Method for distinguishing different gene manipulation cells through in-vivo fluorescence labeling
By combining Cre/loxP, CRISPR/Cas9 and binary expression systems to construct iVGMA, the genome-wide single-cell marking and gene manipulation problems were solved, and in-somatic fluorescent marking and long-term imaging were realized, which promoted the research of genetic chimeric cells and the construction of disease models.
Patent Information
- Application Number
- CN202510504058.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-22
- Publication Date
- 2025-07-25
AI Technical Summary
The prior art has limitations in genome-wide single-cell markers and gene overexpression or knockout studies, and cannot achieve genome-wide gene research and gene overexpression, and lacks real-time imaging capabilities in vivo.
Combined with Cre/loxP-mediated homologous chromosomal recombination, binary expression system and CRISPR/Cas9-mediated gene mutation technology, an in vivo universal genetic chimeric analysis (iVGMA) system was constructed to distinguish wild type, gene overexpression, gene knockout and cells that undergo simultaneous gene overexpression and knockout through different color fluorescent markers.
Accurate fluorescent labeling at the genome-wide and single-cell level is achieved, supporting long-term imaging and detailed lineage tracing at the volume, promoting the research of genetic chimeric cells and disease models, and improving the targeted and effective drug screening.
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Figure CN120366349A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to a method for fluorescently labeling cells, and particularly to a method for fluorescently labeling cells with overexpressed genes or knockout genes, belonging to the fields of cell engineering and genetic engineering. Background Art
[0002] Both gene overexpression and gene knockout belong to gene manipulation methods and are often used to analyze gene functions. The fluorescent labeling technology can intuitively judge the effects of gene overexpression or gene knockout based on the fluorescence signals at the cellular level. The Mosaic Analysis with the Double Markers (MADM) constructed based on Cre / loxP-mediated homologous chromosome recombination enables the precise labeling of mutant cells and wild-type cells with different color fluorescences at the single-cell level. This technology has greatly promoted the research in the fields of neural development, cell-cell interactions, and tumorigenesis and progression, and a series of breakthrough original scientific achievements have been obtained. However, the mouse model used in MADM has limitations in long-term in vivo imaging, restricting its in-depth application in dynamic cell biology research.
[0003] To overcome this challenge, the inventors previously established zebrafish MADM (zMADM), which combines the single-cell labeling precision of MADM with the advantages of zebrafish in vivo real-time imaging, opening up a new way for cell development research and gene mutation phenotype analysis (Bing Xu, Sarah Kucenas and Hui Zong. zMADM (zebrafish Mosaic Analysis with Double Markers) for single-cell gene knockout and dual lineage tracing. Proc Natl Acad Sci U S A. 2022, 119(9): e2122529119.). However, zMADM still has certain limitations: 1. The zMADM element is bound to a specific position on the chromosome, so only genes located between this element and the telomere can be knocked out and labeled at the single-cell level, and genome-wide gene research cannot be carried out; 2. zMADM lacks the ability to achieve gene overexpression, making it difficult to comprehensively analyze gene functions or conduct systematic research.
[0004] In the field of genetic engineering, many powerful gene manipulation techniques have been developed, including Cre / loxP, Gal4 / UAS, tTA / TRE, and CRISPR / Cas9. Cre / loxP is a gene recombination technique that can mediate homologous chromosome recombination; loxP is a DNA sequence containing sites specifically recognized by Cre recombinase; this technique is widely used in gene knockout, gene insertion, gene inversion, and gene translocation. Both Gal4 / UAS and tTA / TRE are binary expression systems, in which the transcription factors Gal4 and tTA can specifically bind to the response elements UAS and TRE, causing the expression of the target gene downstream of UAS and TRE. CRISPR / Cas9 is a gene editing technique that can mediate gene mutation techniques; Cas9 is a nuclease that can cleave double-stranded DNA at specific DNA sequences; after designing sgRNA and introducing it into target cells together with Cas9, sgRNA will guide Cas9 to locate to the target DNA sequence and promote Cas9 to cleave the DNA double strand at this position. When the cell repairs the DNA break generated by the cleavage, mutations can be introduced, thereby achieving gene knockout, insertion, or replacement. Currently, there are studies on combining two of the above existing technologies, but there is no method for combining these three technologies to simultaneously achieve precise labeling and in vivo real-time imaging of cells with specific gene manipulation. Summary of the Invention
[0005] Object of the Invention: The object of the present invention is to provide a method for distinguishing wild-type cells, gene overexpressing cells, gene knockout cells, or cells that have undergone both gene overexpression and gene knockout at the single-cell level within the whole genome in vivo through fluorescence labeling.
[0006] Technical Solution: The present invention provides a general genetic chimeric analysis system, which is characterized in that there is a pair of chimeric reporter genes containing loxP sites at the allelic positions of homologous chromosomes, and one end of one of the chimeric reporter genes has a transcription factor sequence of a binary expression system; the system further includes a target gene connected to the response element of the binary expression system, and the target gene is an overexpressed gene or a Cas9 gene; the chimeric reporter gene is prepared from fluorescent protein genes of different colors.
[0007] The inventors combined Cre / loxP-mediated homologous chromosome recombination, a binary expression system, and CRISPR / Cas9-mediated gene mutation technology to construct a technique that can differentially label wild-type, gene overexpressing, gene mutated, and genes with both overexpression and mutation cells simultaneously using different colors, which was named in vivo versatile genetic mosaic analysis (iVGMA). iVGMA can link the color of cells with the expression of transcription factors in the binary expression system, and then drive the downstream protein expression through response elements: if the target gene driven downstream of the response element is Cas9, gene knockout can be performed by designing gRNAs (one or more genes on the whole genome) targeting any gene of interest; if the target gene driven downstream of the response element is one or several other genes of interest, gene overexpression can be performed. Thus, iVGMA enables precise labeling of gene manipulation (knockout or overexpression) through different fluorescence colors at the whole genome level and single-cell level.
[0008] Preferably, the binary expression system is Gal4 / UAS or tTA / TRE.
[0009] Preferably, one end of the chimeric reporter gene has a strong promoter. The strong promoter can be eab2, and its sequence is as shown in SEQ ID NO.1.
[0010] Preferably, there are several target genes.
[0011] The present invention also provides a preparation method of the system, including the following steps: Step 1: Chimerize fluorescent protein genes of different colors and insert loxP sites at the chimeric positions to obtain a pair of chimeric reporter genes containing loxP sites; Step 2: Connect the transcription factor sequence of the binary expression system to one end of one of the chimeric reporter genes through a linker; Step 3: Insert a pair of chimeric reporter genes into the allelic positions of homologous chromosomes of an organism respectively; Step 4: Mate the organism to obtain fertilized eggs; In Step 3, the organism contains a target gene, and the target gene is connected to the response element of the binary expression system; or in Step 4, introduce the target gene connected to the response element of the binary expression system into the fertilized eggs; The fertilized eggs obtained in Step 4 are the universal genetic chimeric analysis system.
[0012] Preferably, in step three, the organism is transparent or its embryo is transparent in the early stage. Transparent organisms or organisms with transparent embryos in the early stage are convenient for observing the fluorescence color of cells and are the best choice for visualizing gene manipulation.
[0013] Preferably, in step three, the organism is zebrafish. The embryos of zebrafish are transparent in the early stage, and there are also transparent fish lines available for adult zebrafish.
[0014] Preferably, in step three, the chimeric reporter gene is inserted into the chromosome of the organism by CRISPR / Cas9.
[0015] Preferably, in step two, the linker is 2A peptide. The 2A peptide can be P2A.
[0016] The present invention also provides a method for distinguishing different gene-manipulated cells by in vivo fluorescence labeling, including the following steps: When the target gene is an overexpressed gene, Cre recombinase is introduced into the general genetic chimeric analysis system, and the labeling situation of cells is detected by microscopic imaging; When the target gene is Cas9 gene, the sgRNA of the gene to be knocked out and Cre recombinase are introduced into the general chimeric system, and the labeling situation of cells is detected by microscopic imaging.
[0017] After introducing Cre recombinase into cells, Cre recombinase mediates homologous chromosome recombination and different homologous chromatid separation methods. Subsequently, the chimeric reporter gene is recombined to obtain 4 arrangement modes, and finally daughter cells with different fluorescence are formed after the mother cell divides. Also, because the transcription factor expression of the binary expression system is associated with the fluorescence color, gene manipulation (knockout or overexpression) that drives the expression of downstream proteins based on the response element of the binary expression system can be associated with the fluorescence color, thereby achieving precise labeling at the whole genome level and single cell level.
[0018] Preferably, the introduction is carried out by microinjection.
[0019] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages: 1. The present invention has developed a brand-new cell biology research technology iVGMA, which can label cells subjected to different gene manipulations at the single cell level with different colors at the whole genome level, and simultaneously perform gene manipulation and differential labeling to distinguish wild type, gene overexpressed, gene knocked out, and cells with both gene overexpression and knockout.
[0020] 2. The present invention can be combined with research materials with the advantage of in vivo real-time imaging, such as zebrafish, and use different color fluorescence to label daughter cells derived from the same mother cell, so as to achieve in vivo long-term imaging and detailed lineage tracing.
[0021] 3. The present invention has application prospects in multiple fields: (1) Genetic chimerism - cellular and molecular mechanisms: The present invention can achieve simultaneous knockout and overexpression of multiple genes at the whole - genome level, providing a powerful tool for in - depth exploration of the cellular and molecular mechanisms of genetic chimerism, and contributing to understanding genetic variations during cell differentiation, development, and disease occurrence. (2) Disease occurrence and evolution mechanisms: The present invention can be used to construct human disease models originating from single cells, such as tumor models, and track the cellular mechanisms during the occurrence and progression of diseases through long - term in - vivo imaging. In addition, the whole - genome - level gene manipulation ability of iVGMA enables it to be used to study the synergistic effects of different genes in disease occurrence, providing a detailed cellular and molecular map for disease occurrence and evolution. (3) Drug screening. The present invention can label cells with different genotypes using different colors, simultaneously observe and analyze the responses of cells with multiple genotypes to drugs, provide a multi - dimensional reference standard for drug screening, improve the targeting and effectiveness of drug screening, and contribute to the research and application of biomedicine. BRIEF DESCRIPTION OF THE DRAWINGS
[0022] Figure 1 Schematic diagram for distinguishing gene - overexpressing cells by in - vivo fluorescence labeling; Figure 2 Schematic diagram for distinguishing gene - knockout cells by in - vivo fluorescence labeling; Figure 3 Schematic diagram for simultaneously distinguishing gene - overexpressing and gene - knockout cells by in - vivo fluorescence labeling; Figure 4 Results of observing the co - expression of mCerulean with GFP and mApple in zebrafish using confocal imaging (A is the confocal microscopy images of different fluorescent proteins in the same field of view. From left to right are the independent fluorescent signals of mCerulean, GFP, mApple, and the superimposed fluorescent signal of mCerulean, GFP, and mApple; B is the statistical proportion of cells with mCerulean - positive signals in GFP - positive green cells, mApple - positive red cells, and GFP / mApple double - positive yellow cells). DETAILED DESCRIPTION OF THE EMBODIMENTS
[0023] The technical solutions of the present invention will be further described below with reference to the accompanying drawings.
[0024] Sources of experimental materials: (1) Zebrafish: AB / WT is from the School of Life Sciences, Nantong University.
[0025] (2) Chimeric reporter gene, mCerulean gene, sgRNA: All are from gene synthesis companies (GenScript and Genewiz).
[0026] (3) zCas9 gene (Cas9 optimized for zebrafish codons): It is from the research group of Du Jiulin at the Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences. The zCas9 sequence is shown in SEQ ID NO.2.
[0027] Example 1: Preparation method of a general genetic chimeric analysis system In this example, two binary expression systems, Gal4 / UAS and tTA / TRE, are used. The preparation method includes the following steps: Step 1: Interleave the genes of fluorescent proteins mApple and GFP, and insert loxP sites at the interleaved positions to obtain a pair of chimeric reporter genes containing loxP sites: GA (N-GFP-intron-C-mApple) and AG (N-mApple-intron-C-GFP); Step 2: Connect the Gal4 sequence to one end of the chimeric reporter gene GA through the linker P2A to obtain GA-Gal4; connect the tTA sequence to one end of the chimeric reporter gene AG through the linker P2A to obtain AG-tTA; Step 3: Insert AG, GA, GA-Gal4, and AG-tTA into the allelic positions of homologous chromosomes of zebrafish respectively; Step 4: Mate the obtained zebrafish to obtain fertilized eggs; and introduce the target genes mCerulean / zCas9 linked to UAS / TRE into the fertilized eggs (UAS:mCerulean and TRE:zCas9).
[0028] Example 2: A general genetic chimeric analysis system This example is the general genetic chimeric analysis system prepared in Example 1. As Figure 3 shown, in the cell, there is a pair of chimeric reporter genes containing loxP sites at the allelic positions of homologous chromosomes: GA (N-GFP-intron-C-mApple) and AG (N-mApple-intron-C-GFP). One end of the chimeric reporter gene GA has the Gal4 sequence (GA-Gal4), and one end of the chimeric reporter gene AG has the tTA sequence (AG-tTA); the cell also includes the target gene mCerulean linked to UAS and the target gene zCas9 linked to TRE.
[0029] Example 3: Simultaneous in vivo fluorescence labeling to distinguish two gene manipulation cells This example is based on the general genetic chimeric analysis system prepared in Example 1. By using fluorescence labeling to distinguish mCerulean overexpressing cells, zCas9 overexpressing cells (i.e., gene knockout cells), and cells overexpressing both mCerulean and zCas9 (i.e., performing both gene manipulation simultaneously), the following steps are included: Introduce the sgRNA of the gene to be knocked out nf1a and Cre recombinase into the above-mentioned general chimeric system, and detect the labeling of cells through microscopic imaging.
[0030] The principle of this example is as Figure 3 shown. After introducing Cre recombinase into cells, Cre recombinase mediates homologous chromosome recombination and different homologous chromatid separation methods. Subsequently, the chimeric reporter gene is recombined to obtain 4 arrangements, and finally daughter cells with different fluorescences are formed after the mother cell divides. Since the expressions of the transcription factors of the two binary expression systems are respectively associated with the fluorescence colors, the overexpression of the downstream target of gene driven by the response element can be associated with the fluorescence color. Also, because the combination of sgRNA and zCas9 can achieve the functional knockout of specific genes, on the basis of the association between zCas9 gene overexpression and fluorescence color, the gene knockout mediated by zCas9 can be associated with the fluorescence color. Thus, in vivo fluorescence labeling is used to distinguish gene overexpression, gene knockout, and cells performing both gene manipulation simultaneously.
[0031] Example 4: Preparation method of a general genetic chimeric analysis system The difference between this example and Example 1 is that this example only uses one binary expression system tTA / TRE. The preparation method includes the following steps: Step 1: The same as in Example 1; Step 2: Connect the tTA sequence to one end of the chimeric reporter gene AG through the linker P2A to obtain AG-tTA; Step 3: Insert GA and AG-tTA into the allelic positions of homologous chromosomes of zebrafish respectively; Step 4: Mate the obtained zebrafish to obtain fertilized eggs; and introduce the target gene zCas9 connected to TRE into the fertilized eggs (TRE:zCas9).
[0032] Example 5: A general genetic chimeric analysis system This example is the general genetic chimeric analysis system prepared in Example 4.
[0033] Example 6: In vivo fluorescence labeling to distinguish gene knockout cells This example is based on the general genetic chimeric analysis system prepared in Example 4, and distinguishes zCas9-mediated nf1a gene knockout cells through fluorescence labeling, including the following steps: Introduce the sgRNA of the gene to be knocked out nf1a and Cre recombinase into the above-mentioned general genetic chimeric analysis system, and detect the labeling of cells through microscopic imaging.
[0034] The principle of this example is as Figure 2 shown. Overexpression of zCas9 can be associated with the fluorescence color of cells, and zCas9-mediated nf1a gene knockout cells are thus associated with the fluorescence color.
[0035] Example 7: A preparation method of a general genetic chimeric analysis system The difference between this example and Example 1 is that this example only uses a binary expression system Gal4 / UAS. The preparation method includes the following steps: Step 1: The same as Example 1; Step 2: Connect the Gal4 sequence to one end of the chimeric reporter gene GA through the linker P2A to obtain GA-Gal4; Step 3: Insert AG and GA-Gal4 into the allelic positions of homologous chromosomes of zebrafish respectively; Step 4: Mate the obtained zebrafish to obtain fertilized eggs; and introduce the target gene mCerulean linked to UAS into the fertilized eggs (UAS:mCerulean).
[0036] Example 8: A general genetic chimeric analysis system This example is the general genetic chimeric analysis system prepared in Example 7.
[0037] Example 9: In vivo fluorescence labeling to distinguish gene overexpressing cells This example is based on the general genetic chimeric analysis system prepared in Example 7, and distinguishes mCerulean gene overexpressing cells through fluorescence labeling, including the following steps: Introduce Cre recombinase into the above-mentioned general genetic chimeric analysis system, and detect the labeling of cells through microscopic imaging.
[0038] The principle of this example is as Figure 1 shown, and overexpression of mCerulean can be associated with the fluorescence color of cells.
[0039] Example 10: A preparation method of a general genetic chimeric analysis system The difference between this example and Example 7 is that the chimeric reporter genes are AG-Gal4 and GA.
[0040] Example 11: A General Genetic Chimeric Analysis System This example is the general genetic chimeric analysis system prepared in Example 10.
[0041] Example 12: In Vivo Fluorescent Labeling to Distinguish mCerulean Gene Overexpressing Cells This example is based on the general genetic chimeric analysis system prepared in Example 10, and distinguishes mCerulean gene overexpressing cells through fluorescent labeling, including the following steps: Co-inject Cre recombinase into the above-mentioned general chimeric system, and detect the labeling of cells by microscopic imaging at 4 dpf.
[0042] The results are as Figure 4 shown. Cells expressing GFP (green and yellow) achieved mCerulean overexpression, while only red-labeled cells expressing mApple had no mCerulean expression. It shows that the construction of iVGMA-OE can be achieved by introducing the Gal4 / UAS binary expression system.
Claims
1. A general genetic chimerism analysis system, characterized in that, There is a pair of chimeric reporter genes containing loxP sites at the allelic positions of homologous chromosomes. One end of one chimeric reporter gene has a transcription factor sequence of a binary expression system; the system also includes a target gene connected to the response element of the binary expression system, and the target gene is an overexpressed gene or a Cas9 gene; the chimeric reporter gene is prepared from fluorescent protein genes of different colors.
2. The system according to claim 1, characterized in that The binary expression system is Gal4 / UAS or tTA / TRE.
3. The system according to claim 1, wherein One end of the chimeric reporter gene has a strong promoter.
4. The system according to claim 1, characterized in that, There are several target genes.
5. A preparation method of the system according to claim 1, characterized in that, It includes the following steps: Step 1: Intermix fluorescent protein genes of different colors and insert loxP sites at the intermixing positions to obtain a pair of chimeric reporter genes containing loxP sites. Step 2: Connect a transcription factor sequence of a binary expression system to one end of one chimeric reporter gene through a linker. Step 3: Insert a pair of chimeric reporter genes into the allelic positions of homologous chromosomes of an organism respectively. Step 4: Mate the organism to obtain a fertilized egg. In Step 3, the organism contains a target gene, and the target gene is connected to the response element of the binary expression system; or in Step 4, introduce a target gene connected to the response element of the binary expression system into the fertilized egg. The fertilized egg obtained in Step 4 is the system described in Claim 1.
6. The preparation method according to claim 5, characterized in that, In Step 3, the organism is transparent or its early embryo is transparent.
7. The preparation method according to claim 5, wherein In Step 3, the organism is zebrafish.
8. The preparation method according to claim 5, characterized in that, In Step 3, insert the chimeric reporter gene into the chromosome of the organism through CRISPR / Cas9.
9. The preparation method according to claim 5, wherein In Step 2, the linker is 2A peptide.
10. Method for distinguishing cells with different gene manipulations by in vivo fluorescence labeling, characterized in that, It includes the following steps: When the target gene is an overexpressed gene, introduce Cre recombinase into the system described in Claim 1 and detect the labeling of cells through microscopic imaging. When the target gene is a Cas9 gene, introduce the sgRNA of the gene to be knocked out and Cre recombinase into the system described in Claim 1 and detect the labeling of cells through microscopic imaging.