JP7064732B2 - How to identify genes that are specifically expressed using optimized labels - Google Patents

Description

The present invention is a method for identifying a gene specifically expressed in a cell of a specific cell type, and a method for identifying the gene using a label selected as optimal for identifying the gene. Regarding.

Multicellular organisms such as mammals are formed from various different types of cells, and humans, which are said to have more than 210 types of cells, also contain more diverse cell types according to recent studies. Is becoming clear. The diverse cells that make up the body of these animals change from one cell type to another through continuous cell division from a single zygote and the process of "cell fate determination". .. Although it has not yet been completely elucidated how such a process is regulated, it is known that such changes in cell type are involved in changes in cell gene expression. (Non-Patent Document 1). In addition, a wide range of studies have been conducted to classify and isolate various cells that form multicellular organisms according to gene expression patterns, and are specific to various cells in order to identify and isolate cells with specific functions. It is also widely practiced to identify gene products expressed in (Non-Patent Document 2, Patent Document 1, Patent Document 2).

In particular, in recent years, regenerative medicine using stem cells and the relationship between stem cells and diseases such as tumors have been attracting attention. In somatic stem cells such as hematopoietic stem cells, stem cells having various functions depending on the gene expression pattern are mixed. (Non-Patent Document 3, Non-Patent Document 4). In order to identify and isolate stem cells having a specific function, a technique for identifying genes specifically expressed in these cell types is required.

Furthermore, in analyzing the functions of these various cells in vivo, it is extremely useful to be able to observe their gene expression in living cells, and it is extremely useful to observe the gene expression in living cells by flow cytometry or live cell fluorescence observation such as in situ fluorescence observation. Techniques for identifying genes that can analyze the expression state are expected to be widely useful from basic biological research to applied fields such as medicine.

JP-A-2014-233217 JP 2013-146198

Trapnel C, Genome Research, 2015, Vol. 25, pp. 1491-1498 Gazut R et al. , J. Exp. Med. , 2014, Vol. 211, no. 7, pp. 1315-1331 Morrison SJ and Weissman IL, Immunity, 1994, Vol. 1, pp. 661-673 Beerman I et al. , Proc. National Academy of Science USA, 2010, Vol. 107, no. 12, pp. 5465-5470 Christensen and Weissman, Proc. National Academy of Science USA, 2001, Vol. 98, no. 25, pp. 14541-14546)

It is an object of the present invention to provide a method for identifying a gene that is specifically expressed in a cell of a specific cell type and can be observed by fluorescence observation such as flow cytometry and in situ fluorescence observation. ..

The present invention further provides a method for identifying the gene using a fluorescent label selected in advance as optimal for identifying the gene. Furthermore, the present invention is a method for identifying a gene that is specifically expressed in somatic stem cells such as hematopoietic stem cells, particularly long-term hematopoietic stem cells, as cells of a specific cell type, and which enables the above-mentioned fluorescence observation. I will provide a.

As a result of diligent research to solve the above problems, the present inventors have conducted in advance a cell of a specific cell type of interest in identifying a gene specifically expressed in a cell of a specific cell type. The most suitable labeled fluorescent protein for observing fluorescence in the above is searched for, and when the fluorescent protein is used as a fluorescent label for a candidate gene, the transfer amount required for fluorescence to be detectable is obtained, and based on the transfer amount. By identifying a gene that is specifically expressed in a cell of the specific cell type, it is a gene that is specifically expressed in the cell of the specific cell type, and is fluorescent observation such as flow cytometry and in situ fluorescence observation. The present invention has been completed by finding that it is possible to efficiently obtain a gene capable of obtaining a gene.

More specifically, because fluorescent proteins have different excitation and fluorescence wavelengths, they require filters with different optical properties for fluorescence observations such as flow cytometry and in-situ fluorescence observations, thereby causing fluorescent proteins. The intensity of the background fluorescent signal that produces wild-type cells that do not express is also altered. In the above fluorescence observation, it is important that not only the absolute fluorescence intensity but also the difference from the background fluorescence signal intensity (relative fluorescence intensity) is large, and a fluorescent protein that gives a large relative fluorescence intensity is used as a labeling protein. By using it, it becomes possible to observe the expression of a gene having a lower transcription amount by fluorescence.

INDUSTRIAL APPLICABILITY The present invention is a method for identifying a gene that is specifically expressed in a cell of a specific cell type and whose gene expression can be observed by observation of living fluorescent cells, and is optimal for the purpose. A method for identifying the gene using the selected fluorescent label is provided. In particular, in order to analyze the function of these specific cell types in vivo, it is extremely advantageous to be able to observe the gene expression in living cells, and it is extremely advantageous to observe live cell fluorescence such as flow cytometry and in situ fluorescence observation. The present invention, which makes it possible to identify a gene whose expression can be analyzed by an observation / analysis method, is extremely useful.

FIG. 1 shows an outline of an experiment for identifying the optimal labeled fluorescent protein in a cell of interest. A virus having an expression vector encoding various labeled fluorescent proteins is infected with a target cell (for example, NIH3T3 is shown), and the expression of the labeled fluorescent protein is observed in the cells. FIG. 2 shows the results of observing the fluorescence of various labeled fluorescent proteins in NIH3T3 cells with a filter for AlexaFlour488-A. As a control, cells not infected with the expression vector were used. FIG. 3 shows the results of observing the fluorescence of various labeled fluorescent proteins in NIH3T3 cells with a filter for PE-Texas Red-A. As a control, cells not infected with the expression vector were used. FIG. 4 shows the results of single cell quantitative PCR to confirm the atypical geneticity of the Hoxb5, Rnf208 and Smtnl1 genes in HSC. Only the Hoxb5 gene exhibits bimodal signal intensity. FIG. 5 shows a gene targeting strategy for the production of Hoxb5-3-mCherry knock-in mice. UTR means untranslated region and PGK means phosphoglycerate kinase I. FIG. 6 shows an HSC (hereinafter referred to as “pHSC”) identified by an immune phenotype (Lin ^- c-Kit ⁺ Sca ^- 1 ⁺ CD150 ⁺ CD34- ^{/ lo} Flk2-), a pluripotent hematopoietic progenitor cell population. A (Lin ^- c-Kit ⁺ Sca-1 ⁺ CD150 ⁺ CD34 ^{+ Flk2-} ) (hereinafter referred to as "MPPa") and pluripotent hematopoietic progenitor cell population B (Lin ^- c-Kit ⁺ Sca-1 ⁺ CD150) ^-The results of flow cytometry for confirming the expression of the Hoxb 5-3-mCherry reporter gene in CD34 ^{+ Flk2-} ) (hereinafter referred to as "MPPb") are shown. Red indicates the signal of the Hoxb5-3-mCherry reporter gene, and blue indicates the signal generated by wild-type cells. FIG. 7 shows the tissue distribution of long-term hematopoietic stem cells (LT-HSC) expressing the Hoxb5-3-mCherry gene. Localization of Hoxb5-positive cells (red and arrow) and localization of VE-cadherin-positive cells (green) in 3D reconstructed images are shown. The scale bar represents 30 μm. FIG. 8 shows the frequency of Hoxb5-positive cells (n = 287 cells from n = 3 mice) plotted against the distance from VE-cadherin-positive cells, and the frequency of randomly selected positions (n = 287 cells). N = 600 cells from a mouse with n = 3). *** indicates P <0.0001. An independent student's t-test was performed. FIG. 9 shows the average number of Hoxb5-positive cells in the proximal, medial and distal tibia. NS indicates that it is not significant. An independent student's t-test was performed. FIG. 10 shows an outline of a long-term hematopoietic reconstruction test. CD45.1 ⁺ transplant accepting mice were subjected to lethal doses of irradiation and then competitively transplanted with 10 or 3 Hoxb5-3-mCherryHSC and ^2x105 cells of CD45.1 ⁺ / CD45.2 ⁺ sustentacular cells. .. As a secondary transplant, ^1x107 whole bone marrow cells (WBM) or 100 sorted LSK cells were transplanted from the primary transplant recipient animal. FIG. 11 shows a chimeric phenomenon for each cell lineage in the whole bone marrow of a secondary transplant-accepting mouse. Each bar graph represents an individual mouse (n = 6 for Hoxb5 negative mice, n = 7 for Hoxb5 low, and n = 8 for Hoxb5 high).

The present invention is, in one embodiment, a method for identifying a gene specifically expressed in a cell of a specific cell type, (A) a labeled fluorescent protein used for detecting or measuring the expression of the gene in the cell. The step of determining the optimally labeled fluorescent protein by screening using a cell line (B) based on the expression intensity of the reference gene labeled with the fluorescent protein in the cell of the cell type. A step of determining the observable transcription amount required for observing the expression of the gene to be identified with the labeled fluorescent protein by flow cytometry (C) based on the observable transcription amount. Step to obtain the required lower limit transfer amount and the smaller exclusion upper limit transfer amount,
(D) A gene having an expression exceeding the required lower limit transcription amount in a cell group containing the cell of the specific cell type and having an expression lower than the exclusion upper limit transcription amount in cells of another cell type. Process to identify,
The above-mentioned method is provided.

The exclusion upper limit transcription amount is based on the background fluorescence signal intensity produced by cells of the wild type, that is, the specific cell type that does not express fluorescent protein, and the gene expression level that is expected to bring about the same degree of fluorescence signal. Can be set.

The exclusion upper limit transcription amount may be 0.1 to 2.0 times the gene expression level expected to bring about the background fluorescence signal intensity. In addition, the exclusion upper limit transcription amount is 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, of the gene expression level expected to bring about the background fluorescence signal intensity. It may be 1.1, 1.2, 1.3, 1.4, 1.5 times.

In yet another embodiment, the invention determines the observable transcription volume in step (B) as "the number of fragments per 1 million read-out fragments and 1 kilobase of transcript" of the reference gene (. Provided is a method for identifying the above gene, which is obtained based on FPKM).

In still another embodiment, the present invention determines the required lower limit transcription amount in the step (C) by the following formula: required lower limit transcription amount> = observable transcription amount / number of copies of the labeled fluorescent protein encoded by the labeled nucleic acid. Provided is a method for identifying a gene.

The required lower limit transcription amount may be 1 to 10 times the number of copies of the observable transcription amount / labeled fluorescent protein encoded by the labeled nucleic acid. In addition, the exclusion upper limit transcription amount is 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, of the gene expression level expected to bring about the background fluorescence signal intensity. It was 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 times. You may.

In still another embodiment, the present invention provides a method for identifying the above gene, which comprises a step of further identifying the gene based on (E) the expression pattern of the gene obtained in the first step (D) for each cell. do.
The cell-by-cell expression pattern includes, but is not limited to, for example, those confirmed by fluorescence microscopy and variations in cell-by-cell gene expression within a cell population using single-cell quantitative PCR. ..

In still another embodiment, the present invention is a method for identifying the above-mentioned gene, which is applied to a group of candidate genes that have been subjected to primary screening in advance.
The primary screening is
a) A screening method for comparing gene expression in multiple cell groups belonging to the same cell lineage,
b) a screening method for comparing gene expression in cell groups of a plurality of different cell types belonging to the same anatomical space, or c) a screening method for combining the above two methods is provided.

In still another embodiment, the present invention increases the number of copies of the labeled fluorescent protein or the fluorescent protein when the expression level of the candidate gene is lower than the required lower limit transcription amount in the previous term in the step (C). Provided is a method for identifying the gene, which amplifies the intracellular signal of the protein encoded by the gene and lowers the required lower limit transcription amount by adding a cell localization signal sequence to the gene.

In yet another embodiment, the invention is that the particular cell type is a stem cell and the other cell type in step (D) is a pluripotent progenitor cell derived from the stem cell, or within the same cell lineage. Provided is a method for identifying the above-mentioned gene, which is a differentiated cell of the above.

In yet another embodiment, the invention is that the particular cell type is a long-term hematopoietic stem cell and the other cell type in step (D) is a pluripotent hematopoietic progenitor cell or differentiation within the same cell lineage. Provided is a method for identifying the above gene, which is a cell.

In yet another embodiment, the present invention is a method for identifying the gene, and further (F) a step (H) of transplanting a stem cell expressing the gene identified in the step (E) into a primary host animal. A step of transplanting a stem cell collected from the primary host animal into a secondary host animal, and (I) in the secondary host animal, a chimeric state in which the gene is expressed in a plurality of differentiated cells derived from the stem cell. The process of confirming that it is maintained,
The above-mentioned method is provided.

In yet another embodiment, the invention provides a method of identifying the gene in step (I), wherein the chimeric state is maintained for at least 16 weeks after transplantation.

In still another embodiment, the present invention is a method for identifying the gene, wherein the stem cells to be transplanted into the secondary host animal in the step (H) are obtained from the primary host animal 16 weeks or later after transplantation. I will provide a.

In still another embodiment, in the step (H), the stem cells are hematopoietic stem cells selected based on the Lin ^- c-Kit ⁺ Sca-1 ⁺ marker profile, and stem cells and tissue stem cells, respectively. Provided is a method for identifying the above gene, which is a cell population containing the above.

In still another embodiment, in the present invention, the cell group containing cells of a specific cell type in the step (D) is a marker profile of Lin ^- c-Kit ⁺ Sca ^- 1 ⁺ CD150 ⁺ CD34- ^{/ lo} FLK2-. Provided is a method for identifying the above-mentioned gene, which is a hematopoietic stem cell selected based on the above, and a cell population containing each of the stem cell and the tissue stem cell.

Explanation of Terms In the present invention, the “cell of a specific cell type” includes, but is not limited to, various differentiated cells, stem cells, tumor cells, and the like.

In the present invention, the "cells differentiated into various tissues" are, for example, epidermal cells, pancreatic parenchymal cells, pancreatic duct cells, hepatocytes, blood cells, myocardial cells, skeletal muscle cells, osteoblasts, skeletal myoblasts, and nerves. It includes, but is not limited to, cells, vascular endothelial cells, pigment cells, smooth muscle cells, fat cells, bone cells and the like.

In the present invention, "stem cells" include, but are not limited to, for example, embryonic stem cells, somatic stem cells, interstitial stem cells, hematopoietic stem cells, neural stem cells, cancer stem cells, artificial pluripotent stem cells and the like.

In the present invention, "somatic stem cells" include, but are limited to, for example, embryonic stem cells, somatic stem cells, interstitial stem cells, hematopoietic stem cells, neural stem cells, cancer stem cells, artificial pluripotent stem cells, and the like. Not done. In particular, hematopoietic stem cells, especially long-term hematopoietic stem cells, are preferred.

In the present invention, the "pluripotent progenitor cell" includes, but is not limited to, pluripotent glial progenitor cell, skin-derived pluripotent progenitor cell, pluripotent hematopoietic progenitor cell, and the like.

In the present invention, the "differentiated cell in the same cell lineage as the pluripotent hematopoietic progenitor cell" includes, but is not limited to, for example, leukocytes, erythrocytes, platelets and the like.

In the present invention, genes of various fluorescent proteins can be used as "fluorescent labels", for example, GFP, variants of GFP such as EGFP, hrGFP (Agilent), mCherry (Clontech), mKate2 (Wako), and the like. It includes, but is not limited to, AmCyan, ZsGreen, AsRedp, ZsYellow, mOrange, CFP-, mYFP, mRFP, mBFP, mPlum and the like.

In the present invention, "FPKM" means "the number of fragments per one million read fragments positioned and the number of fragments per kilobase of transcript", and when measuring the expression level of a gene by RNAseq using RNA. , A value commonly used by those of skill in the art in assessing the amount of transcription of a particular genomic region between different samples by normalizing total RNA expression and mapped readout fragments.

In the present invention, the "cell localization signal sequence" includes, but is not limited to, peptides that direct protein transport, such as nuclear translocation signal peptides and cell penetrating peptides.

Hereinafter, the present invention will be described in more detail by way of examples, but these are merely intended for illustration purposes, and the present invention is not limited to the examples described below. The contents of each of the documents cited below are incorporated herein by reference.

Example: Identification of a gene specifically expressed in long-term hematopoietic stem cells In the following examples, from the cell population contained in the bone marrow of a mouse, specifically among hematopoietic stem cells, especially long-term hematopoietic stem cells (LT-HSC). We identified genes that are expressed and whose expression can be observed by flow cytometry and institut fluorescence.

Selection of candidate genes By microarray analysis using the Gene Expression Commons platform, genes that are specifically expressed in hematopoietic stem cells (HSC) and whose expression is not observed in other cell types such as epithelial cells were obtained. As a result, 45 candidate genes were obtained.

For microarrays All microarray data used can be referenced in Gene Expression COMMONS (http://gexx.stanford.edu) and GEO GSE77078. Two to four microarray replications were evaluated for each individual cell population (Seita J et al., PLOS One, 2012, Vol. 7, e40321, Chan CK et al., Cell, 2015, Vol. 160. , Pp. 285-298, Chan CK et al., Proc. National Academia of Science. USA, 2013, Vol. 110, pp. 12643-12648).

Determining the Optimal Labeled Fluorescent Protein In order to obtain a gene specifically expressed in long-term hematopoietic stem cells (LT-HSC), we searched for the optimal fluorescent protein for gene identification in HSC.
The relative fluorescence intensity of each fluorescent protein was determined as follows.
Relative fluorescence intensity of fluorescent protein A and fluorescent protein B = Fluorescent intensity of fluorescent protein A / Fluorescent intensity of fluorescent protein B Fluorescent intensity of each fluorescent protein = Fluorescent intensity of cells into which each fluorescent protein of 1 copy has been introduced / Introduced the protein Fluorescence intensity of cells not used (wild strain) At this time, the characteristics of the filter used for observation differ depending on the excitation light of the fluorescent protein and the wavelength of fluorescence. Therefore, the fluorescence intensity of the wild strain differs depending on the type of fluorescent protein. Please be careful.
The outline of the experiment is shown in FIG. 1. Specifically, a plasmid in which the gene sequences encoding various fluorescent proteins were inserted into a lentiviral vector was prepared and purified by a usual method. These were gene-introduced into 293T cells together with a virus packaging plasmid and cultured at 37 ° C. for 2 days. The culture supernatant containing the produced virus particles was collected and used as a virus solution. These virus solutions were added to the NIH 3T3 cell culture medium to infect them, and the conditions under which the infection efficiency was 10% or less by the continuous dilution method were examined using a flow cytometer. Under these conditions, one copy of each cell will be integrated into the genome of the host cell. In this state, the intracellular biological fluorescence intensities of various fluorescent proteins were compared and examined by measuring the fluorescence intensity using a flow cytometer.
As shown in FIGS. 2 and 3, various fluorescent signals were obtained by combining various labeled fluorescent proteins and an observation filter.
The vector encoding the fluorescent protein "eGFP" (Cormac et al., Gene, 1996) was purchased from Addgene. The vector encoding the fluorescent protein "Venus" (Nagai et al., Nature Biotechnology, 2002) was purchased from RIKEN. The vector encoding the fluorescent protein "ZsGreen" (Welm et al., Cell Stem Cell, 2008) was purchased from Addgene. The vector encoding the fluorescent protein "Ai9" (Madisen et al., Nature Neuroscience, 2010) was purchased from Addgene. The vector encoding the fluorescent protein "mRubi2" (Lam et al., Nature Methods, 2012) was purchased from Addgene. The vector encoding the fluorescent protein "mCherry" (Shaner et al., Nature Biotechnology, 2004) was purchased from Addgene.
A BD flow cytometer (BD FACSAria) is used to measure the fluorescence intensity, and a BD filter (Alexa Floor 488-A filter: 530/30 BP) and a BD filter (PE-Texas Red-A filter) are used. : 610/20BP) was used for observation, and the fluorescence intensity was measured using the FlowJo software "FlowJo".
The results are summarized in Table 1.

As shown in Table 1, it was found that mCherry yielded the highest signal-to-noise ratio and the highest signal.

Determining the amount of observable transcription When the Bmi-1 gene is selected as the reference gene and the eGFP is used as a fluorescent label using mice in which Bmi-1-eGFP in which the gene of the fluorescent protein eGFP is fused to Bmi-1 is knocked in. It was confirmed that the expression of the Bmi-1 gene was observable in HSC by flow cytometry and in-situ fluorescence analysis. Next, the gene expression level of Bmi-1 in HSC was confirmed as the transcription amount expressed by "the number of fragments per positioned 1 million read fragments and the number of fragments per kilobase of the transcript" (FPKM). Based on the difference between the transcription amount and the relative fluorescence intensity of eGFP and mCherry, when a single mCherry protein is used as a fluorescent label for the product of the gene of interest in HSC cells, the gene can be observed fluorescently. It was clarified that the expression level needs to be 20 FPKM or more in order to be present.

Next, in order to reduce the expression level of the gene required for fluorescence observation, by using three mCherry as fluorescent labels, even if the gene has an expression level of about 7 FPKM, the fluorescence observation is theoretically performed. It was possible.

About RNA sequence reading RNA sequence reading was performed according to a known method (Moraga et al., Cell, 2015, Vol. 160, pp. 1196-1208). Briefly, total RNA isolated with trizol is treated with RQ1RNase-free DNase (manufactured by Promega) to remove trace amounts of DNA that may remain, and the RNeasy minorute column (QIAGEN). Was washed using (manufactured by). A cDNA library for pHSC, MPPa and MPPb was prepared using Ovation RNA-Seq System V2 (NuGen) and a 2x150 base pair (bp) paired end read fragment using HiSeq 2500 (Illumina), respectively. Got Using OLego (Wu J, et al., Nucleic Acids Research, 2013, Vol. 41, pp. 5149-5163), the raw transcriptome sequence data was mapped to the Mus musculus reference mRNA sequence to support the reference sequence. Transcript assembly was performed. Four replication experiments were performed on each cell population. The data can be referred to as NCBI SRP068593.

Determination of the required lower limit transfer amount and the exclusion upper limit transfer amount Among the HSCs, LT-HSC (Christensen and Weissman, PNAS, 2001, Vol. 98, no. 25, pp. In order to identify a gene specifically expressed in (14541-14546)), the minimum transcription amount that the candidate gene should have in LT-HSC is the "required minimum transcription amount", the maximum that may have in other HSC series. The transfer amount was determined as the "exclusion upper limit transfer amount". In this example, as HSC lineage cells, HSC (“pHSC”) identified by immune phenotype (Lin ^- c-Kit ⁺ Sca ^- 1 ⁺ CD150 ⁺ CD34- ^{/ lo} Flk2-), pluripotent hematopoiesis. Progenitor cell population A (Lin ^- c-Kit ⁺ Sca-1 ⁺ CD150 ⁺ CD34 ^{+ Flk2-} ) ("MPPa") and pluripotent hematopoietic progenitor cell population B (Lin ^- c-Kit ⁺ Sca-1 ⁺ CD150 ^- CD34 Using ^{+ Flk2-} ) (“MPPb”), the minimum transfer amount “exclusion upper limit transfer amount” that should be possessed in LT-HSC was set to 7 FPKM based on the above-mentioned observable transfer amount. Further, the maximum transfer amount "exclusion upper limit transfer amount" allowed in pHSC and MPPb may give the same fluorescence intensity as the background fluorescence intensity in which the wild type occurs when the observable transfer amount is determined. Based on the value calculated as the expected expression level, it was set to 2.5 FPKM.

Gene Identification Based on Expression Level Three genes, Hoxb5, Rnf208, and Smtnl1, were obtained as a result of screening genes using the Gene Expression Comics platform based on the above-mentioned exclusion upper limit transcription amount and exclusion upper limit transcription amount.

Identification of genes based on further expression levels Atypical geneticity has been reported for pHSC cells (Breaman I et al., Proc. National Academia of Science USA, 2010, Vol. 107, no. 12, pp. 5465. -5470, Yamamoto R et al., Cell, 2013, Vol. 154, pp. 1112-1126, Fathman JW et al., Stem Cell Reports, 2014, Vol. 3, pp. 707-715, Oguro H et al. , Cell Stem Cell, 2013, Vol. 13, pp. 102-116). Therefore, the candidate genes that are generally expressed in pHSC are expected to have various different expression levels in pHSC, and single-cell quantitative PCR was performed to confirm the expression status of the above three genes in each cell (Fig.). 4). As shown in FIG. 4, since only the Hoxb5 gene showed bimodal signal intensity, the Hoxb5 gene was further analyzed below.

About Single Cell qPCR Single cells for qPCR were treated with the Single Cell-to-CT qRT-PCR kit (manufactured by Life Technologies) as instructed by the kit. Cells were placed directly in degradation solutions on 96-well plates, subjected to reverse transcription reaction for cDNA synthesis, amplified by 14 cycles of TaqMan Gene Expression Assay and diluted with 1xTE buffer (pH 8.0). For real-time PCR, 50 cycles of 95 ° C., 3 seconds and 60 ° C., 30 seconds were performed using the following TaqMan probe to amplify the sample. Hoxb5: Mm00657672, Rnf208: Mm03039759-s1, Smtnl1: Mm00470338-m1, Gapdh: Mm99999915-g1. All thermocycles were performed by the 7900HT Fasr Real-Time PCT System (Applied Biosystems). The presence of a single cell was confirmed by a _Gapdh Ct value of 30 or less.

Flow cytometry and in situ fluorescence observation using Hoxb5 gene expression product To add a nucleic acid sequence encoding 3 copies of mCherry fluorescent protein to the Hoxb5 gene obtained by gene identification based on the above expression level by gene targeting. , A nucleic acid sequence containing a gene encoding a 3-copy mCherry fluorescent protein was inserted into the carboxy terminus of the endogenous Hoxb5 gene in ES cells (BRUCE-4) using a CRISPR vector (FIG. 5).

The nucleic acid sequence had a gene encoding 3 copies of mCherry fluorescent protein isolated by Porcine teschovirus-I 2A (P2A) and a CAAX membrane localized sequence (FIG. 5).

As a result of creating a chimeric mouse from the ES cells and observing the bone marrow cells obtained from the chimeric mice by flow cytometry, as expected, cells having fluorescence by the Hoxb5-3-mCherry fluorescent protein were found specifically in the HSC cells. Observed (Fig. 6).

In addition, as a result of in-situ fluorescence observation of the tibia of the chimeric mouse, it was confirmed that the cells expressing the Hoxb5-3-mCherry protein were uniformly distributed in the tibia along the blood vessels (FIGS. 7-9). ).

Gene Targeting and Creation of Chimeric Mice The gene constructs for targeting were pjYC vector (derived from pUC19. Golden gate insertion of TALE vector and removal of LacZ sequence (Sanjana et al., Nature Protocols, 2012, Vol. 7, pp.171-192)). Homological regions consisting of about 700 base pairs on the right and left sides were cloned from the genomic DNA of C57BL / 6J by PCR, and the entire sequence was confirmed by sequence reading. BRUCE-4 embryonic stem cells (ES cells) derived from C57BL / 6J (manufactured by Millipore), bicistronic containing a targeted construct and Cas9 and Hoxb5-specific single-stranded guide RNAs (5'-GGCUCCUCCGGAUGGCGCUCA-3'). The CRISPR vector PX330 (Cong Let al., Science, 2013, Vol. 339, pp. 819-823) was transduced. After homologous recombination, recombinant clones transduced with the EF1α-Cre-puromycin vector were positively selected with puromycin (1 μgml ^-1 ) for 2 weeks. Serially diluted ES cell colonies are collected, expanded and proliferated, and PCR and qPCR are used to confirm the presence or absence of site-specific gene transfer, eliminate those showing off-target effects, and select the correct copy number. rice field. After confirming the target site by sequence reading, chimeric mice were prepared using the correctly obtained ES cells. Chimeric mice were germline-transmitted by crossing with female C57BL / 6-Tyr ^c-2j / J mice.

Flow cytometry Flow cytometry and cell sorting were performed using FACS Maria II cell sator (manufactured by BD Bioscience) and analyzed using FlowJp software (manufactured by Tree Star). Bone marrow cells are 2% heat-treated inactivated fetal bovine serum (manufactured by Gibco) from both tibia, femur, humerus and pelvis, with Ca ²⁺ free and Mg ²⁺ free PBS added, generally using a dairy bowl and pelvis. And obtained by disrupting these in 2 mM EDTA. Cells were passed through 100 μm, 70 μm and 40 μm filters prior to measurement and sorting. To enrich the HSC and precursor cell population, cells were stained with APC-binding anti-c-Kit (2B8) and fractionated using anti-APC-magnetic beads and an LS column (both manufactured by Miltenyi Biotec). .. The c-Kit positive cells were then stained with a combination of antibodies against the following cell surface markers and cell lineage markers. Cell surface markers: Sca-1, Flk2, CD150, CD34, IL-7R and CD16 / 32. Cell lineage markers: Ter-119, B220, CD2, CD3, CD4, CD5, CD8a, Gr-1, CD11a, CD11b, CD41, CD48, CD229 and CD244. For lymph cell populations, bone marrow cells were stained with antibodies against CD3, CD4, CD5, CD8a, CD11b, CD11c, Gr-1, NH-1.1, Ter119, B220, c-Kit and F4 / 80. The myeloid cell (myeloid) population was stained with antibodies against CD3, CD3-, CD11b, CD11c, CD19, Gr-1, NH-1.1, Ter119 and F4 / 80. Staining with the antibody was performed at 4 ° C. and the cells were incubated for 30 minutes. Cells stained with CD34 were incubated for 90 minutes. Prior to cell analysis or sorting, cells were stained with SYSTEMX Red Dead Cell Stein (Life Technologies) according to the manufacturer's recommendations to assess cell viability. The transplanted cells were sorted twice to increase their purity.

About CUBIC Bone Marrow Imaging The bone removal protocol was modified from the original CUBIC protocol (Susaki EA et al., Cell, 2014 Vol. 157, pp. 726-739). Specifically, the tibia was collected, fixed in a 4% PFA solution for 2 days, and then the bone marrow plug was extracted from the distal side by an outflow method using a 25 gauge syringe. For nuclear staining, bone marrow plugs were dipped in DAPI / PBS solution and gently shaken at 35 ° C. for 3 days. For washing, the bone marrow plug was immersed in ScaleCUBIC-1 (Reagent-1) and gently shaken at 37 ° C. for 2 weeks. The solution was changed every 48 hours. In order to visualize the blood vessels, Alexa488-bound anti-mouse VE-cadherin antibody (BV13) was injected via a vein (posterior orbit), and the tibia was collected 30 minutes later. For imaging, the treated bone marrow plug was implanted in a 4 mm diameter glass capillary containing 2% agarose. The images were acquired by Zeiss Z1 lightsheet microscope (manufactured by Zeiss) and reconstructed into 3D images by Zen software (manufactured by Zeiss). The acquired 3D image was analyzed by Imaris software (manufactured by Bitplane). After eliminating abnormal values including events such as abnormal size (> 30 μm) or signal intensity, all other mCherry ⁺ (Hoxb5 ⁺ ) cells were analyzed in the tibia (n = total n = from 3 mice). 287 cells). The mCherry negative threshold was determined based on the signal intensity in the wild-type control tibial plug. These mCherry negative events with signal intensities of 0.002 to 75.998 are converted into a list of integers consisting of 75,000 values (75,000 is the length of the list), 600 of which are Excel 2015. It was randomly selected using the randomevent (175000) function (manufactured by Microsoft). Using a list of random points identified by these signal intensities, cell locations and distances to VE-cadherin-positive cells were measured using Imalis software. All CUBIC imaging experiments were performed by 3 replications using 3 mice.

Confirmation that the cells expressing the Hoxb5 gene are LT-HSC Bone marrow cell populations obtained from chimeric mice having Hoxb5 supplemented with the above 3 copies of mCherry fluorescent protein were transplanted into radiation-treated mice and further. A limit dilution analysis was performed in which the bone marrow cell population obtained from the transplanted mouse was transplanted to another radiation-treated mouse for two generations of transplantation (FIG. 10). As a result of peripheral blood analysis in the second generation transplant-accepting mouse, it was confirmed that the cells expressing the Hoxb5 gene were differentiated into various cells such as NK cells, B cells, T cells, granule leukocytes, and monocytes. (FIG. 11), it was confirmed that the cells expressing the Hoxb5 gene are LT-HSCs that function as hematopoietic stem cells for a long period of time.

Mice 8-12 week old male C57BL / 6J mice (Jackson Lab) were used as wild-type controls. B6 of 8-12 week old males. SJL-Ptprc ^a Pepc ^b / BoyJ mice were used as transplant-accepting mice. The sustentacular cells used in the competitive reconstitution assay were B6. Obtained from the crossing of SJL-Ptprc ^a Pepc ^b / _BoyJxC57BL / 6J (F1 mouse CD45.1 ⁺ / CD45.2 ⁺ ). Hoxb5-3-mCherry (C57B / 6J background) mice were used as donors for transplantation experiments.

Transplantation and peripheral blood analysis B6. Transplant-accepting mice of SJL-Ptprc ^a Pepc ^b / BoyJ mice (Jackson laboratory) were subjected to a single lethal irradiation of 9.1 Gy. For reconstitution analysis, transplant donated cells were placed in 200 μl PBS containing 2% FBS in ^2x105 bone marrow sustentacular cells (B6.SJL-Ptprc ^a Pepc ^b / _BoyJxC57BL / 6J F1 mouse CD45.1 ⁺ / CD45. It was mixed with .2 ⁺ ) and injected into the posterior orbital venous plexus. Peripheral blood analysis was performed 4, 8, 12 and 16 weeks after the primary and secondary transplants. At each time point, 50 μl of blood was drawn from the tail vessel and added to 100 μl PBS containing 2 mM EDTA. Red blood cells were then lysed using BD Pharma Lyse Buffer (manufactured by BD Pharmaningen) for 3 minutes on ice and subsequently by blocking with 5 μg / ml rat IgG according to the manufacturer's protocol. Leukocytes were stained with the following antibodies. CD45.1 (FITC), CD45.2 (PE), CD11b (BUV395), Gr-1 (Alexa-Flour700), B220 (BV786), CD3 (BV421), TCRβ (BV421) and NK-1.1 (PerCP) -Cy5.5). In each mouse, the percentage of chimeric phenomena in the peripheral blood of transplant-accepting mice was taken as the percentage of CD45.1 ^- CD45.2 ⁺ cells to the sum of CD45.1 ^- CD45.2 ⁺ and CD45.1 ⁺ CD45.2 ⁺ . Defined. Cells exhibiting more than 50% host chimera after 16 weeks of transplantation were excluded from the analysis to control the survival of host animals against lethal irradiation. The frequency of chimeric phenomena in peripheral blood was analyzed as follows. In order to evaluate the dynamics of the transplant-donated animal cells (CD45.1 ^- CD45.2 ⁺ ), after removing the fraction of the transplant-receptor animal cells (CD45.1 ^{- CD45.2-} ), the fraction of the transplant-donated animal cells. The frequency of the picture was calculated. The frequency of each cell line (NK cells, B cells, T cells, granulocytes and monocytes) was measured in the entire fraction of the transplanted animal cells. To assess the dynamics of contributions to the cell lineage, the frequency of transplant donor animal-derived cells (CD45.1 ^- CD45.2 ⁺ ) was calculated after fractionating each cell lineage. To rule out ambiguous cases, transplant recipients showing less than 1% chimeric phenomenon were treated as negative.

Genotyping Genomic DNA of Hoxb5-3-mCherry mice was obtained from a tail biopsy using QuickExtract solution (manufactured by EpiCenter). To distinguish between Hoxb5-3-mCherry mice and wild-type alleles, the same forward primer (5'-GACGTATCGAGAGTCGCCCAC-3') and two reverse primers (Hoxb5-3-mCherry mice: 5'-CCTTGGTCACCTCTCTCTG3') ´, Wild type: 5 ´-AGATTGGAAGGGTCGAGCTG-3 ´)

For critical dilution analysis, the frequency of long-term and short-term HSCs was calculated using transplantation data from 10 and 3 pHSC transplanted cells with high Hoxb5, low Hoxb5 or Hoxb5 negative. Mice that showed multi-series rearrangements (containing more than 1% of each cell line) over a long period of time (longer than 16 weeks) were counted as positive transplant recipients. By using the non-linear regression quasi-log fit straight line, the frequency of long-term HSC / short-term HSC at F0 was calculated to be _0.368 (using Graph Pad Prism 6).

Statistical analysis All comparative analyzes were performed using the independent student's t-test. Pearson's chi-square test was performed using online software (http://vassarstats.net/).

The present invention is a method for identifying a gene specifically expressed in a cell of a specific cell type, and the method for identifying the gene using a label selected as optimal for identifying the gene. I will provide a. In particular, in order to analyze the function of these specific cell types in vivo, it is extremely advantageous to be able to observe the gene expression in living cells, and it is extremely advantageous to use analysis methods such as flow cytometry and in situ fluorescence observation. The present invention, which makes it possible to identify a gene whose expression can be analyzed, can be applied to a wide range of cells as a method for identifying a gene that can be used for such analysis, and is extremely useful. ..
The present invention provides the following in one aspect.
[Item 1]
A method for identifying a gene that is specifically expressed in a cell of a specific cell type and whose expression of the gene can be observed by observing living fluorescent cells.
(A) The step of determining a labeled fluorescent protein used for detecting or measuring the expression of the gene in a cell, wherein the optimal labeled fluorescent protein is determined by screening using a cell line.
(B) Necessary for observing the expression of the gene to be identified by fluorescent observation when the gene to be identified is labeled with the labeled fluorescent protein based on the expression intensity of the reference gene labeled with the fluorescent protein in the cell of the cell type. The process of determining the observable transfer amount,
(C) A step of obtaining a required lower limit transfer amount and a smaller exclusion upper limit transfer amount based on the observable transfer amount.
(D) A gene having an expression exceeding the required lower limit transcription amount in a cell group containing the cell of the specific cell type and having an expression lower than the exclusion upper limit transcription amount in cells of another cell type. Process to identify,
The method described above.
[Item 2]
In item 1 in the step (B), the observable transcription amount is determined based on the "number of fragments per 1 million read fragments positioned and the number of fragments per kilobase of the transcript" (FPKM) of the reference gene. The method described.
[Item 3]
The method according to item 1 or 2, wherein in the step (C), the required lower limit transfer amount is obtained by the following formula.

Required lower limit transfer amount
> = Observable transcription amount / number of copies of labeled fluorescent protein encoded by the labeled nucleic acid

[Item 4]
(E) The method according to any one of items 1 to 3, further comprising a step of specifying the gene based on the expression pattern of the gene obtained in the first step (D) for each cell.
[Item 5]
The method according to any one of items 1 to 4, which is applied to a group of candidate genes for which primary screening has been performed in advance.
The primary screening is
a) A screening method for comparing gene expression in multiple cell groups belonging to the same cell lineage,
b) A screening method that compares gene expression in cell groups of different cell types belonging to the same anatomical space, or
c) The method, which is performed by a screening method that combines the above two methods.
[Item 6]
In the step (C), when the expression level of the candidate gene is lower than the required lower limit transcription amount in the previous term, the number of copies of the labeled fluorescent protein is increased, or the cell-localized signal sequence is added to the fluorescent protein. The method according to item 5, wherein the intracellular signal of the protein encoded by the gene is amplified and the required lower limit transcription amount is lowered.
[Item 7]
Items 1 to 6 wherein the specific cell type is a stem cell and the other cell type in the step (D) is a pluripotent progenitor cell derived from the stem cell or a differentiated cell within the same cell lineage. The method described in any one of the above.
[Item 8]
Items 1 to 7, wherein the specific cell type is a long-term hematopoietic stem cell, and the other cell type in the step (D) is a pluripotent hematopoietic progenitor cell or a differentiated cell within the same cell lineage. The method described in item 1.
[Item 9]
The method described in item 7 or 8.
moreover
(F) A step of transplanting a stem cell expressing the gene specified in the step (D) or (E) into a primary host animal.
(G) A step of transplanting stem cells collected from the primary host animal into a secondary host animal, and
(H) A step of confirming that the chimeric state in which the gene is expressed in a plurality of differentiated cells derived from the stem cells in the secondary host animal is maintained.
The method described above.
[Item 10]
9. The method of item 9, wherein in step (H), the chimeric state is maintained for at least 16 weeks after transplantation.
[Item 11]
The method according to item 9 or 10, wherein in the step (G), the stem cells to be transplanted into the secondary host animal are obtained from the primary host animal 16 weeks or later after transplantation.
[Item 12]
In the step (G), the stem cells are hematopoietic stem cells selected based on the marker profile of Lin ^- c-Kit ⁺ Sca-1 ⁺ , and a cell population containing stem cells and tissue stem cells, respectively, items 9 to 11. The method according to any one of
[Item 13]
The cell group containing cells of a specific cell type in the step (D) is a hematopoietic stem cell selected based on the marker profile of Lin ^- c -Kit ⁺ Sca-1 ^{+ CD150} + ^CD34- / ^lo FLK2- . The method according to any one of items 1 to 12, which is a cell population containing each of stem cells and tissue stem cells.

Claims (9)

A method for identifying a gene that is specifically expressed in a cell of a specific cell type and whose expression can be observed by observing live fluorescent cells. (A) Detection of expression of the gene in the cell. Alternatively, a step of determining a labeled fluorescent protein to be used for measurement, wherein the optimal labeled fluorescent protein is determined by screening using a cell line.
(B) Necessary for observing the expression of the gene to be identified by fluorescent observation when the gene to be identified is labeled with the labeled fluorescent protein based on the expression intensity of the reference gene labeled with the fluorescent protein in the cell of the cell type. The process of determining the observable transfer amount,
(C) A step of obtaining a required lower limit transfer amount and a smaller exclusion upper limit transfer amount based on the observable transfer amount.
(D) A gene having an expression exceeding the required lower limit transcription amount in a cell group containing the cell of the specific cell type and having an expression lower than the exclusion upper limit transcription amount in cells of another cell type. Process to identify,
Including
The method for determining a labeled fluorescent protein having a fluorescence signal intensity that causes a large difference with respect to the background fluorescence signal intensity in the step (A) as the optimal labeled fluorescent protein. In the step (B), the observable transcription amount is determined based on "the number of fragments per 1 million read fragments positioned and the number of fragments per kilobase of the transcript" (FPKM) of the reference gene, claim 1. The method described in. The method according to claim 1 or 2, wherein in the step (C), the required lower limit transfer amount is obtained by the following formula.

Required lower limit transcription amount> = Observable transcription amount / Number of copies of labeled fluorescent protein encoded by the labeled nucleic acid
(E) The method according to any one of claims 1 to 3, further comprising a step of specifying the gene based on the expression pattern of the gene obtained in the first step (D) for each cell. The method according to any one of claims 1 to 4, which is applied to a group of candidate genes for which primary screening has been performed in advance.
The primary screening is
a) A screening method for comparing gene expression in multiple cell groups belonging to the same cell lineage,
b) A screening method for comparing gene expression in cell groups of a plurality of different cell types belonging to the same anatomical space, or c) a screening method for combining the above two methods. In the step (C), when the expression level of the candidate gene is lower than the required lower limit transcription amount in the previous term, the number of copies of the labeled fluorescent protein is increased, or the cell-localized signal sequence is added to the fluorescent protein. The method according to claim 5, wherein the intracellular signal of the protein encoded by the gene is amplified to reduce the required lower limit transcription amount. Claims 1 to 1, wherein the specific cell type is a stem cell, and the other cell type in the step (D) is a pluripotent progenitor cell derived from the stem cell or a differentiated cell within the same cell lineage. The method according to any one of 6. 13. The method described in any one of the items. The cell group containing cells of a specific cell type in the step (D) is a hematopoietic stem cell selected based on the marker profile of Lin ^- c-Kit ⁺ Sca ^- 1 ⁺ CD150 ⁺ CD34- ^{/ lo} FLK2-. The method according to any one of claims 1 to 8 , which is a cell population containing each of stem cells and tissue stem cells.

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