the pluripotent stem cell is a naive pluripotent stem cell or a primed pluripotent stem cell. [5] The method according to claim 4, wherein the embryonic stem cells (ESCs) derived from the double fluorescent marker transfected mouse are GFP ⁺ RFP ^- cells, which are considered to be naive pluripotent stem cells A method for distinguishing allogenic stem cells. The method according to claim 4, wherein the pluripotent stem cell differentiation method comprises the step of culturing EpiSC-like cells differentiated from epiblast stem cells (EpiSCs) or embryonic stem cells (ESCs) Is a primed pluripotent stem cell when it is a GFP ^- RFP ⁺ cell.

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