阅读说明：本技术 一种粒细胞或单核细胞标记系统及其标记方法和应用 (granulocyte or monocyte marking system and marking method and application thereof ) 是由 弗洛伦特·津浩克斯 刘兆远 于 2018-07-20 设计创作，主要内容包括：本发明提出了一种粒细胞和单核细胞标记系统,所述标记系统包括Ms4a3基因、IRES-Cre重组酶和LoxP-Stop-LoxP-reporter报告系统。所述Ms4a3基因为粒细胞和单核细胞前体所特异性表达的基因,其序列如SEQ ID NO.1所示。本发明还提出了所述标记系统的构建方法以及检测方法。本发明还提出所述系统在造血系统发育、免疫细胞发育和功能、肿瘤学研究等方面的广泛应用。(The invention provides a marking system for granulocytes and monocytes, which comprises an Ms4a3 gene, an IRES-Cre recombinase and a LoxP-Stop-LoxP-reporter reporting system, wherein the Ms4a3 gene is a gene specifically expressed by granulocytes and monocyte precursors, and the sequence of the gene is shown as SEQ ID NO. 1.)

Use of the Ms4a3 gene as a granulocyte or monocyte precursor marker.

Use of the Ms4a3 gene as a marker in the process of labeling/detecting granulocyte or monocyte precursors.

3. Application of the reagent for detecting Ms4a3 gene in preparing in vitro diagnostic reagent for marking/detecting granulocyte or monocyte precursor.

A granulocyte or monocyte precursor marker system, wherein the marker system comprises a 5 'homology arm-IRES-Cre recombinase-3' homology arm, a LoxP-Stop-LoxP-reporter gene, wherein the sequence of the 5 'homology arm is shown as SEQ ID NO.2, and the sequence of the 3' homology arm is shown as SEQ ID NO. 3.

A seed cell or monocyte precursor marker system, wherein the marker system comprises Ms4a3 gene-IRES-Cre recombinase, LoxP-Stop-LoxP-reporter gene.

6. The granulocyte or monocyte precursor marker system of claim 5, wherein the Ms4a3 gene drives the expression of Cre recombinase, the Cre recombinase cleaves the Stop signal Stop sequence between two LoxP upstream of the reporter gene, thereby releasing the Stop signal Stop from the expression of the reporter gene, and the reporter gene is expressed, thereby completing the marking of the specific granulocyte or monocyte precursor.

7. The granulocyte or monocyte precursor marker system of claim 5, wherein the Ms4a3 gene is a gene specifically expressed by granulocyte or monocyte precursor GMP and has the sequence shown in SEQ ID No. 1.

8. The granulocyte or monocyte precursor marker system of claim 5, wherein the Cre recombinase is Cre recombinase derived from the P1 phage and has the sequence shown in SEQ ID No. 4.

A labeling composition of claim 9, , comprising the granulocyte or monocyte labeling system of claim 4.

An in vitro diagnostic reagent of comprising the granulocyte or monocyte marker system of claim 4 or 5 or the marker composition of claim 9.

11. Use of a granulocyte or monocyte precursor marker system as claimed in claim 4, or a marker composition as claimed in claim 9 for marking granulocytes or monocytes.

12/ method for labelling granulocytes or monocytic precursors, characterized in that it comprises the following steps:

step 1: introducing a granulocyte or monocyte marker system according to claim 4 or 5 into granulocytes or monocyte precursors;

step 2: the Ms4a3 gene drives the Cre recombinase to be specifically expressed in the cell;

and step 3: the Cre recombinase recognizes and cuts the Stop signal Stop sequence at the upstream of the reporter gene, thereby releasing the inhibition of the Stop signal Stop on the expression of the reporter gene;

and 4, step 4: reporter gene expression to complete the labeling of the cell;

and 5: detection by flow cytometry or fluorescence microscopy.

13.. method for constructing a marking system for granulocytes and monocyte precursors, said method comprising the steps of:

(1) the IRES-Cre sequence is grafted after the Ms4a3 gene of the granulocyte and monocyte precursor to form Ms4a3-IRES-Cre, and the expression of Cre recombinase driven by Ms4a3 is realized;

(2) the granulocyte and monocyte precursor marker system can be constructed by integrating Ms4a3-IRES-Cre with a LoxP reporter system.

Technical Field

The invention relates to the fields of hematopoietic system development, tissue macrophage renewal and tumor research related to granulocytes or monocytes, in particular to a marker system of granulocytes or monocytes, a marking method and application.

Background

Myeloid cells (Myeloid cells) including Granulocytes (Granulocytes), Monocytes (Monocytes), Macrophages (Macrophages), Dendritic cells (Dendritic cells) play an important role in anti-infectious immunity, inflammation, tissue repair and reconstruction, and tumors. Research on their origin and development will help to improve the understanding of the function of myeloid lineage cells in both physiological and pathological states. Manipulation of myeloid lineage cells is helpful in combating infection, alleviating inflammation, and treating cancer.

However, hematopoietic stem cells in bone marrow are mainly defined by hematologists, while terminally differentiated immune cells are defined by immunologists, gray areas exist between hematopoietic stem cells and terminally differentiated immune cells, where much of the developmental differentiation process is still not well defined. generally accepted view that Myeloid Progenitor Cells (CMP) develop in bone marrow producing Granulocyte-Monocyte precursors (GMP), GMP proceeds to producing Granulocyte precursors and Monocyte precursors (Monocytes and DC precursors, MDP) which in turn produce granulocytes, MDP producing Monocytes and dendritic cells, while development of sequencing techniques and new single cell dendritic cell models have been helpful for establishing a high-efficiency model of Myeloid development.

In addition, although the contribution of circulating monocytes to the macrophage population of tissues seems to be limited to specific tissues such as the intestine and dermis, the degree and dynamic changes of the mononuclear cells of bone marrow origin to the macrophages of different tissues remain controversial.

The marker for cells is the basis of studying cell development and function, and various marker systems have been developed since long, wherein the Cre/LoxP system is widely used for cell tracing and gene knockout studies by , which is a basic technology in the field, the technology comprises two parts of a Cre recombinase and a LoxP reporter system, the Cre recombinase is a DNA recombinase found in P1 bacteriophage, the Cre recombinase can recognize a specific DNA sequence, i.e., a LoxP sequence, and mediate deletion of a DNA fragment between two homologus LoxP sequences, the LoxP reporter system comprises a LoxP sequence, a Stop sequence and a reporter gene, the Stop sequence consists of a plurality of small Cre intron and SV40poly (a) Signal elements, which can inhibit expression of the downstream reporter gene, two LoxP sites exist at both ends of the Stop sequence, the Stop sequence inhibits expression of the downstream gene marker gene when no Cre recombinase exists, the cells cannot be identified by the Cre recombinase, the presence of two LoxP sites at both ends of the Cre sequence, and the fluorescent marker sequences can be inserted into cells, which can inhibit expression of the downstream marker gene, thus the expression of the red marker protein in cells, the fluorescent marker recombinase can be detected, and the fluorescent marker sequences can be detected when the expression of the red marker recombinase is detected, the fluorescent marker system, which can be detected by the fluorescent marker recombinase, which marker recombinase can be detected when the fluorescent marker recombinase is detected, which marker sequences can be detected by the fluorescent marker recombinase, which marker recombinase can be detected by the fluorescent marker recombinase is detected, which marker recombinase is detected by the fluorescent marker recombinase, which marker recombinase is detected by the cells, which marker recombinase is detected by the fluorescent marker recombinase, which is detected when the fluorescent marker recombinase is detected, which marker recombinase is detected by the fluorescent marker, the fluorescent marker recombinase is detected, the fluorescent marker, which marker, the fluorescent marker recombinase is detected by the fluorescent marker, which marker recombinase is detected by the fluorescent marker recombinase is detected by which marker, and which marker, the fluorescent marker recombinase is detected by which.

In the prior art, there is a major problem in that it is difficult to achieve control of the specific expression of Cre recombinase in granulocytes or monocytes. The main disadvantage of the existing labeling method is poor specificity, such as a Cx3cr1 tracing system, which labels monocytes and also labels dendritic cells and partial lymphocytes.

Disclosure of Invention

The present invention combines the specific promoter expressed in granulocyte or monocyte precursor (the gene specifically expressed in granulocyte or monocyte precursor) with the Cre/LoxP system to establish a high-efficiency specific monocyte and granulocyte tracing model, and the present invention combines the specific Ms4a3 gene expressed in monocyte-granulocyte precursor with the Cre/LoxP system for the first time, so as to successfully solve the problem that the existing technology can not realize the specific expression of Cre in monocyte and granulocyte, thereby realizing the specific recombinase marking on the two lineages of cells.

The Ms4a3 gene can be specifically expressed in granulocytes or monocytes, and based on the specific expression, the invention provides application of the Ms4a3 gene as a granulocyte or monocyte marker.

The invention provides application of Ms4a3 gene as a marker in the process of marking/detecting granulocytes or monocytes.

The invention provides application of a reagent of Ms4a3 gene in preparing an in vitro diagnostic reagent for marking/detecting granulocytes or monocytes.

The invention provides an seed cell or monocyte marking system, which comprises a 5 'homology arm-IRES-Cre recombinase-3' homology arm and a LoxP-Stop-LoxP-reporter gene, wherein the sequence of the 5 'homology arm is shown as SEQ ID NO.2, and the sequence of the 3' homology arm is shown as SEQ ID NO. 3.

The invention also provides an seed cell or monocyte precursor marker system, which comprises Ms4a3-IRES-Cre and LoxP-Stop-LoxP-reporter gene, wherein the Ms4a3-IRES-Cre recombinase is formed by fusing Ms4a3 gene sequence and IRES-Cre recombinase sequence.

Preferably, the granulocyte or monocyte marking system according to the present invention comprises Ms4a3 gene-IRES-Cre recombinase, LoxP-Stop-LoxP-tdTomato reporter gene.

In the cell expressing Ms4a3, Ms4a3 gene drives the expression of Cre recombinase, and Cre recombinase cuts a termination signal Stop before (upstream) of a reporter gene tdTomato, so that the termination signal Stop is released from preventing the tdTomato expression, and the tdTomato is expressed to generate red fluorescent protein, thereby completing the marking of the cell.

Preferably, the marking system can comprise a vector, wherein the vector comprises a Cre recombinase coding gene and LoxP-Stop-LoxP-tdTomato, or the vector comprises Cre-LoxP-Stop-LoxP-tdTomato, or preferably the marking system comprises th vector and a second vector, wherein the th vector comprises a Cre recombinase coding gene, and the second vector comprises LoxP-Stop-LoxP-tdTomato.

Wherein the Ms4a3 gene is specifically expressed in myelogranulocyte and monocyte precursors, and the sequence of the Ms4a3 gene is shown as SEQ ID NO. 1.

Wherein the Cre recombinase is Cre recombinase derived from P1 phage, and the sequence of the Cre recombinase is shown as SEQ ID NO. 4. The expression of Cre recombinase is regulated by Ms4a3 gene and is synchronous with the expression of Ms4a3, the Cre recombinase is specifically expressed in granulocyte or monocyte precursor GMP, and the Cre recombinase can be fused with estrogen receptor to form a creERT2 inducible recombination system.

Wherein the IRES is a ribosome internal entry site, and the sequence is shown as SEQ ID NO. 5.

The LoxP-Stop-LoxP-reporter gene comprises LoxP, Stop and a reporter gene element, wherein the LoxP sequence is a DNA sequence which can be recognized by Cre recombinase, the Cre recombinase can cut off the DNA sequence between two homonymous LoxP, the Stop is DNA sequences, the Stop can prevent the expression of the following gene, the expression of the reporter gene is inhibited under normal conditions due to the existence of the Stop sequence, when the Cre recombinase exists, the Stop sequence between two LoxP is cut off, the inhibition of the reporter gene by the Stop is released, and the reporter gene begins to be expressed, wherein the reporter gene can be tdTomato or a gene coding for other fluorescent proteins, such as GFP, YFP and the like, and preferably, the LoxP-Stop-LoxP-reporter gene, wherein the reporter gene is tdTomato element.

Wherein the sequence of the LoxP is shown as SEQ ID NO. 6.

Wherein the sequence of Stop is shown as SEQ ID NO. 7.

Wherein the sequence of tdTomato is shown as SEQ ID NO. 8.

Wherein the sequence of the Rosa gene (Gt (ROSA)26Sor) is shown in SEQ ID NO. 9.

Preferably, the present invention proposes a technical solution for selecting the labeling of granulocytes and monocytic precursors, by which nearly all granulocytes, monocytes and other cells of monocytic origin, including dendritic cells and macrophages, can be efficiently labeled.

The present invention also proposes marker compositions comprising a granulocyte or monocyte marker system as described above.

The invention also provides in vitro diagnostic reagents, which comprise the granulocyte or monocyte marking system or the marking composition.

The invention also proposes the use of a granulocyte or monocyte precursor marker system as described above, or of a marker composition as described above, for marking granulocytes or monocytes.

The invention also provides a method for labeling granulocyte or monocyte precursor, which comprises the following steps:

step 1: introducing a granulocyte or monocyte marker system according to claim 4 into a granulocyte or monocyte precursor;

step 2: the Ms4a3 gene drives the Cre recombinase to be specifically expressed in the cell;

and step 3: the Cre recombinase recognizes and cuts the Stop signal Stop sequence at the upstream of the reporter gene, thereby releasing the inhibition of the Stop signal Stop on the expression of the reporter gene;

and 4, step 4: reporter gene expression to complete the labeling of the cell;

and 5: detection is performed by flow cytometry, fluorescence imaging or other corresponding detection means.

The invention also provides a construction method of the seed cell and monocyte precursor marker system, which comprises the following steps:

(1) determining the Ms4a3 gene as a gene specifically expressed in granulocytes and monocyte precursors;

(2) an IRES-Cre sequence is grafted after the Ms4a3 gene to form Ms4a3-IRES-Cre, so that Ms4a3 is driven

Expression of the Cre recombinase;

(3) integrating Ms4a3-IRES-Cre with a LoxP reporter system (LoxP-Stop-LoxP-tdTomato reporter gene), the granulocyte and monocyte marker system can be constructed to realize cell tracing.

Wherein, in the step (2), the IRES-Cre sequence may be constructed on the th vector, and the granulocyte and monocyte precursor may be introduced.

In step (3), the LoxP reporter system may be constructed on -th vector, and the granulocyte and monocyte precursor may be introduced.

Alternatively, in the steps (2) to (3), the IRES-Cre sequence and the LoxP reporter system may be constructed on the same vector , and the granulocyte and monocyte precursor may be introduced.

The vector may be any vector used for gene targeting, such as pUC19, among others.

Specifically, the construction method comprises the following steps:

(1) determining the Ms4a3 gene as a gene specifically expressed in granulocytes and monocyte precursors;

through analysis of the sequencing results of hematopoietic single cells in bone marrow, and search analysis across databases, the present invention proposes a gene specifically expressed by granulocytes and monocytic precursors (Ms4a3), i.e., determined Ms4a3 gene as a gene specifically expressed in granulocytes and monocytic precursors.

(2) Constructing Ms4a 3-Cre; Rosa-tdTomato mouse tracking model/marking system

Construction of a granulocyte and monocyte tracking model/marker system based on the Cre/LoxP system, comprising the steps of inserting pieces of IRES-Cre (IRES, internal ribosome entry site; Cre, recombinase) into the mouse Ms4a3 Stop codon, in which mouse all cells expressing Ms4a3 express Cre recombinase simultaneously, then crossing the mouse with a Rosa-tdTomato reporter mouse to obtain Ms4a3-Cre, and Rosa-tdTomato mouse in which Ms4a3-Cre, and Rosa-tdTomato mouse in which the Cre recombinase recognizes two LoxP sequences of the Rosa26 site and cuts off the intermediate Stop signal sequence (LoxP-Stop-LoxP), when the Stop signal sequence is cut, the cell and its progeny will continue to express Tomato, producing red fluorescence, which can be detected by flow cytometry and microscopic imaging of the daughter cells and the nuclei.

(3) Verifying Ms4a 3-Cre; Rosa-tdTomato granulocyte and monocyte tracking system

To verify the performance of the tracing system, the invention detects the marking condition of each cell lineage in the blood and spleen of the mice, and finds that the Ms4a 3-Cre; the Rosa-tdTomato granulocyte and monocyte tracking system can efficiently and specifically mark monocytes (97%) and granulocytes (99%), and does not mark or hardly mark lymphocytes and dendritic cells (including traditional dendritic cells and plasmacytoid dendritic cells) such as T cells, B cells and NK cells.

In conclusion, the granulocyte and monocyte tracing system can efficiently and specifically mark/detect the granulocyte and the monocyte.

The invention also provides the application of the granulocyte and monocyte marking system in the fields of myeloid cell development, macrophage source and updating, tumor treatment, tumor efficacy evaluation and the like. Such as

When Ms4a3-Cre strain was crossed with LoxP reporter strain mice, to study the contribution of granulocyte-monocyte precursors to peripheral immune cells, and to tumor infiltrating cells (either systemic flox or specific flox model);

when the Ms4a3-Cre strain was used in combination with a flox mouse model (cell lineage specific flox DTA or cell specific floxDTR) to eliminate GMP, monocytes, monocyte derived cells (dendritic cells or macrophages);

the Ms4a3-Cre mouse model was used in combination with a flox mouse model targeting a specific gene to test the function of the corresponding gene in GMP, monocytes, monocyte derived cells (dendritic cells or macrophages) and granulocytes (including under both steady state and inflammatory environments), and the role of the corresponding gene in tumor infiltrating myeloid cells.

The granulocyte or monocyte marking system can specifically mark the monocyte or granulocyte, thereby being beneficial to research or application related to the monocyte or granulocyte.

Drawings

FIG. 1 Single cell transcriptome analysis showed that Ms4a3 was specifically highly expressed in cMyP. and a, performing tSNE dimensionality reduction analysis on BM cMyP, BM CDP, BM MDP, BM predC, BM Monocytes and blood Monocytes. b, heat map and Violin graph analysis using semuat was performed on the cell population in a above.

FIG. 2Ms4a 3-schematic diagram of the construction of the Cre granulocyte-monocyte labeling system. fragments of IRES-Cre (IRES, internal ribosome entry site; Cre, recombinase) were inserted after the Stop codon of mouse Ms4a 3. in this mouse, all cells expressing Ms4a3 would express Cre recombinase simultaneously (FIG. 2 a). then, this mouse was hybridized with a Rosa-tdTomato reporter mouse to obtain Ms4a 3-Cre; Rosa-tdTomato mouse (FIG. 2 b). in this Ms4a 3-Cre; Rosa-tdTomato mouse, the Cre recombinase recognizes two LoxP sequences of the Rosa26 site and removes the Stop signal sequence in between (LoxP-Stop-XP). when the Stop signal sequence is cleaved, the cells and their cells would continue to express the ToxP excision, the progeny cells would generate red fluorescent cells and the fluorescent single-monocyte labeling map (fluorescent image of the cells) which can be detected by flow cytometry.

FIG. 3Ms4a3-Cre trace model mouse blood markers and spleen dendritic cells analysis diagram. a, b, in peripheral blood, only granulocytes and monocytes of the myeloid lineage are labeled. And c, counting the marking condition of each lineage cell.

FIG. 4Ms4a3-Cre tracer model mice were used to study the contribution of monocytes to tissue macrophages. a, shows that monocytes contribute very little to cerebral Microglia (Microglia), epidermal Langerhans cells (Langerhans cells), and liver Kupffer cells (Kupffer cells). b, monocytes have a fixed contribution to alveolar/spleen/kidney and abdominal macrophages. c, monocytes contribute significantly and rapidly to intestinal and dermal macrophages.

FIG. 5 shows the contribution of monocytes to macrophages by fluorescence imaging. A positive F4/80 indicates all tissue macrophages. F4/80 positive, whereas tdTomato negative indicates embryonic-derived macrophages. F4/80 and tdTomato double positives indicate macrophages derived from bone marrow monocytes. In the liver, the vast majority of F4/80 positive cells were tdTomato negative, indicating that these macrophages were derived from embryonic stages. In the intestinal tract and skin, the F4/80 positive cells were tdTomato positive, indicating that these cells were derived from bone marrow mononuclear cells.

FIG. 6 is a schematic representation of the tracer model of the invention used to study tumor infiltrating myeloid lineage cells. a shows the position of different cell subsets on the tSNE map; b, marking the tdTomato of different cell populations, wherein gray represents high expression level, and black represents low expression level; c is the expression of CD11b, CD172a, MHCII, Ly6C, F4/80 and CD64 in tumor infiltrating cells, gray indicates high expression level, and black indicates low expression level.

Detailed Description

The present invention will be described in further detail in with reference to the following specific examples and accompanying drawings, and the processes, conditions, experimental methods, etc. for carrying out the invention are those generally known in the art and are not intended to be limited except as set forth in the following patent No. .