A MSC-specific promoter FMO1, vector, cell, and screening method and application thereof

Abstract

The present invention belongs to the field of gene engineering technology, and provides an MSC-specific promoter and its application, wherein the MSC-specific promoter FMO1 is selected from one of SEQ ID NO: 1 to SEQ ID NO: 9, wherein SEQ ID NO: 7 is preferred, or a sequence having at least 83.1% similarity to its sequence, or a sequence having at least 73.4% similarity to its truncated fragment. The MSC-specific promoter provided by the present invention can carry exogenous genes into cells at the pluripotent stem cell stage, and only initiates transcriptional activity after the pluripotent stem cells differentiate into MSCs, which is of great significance to MSC engineering transformation and research on MSC-based precision therapy.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering and provides a MSC-specific promoter FMO1, a vector, a cell and a screening method and application thereof. Background Art

[0002] MSC is an adult stem cell with biological characteristics such as multidirectional differentiation, immunomodulation, anti-fibrosis, anti-inflammation, anti-apoptosis and angiogenesis. It plays an important role in tissue repair, cancer treatment and immune regulation, and is widely used in disease research and regenerative medicine. However, the use of MSC for treatment also has the disadvantages of difficult preparation and unstable effects. For example, the sources of MSC from umbilical cord tissue and placenta are limited, the preservation cost is high, and there are logistical challenges; MSC from bone marrow, fat, and peripheral blood require volunteers to be injured, and MSC from natural tissues are limited by their own limited expansion capacity, so they may need to be recruited repeatedly. In this process, due to different donors, different tissue sources, and differences in cell passage operations, cell morphology changes, DNA abnormalities, cell aging, decreased proliferation and differentiation ability, and changes in cell plasticity may occur, thereby affecting the quality and life of MSC, and further affecting the repeatability of the experiment, resulting in inconsistent clinical research results. In order to overcome these obstacles, researchers have designed many efficient culture systems to produce high-quality MSCs, thereby achieving reproducible results in regenerative medicine. Among these methods, deriving MSCs from pluripotent stem cells is a key alternative method. MSCs induced by pluripotent stem cells have the same multi-lineage differentiation potential and secretory factor function in vitro and in vivo as natural MSCs, and can be prepared on a large scale with more stable and uniform characteristics. In addition, a large number of studies have also confirmed that natural MSCs have some adverse effects in the treatment process. For example, interleukin-4, one of the most effective anti-inflammatory cytokines in inflammatory response, is expressed at a low level in natural MSCs, so the therapeutic effect is limited; adipose-derived MSCs lack the ability to differentiate into cartilage because they do not express TGF-β1 receptors and low expression of BMPs. Therefore, how to improve the therapeutic ability of MSCs is also a development trend of current research. In this sense, it is a very important strategy to develop suitable engineered MSCs based on engineering methods such as iPSC technology, CRISPR / Cas 9 gene editing technology, and iPSC induced differentiation technology to provide suitable cells for human disease research and regenerative medicine. Engineering modification of MSCs includes the insertion of exogenous genes. Considering that iPSCs have better proliferation capabilities than MSCs, it is a better choice to place the genetic modification stage in the iPSC cell stage. However, most of the existing mammalian promoters are constitutive expression promoters, which will maintain a certain degree of expression after being transferred into cells and are not affected by cell types. Premature gene expression may affect the proliferation and differentiation capabilities of iPSCs. Therefore, it is very important to develop a method that can accurately control the expression of exogenous genes. Summary of the invention

[0003] In order to prevent the expression of inserted genes from affecting the pluripotency and differentiation ability of pluripotent stem cells after exogenous genes are knocked into pluripotent stem cells, the present invention provides an MSC-specific promoter, which only starts transcription activity after pluripotent stem cells differentiate into MSCs, thereby achieving the purpose of precise expression.

[0004] In order to achieve the above-mentioned purpose, one embodiment of the present invention provides a method for screening MSC-specific promoters, and the specific steps are: performing transcriptome sequencing analysis on pluripotent stem cells and MSC cells, screening MSC-specific highly expressed genes, predicting and synthesizing promoter fragments, constructing a fluorescence verification vector to verify the promoter function, optimizing the promoter sequence, and finally screening out promoters that are specifically expressed only in MSCs.

[0005] An object of the present invention is to provide an MSC-specific promoter, characterized in that its DNA sequence is selected from one of SEQ ID NO: 1 to SEQ ID NO: 9, wherein SEQ ID NO: 7 is preferred, or a sequence having at least 83.1% similarity thereto, or a sequence having at least 73.4% similarity thereto to a truncated fragment thereof.

[0006] In another aspect, the present invention also relates to a vector having the above MSC-specific promoter.

[0007] In another aspect, the present invention also relates to cells containing the above-mentioned vector.

[0008] In another aspect of the present invention, the present invention also provides the use of the above-mentioned MSC-specific promoter in the preparation of a product in which exogenous genes are specifically expressed in MSC cells derived from pluripotent stem cells, characterized in that the promoter is used to carry exogenous genes into pluripotent stem cells, where they are not expressed in the pluripotent stem cell stage, and then transcription activity is initiated only after the pluripotent stem cells differentiate into MSCs.

[0009] The present invention has the following advantages: The present invention provides a promoter with MSC cell-specific expression, which can ensure that the target gene is expressed only in successfully differentiated MSC cells, avoiding interference with pluripotent stem cells. The present invention greatly enriches the MSC-specific promoter resources, and is of great significance for MSC engineering transformation and research on the differentiation mechanism of pluripotent stem cells into MSC. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 For iPSC and MSC cells FMO1 Comparison chart of gene FPKM values.

[0011] Figure 2 This is a comparison chart of the RNA expression levels of fluorescence-verified vectors with different truncated specific promoters in MSC cells.

[0012] Figure 3 Schematic diagram of the pSP2000-EGFP plasmid map.

[0013] Figure 4 Comparison of RNA expression levels of fluorescence-verified vectors with different sequence-optimized promoters in MSC cells.

[0014] Figure 5 This is a comparison chart of RNA expression levels of fluorescence-verified vectors with specific promoters from different sources in MSC cells.

[0015] Figure 6 This is a comparison chart of the RNA expression levels of fluorescent genes at different stages of ESC differentiation into MSC.

[0016] Figure 7 The graph shows the proportion of CD73, CD90 and CD105 positive cells in MSC cells differentiated by ESC induced by different promoters. DETAILED DESCRIPTION

[0017] The present invention will be further described below in conjunction with embodiments and drawings, but the present invention is not limited by the following embodiments.

[0018] The expression of exogenous genes in cells is affected by promoters, which regulate the intensity, time and space characteristics of gene expression. Using this specific promoter, exogenous genes can be targeted and fixed in a certain tissue or cell for expression, reducing the loss of energy and material in the body and the impact on the body's metabolic activities, while also being able to improve the characteristics of a certain cell in a targeted manner. However, so far, there have been no reports on MSC cell-specific promoters derived from pluripotent stem cells in the industry, so the present invention provides an MSC-specific promoter and its application to help the engineering transformation of MSC.

[0019] Example 1: Transcriptome sequencing to identify genes that are not expressed in iPSC cells but are highly expressed in MSCs FMO1 Gene.

[0020] (1) Use iPSC medium to expand and culture iPSC cells until the number of cells reaches 1 × 10 6 At 4 pm, cells were collected using TRNzol Universal total RNA extraction reagent.

[0021] (2) Treat iPSCs with MSC induction medium to induce them to differentiate into MSCs. Use an MSC detection kit to detect the expression of MSC markers. Continue to culture MSCs until the number of cells reaches 1 × 10 6At 4 pm, cells were collected using TRNzol Universal total RNA extraction reagent.

[0022] (3) Total RNA was extracted from the above iPSC samples and iPSC-induced MSC samples, and then transcriptome sequencing was performed. The depth of transcriptome sequencing was at least 10 G to ensure that low-abundance genes in both cell types could also be detected.

[0023] (4) The differential expression analysis of the expressed genes between the two groups of cells was performed using the DESeq2 R software package. DESeq 2R provides statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The P value was adjusted using the Benjamini and Hochberg method to control the false discovery rate. Genes with FDR < 0.05 and log2(foldchange) ≥ 2 found by DESeq2 R were designated as differentially expressed genes.

[0024] (5) The differentially expressed genes between iPSC and MSC were rescreened according to the FPKM value of the iPSC group less than 0.1, i.e., the no expression threshold, and then the FPKM value of the MSC group was arranged in descending order, i.e., the genes that were MSC-specific and had a higher expression level. Among them, FMO1 The gene was not detected in iPSC cells, with an average FPKM value of 0, and was highly expressed in MSCs, with an average FPKM value of 52.1. Figure 1 shown.

[0025] Example 2: Prediction of promoter regions and synthesis of promoter fragments.

[0026] (1) According to the selected FMO1 Gene name, gene sequence was searched in the human genome on the NCBI website, a fragment of about 3000 bp upstream of the transcription start site was selected, and then cis-acting element analysis was performed using the online website plantCARE to predict the promoter region.

[0027] (2) Based on the prediction results, 5′-end truncated fragments were designed, with the first base upstream of the transcription start site as position -1. Six groups of truncated fragments were designed, namely -3000 to -1, -2500 to -1, -2000 to -1, -1500 to -1, -1000 to -1, and -500 to -1. Mlu Ⅰ restriction site ACGCGT, add the fragment downstream Nhe I restriction site GCTAGC is used to facilitate subsequent vector construction, and then each promoter truncated fragment is synthesized. The sequences of each truncated fragment are shown in SEQ ID NO: 1 to SEQ ID NO: 6, respectively.

[0028] Example 3: Construction of fluorescence verification vector.

[0029] (1) First, synthesize the pCDNA3.1-EGFP vector and clone the vector at the multiple cloning restriction site of the pCDNA3.1 plasmid. Hind III and Bm The "EGFP" fragment was inserted into Ⅰ as a fluorescence verification vector.

[0030] (2) Using conventional molecular cloning methods, the sequences of the promoters of the different truncated length fragments in (2) of Example 2 were replaced in the fluorescence verification vector. Mlu Ⅰ and Nhe The "CMV" promoter in region Ⅰ was used to obtain the specific promoter fluorescence verification vectors pSP (Specific Promoter) 3000-EGFP, pSP2500-EGFP, pSP2000-EGFP, pSP1500-EGFP, pSP1000-EGFP, and pSP500-EGFP.

[0031] Example 4: Verification of promoter function.

[0032] (1) iPSC cells and MSC cells were cultured to 90% confluence using standard methods. The cells were digested and counted, and 1× 10 6 Centrifuge the cells at low speed, take out the centrifuge tube into the biosafety cabinet, open the tube cover, and discard the supernatant.

[0033] (2) Take out 2 μg of each of the above-mentioned specific promoter fluorescence verification vectors pSP3000-EGFP, pSP2500-EGFP, pSP2000-EGFP, pSP1500-EGFP, pSP1000-EGFP, and pSP500-EGFP, add them to the electroporation buffer, pipette and mix them several times, then aspirate all the liquid and add it to the cell pellet obtained above. After resuspending the cells, transfer them to the electroporation cup, and then place it in the transfection chamber of the electroporator for electroporation. After the electroporation operation is completed, take out the electroporation cup and place it in the biosafety cabinet, and transfer the cells to the culture dish.

[0034] (3) After 48 h of culture, some cells were taken to extract total RNA, and after reverse transcription to obtain cDNA, the expression level of the target EGFP gene was determined by qPCR. The results showed that in iPSC cells, no EGFP RNA transcription was detected in all plasmid groups, while in MSC cells, RNA transcription could be detected in all plasmid groups. The quantitative results of EGFP in MSC cells are shown in Figure 2. Figure 2 As shown, the RNA transcription level of EGFP gene in cells of pSP2000-EGFP group is higher. The picture of pSP2000-EGFP plasmid is shown in Figure 3As shown, the corresponding sequence corresponds to SEQ ID NO: 3.

[0035] Example 5: Optimization of promoter SEQ ID NO: 3 sequence.

[0036] (1) Based on the data results of (1) in Example 2, the sequence of the transcription factor binding site was kept unchanged, and the flanking sequence of the transcription factor binding site was optimized. The transcriptional activity and specificity of the promoter were improved by changing the GC content and CpG island, and a total of three groups of promoter optimized sequence fluorescent vectors were designed, namely pe1 (enhance) SP2000-EGFP, pe2SP2000-EGFP, and pe3SP2000-EGFP.

[0037] (2) The promoter activity was verified using the same promoter function verification method as in Example 2. The results are as follows: Figure 4 As shown, the RNA transcription levels of the pe2SP2000-EGFP and pe3SP2000-EGFP structure optimized groups had no significant difference compared with the wild type group, while the pe1SP2000-EGFP group was higher, and its promoter sequence is shown in SEQ ID NO: 7.

[0038] Example 6: Verification of homologous promoter function.

[0039] (1) Considering that gene sequences of different species have homology, we selected homologous promoters from rhesus monkeys, which are primates like humans, and pigs, which are mammals, to verify their biological functions. We searched for promoters of rhesus monkeys and pigs on the NCBI website. FMO1 For the gene sequence, a fragment of about 2000 bp upstream of the transcription start site was selected. After analysis by nucleic acid sequence comparison software, it was found that the sequence of the rhesus monkey had a sequence similarity of 83.1% with SEQ ID NO: 3, and its sequence is shown in SEQ ID NO: 8. The sequence of the pig had a sequence similarity of 73.4% after being shortened by 1500 bp, and its sequence is shown in SEQ ID NO: 9.

[0040] (2) Use conventional molecular cloning methods to replace the sequences of SEQ ID NO: 8 and SEQ ID NO: 9 in the fluorescence verification vector Mlu Ⅰ and Nhe The "CMV" promoter in region Ⅰ was used to obtain the homologous specific promoter fluorescence verification vectors pR (Rhesusmonkey) SP2000-EGFP and pp (pig) SP2000-EGFP.

[0041] (3) Using the same cell culture, plasmid electroporation, and qPCR assay methods as in Example 4 (1), (2), and (3), similar to the results with the human promoter sequence, in iPSC cells, no EGFP RNA transcription was detected in the pRSP2000-EGFP and ppSP2000-EGFP plasmid groups. In MSC cells, RNA transcription was detected in all three source groups. The quantitative results of EGFP in MSC cells are shown in Figure 2. Figure 5 As shown, the EGFP gene RNA transcription level in the rhesus monkey-derived promoter plasmid pRSP2000-EGFP group was similar to that in the human-derived group, while that in the pig-derived ppSP2000-EGFP group was lower.

[0042] Example 7: Application of exogenous genes for specific expression in MSC cells derived from pluripotent stem cells.

[0043] (1) The human sequence optimized MSC-specific high expression promoter sequence SEQ ID NO: 7, rhesus monkey SEQ ID NO: 8 and porcine SEQ ID NO: 9 selected in Example 5 were constructed into the transposon stable integration vector PBCAG-EGFP by molecular cloning, replacing the original CAG promoter in the vector, and obtaining PBe12000-EGFP, PBR2000-EGFP, PBp2000-EGFP stable integration vectors, which were then co-electroporated into ESC cells with the transposon vector. The culture medium of the cells after electroporation was changed every day. After two days of culture, the cells were digested, counted, and 5000 cells were inoculated in a 10 cm culture dish.

[0044] (2) Continue culturing for 7 days, pick single clones in a laminar flow bench, and inoculate them into 24-well plates. When the number of clones is sufficient, take some cells for PCR detection, use a DNA extraction kit to extract the genomic DNA of the cells in each well, perform PCR amplification and sequencing, and select cell clones with copies of the target gene.

[0045] (3) The ESC cells of the positive clones in Example 7 (2) were treated with MSC induction medium to induce differentiation into MSC. Parts of the cells were taken at different stages of culture for qPCR detection. The results were as follows: Figure 6 As shown, in the early stage of continuous culture, the cells are still in the ESC stage and the promoter is in a silent state, so RNA transcription cannot be detected. When cultured to the 16th day, some cells in the three experimental groups were successfully transformed into MSCs, and the specific promoter initiated transcription activities. As the culture time increased, the proportion of MSCs in the cells increased, so the RNA level gradually increased.

[0046] Example 8: Effects of exogenous expression of specific promoters and constitutive promoters on the differentiation ability of pluripotent stem cells MSCs.

[0047] (1) The natural PBCAG-EGFP plasmid and the transposon vector were co-electroporated into ESC cells, and then the PBCAG-EGFP-ESC cells were treated using the same clone picking, positive cell identification, MSC induction differentiation methods as in Example 7 (1), (2), and (3) to obtain exogenous expression cells carrying a constitutive promoter.

[0048] (2) On the 24th day of MSC differentiation, MSCs differentiated by PBCAG-EGFP-ESC, PBe12000 (human promoter)-EGFP-ESC, PBR2000 (rhesus promoter)-EGFP-ESC, and PBp2000 (pig promoter)-EGFP-ESC were collected and treated with flow cytometry antibodies for MSC surface markers CD73, CD90, and CD105. The proportion of CD73, CD90, and CD105 fluorescence positive cells in the four groups were then analyzed by flow cytometry. The results are shown in Figure 2. Figure 7 As shown, the proportion of CD73, CD90 and CD105 fluorescence positive cells in the MSC cells induced by PBCAG-EGFP-ESC was lower than that in the MSC cell groups induced by PBe12000-EGFP-ESC, PBR2000-EGFP-ESC and PBp2000-EGFP-ESC, indicating that among the cells expressing exogenous genes using a constitutive expression promoter, there are still some cells that fail to successfully differentiate into MSCs, further confirming that the MSC-specific promoter of the present invention, including rhesus monkey-derived and pig-derived promoters, will not interfere with the differentiation ability of pluripotent stem cells.

[0049] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims

1. A MSC-specific promoter FMO1, characterized in that: The DNA sequence is selected from one of SEQ ID NO: 1 to SEQ ID NO: 9, wherein SEQ ID NO: 7, or a sequence having at least 83.1% similarity thereto, or a sequence having at least 73.4% similarity thereto to a truncated fragment thereof is preferred.

2. A vector comprising the promoter according to claim 1.

3. A cell containing the vector according to claim 2.

4. The method for screening the MSC-specific promoter FMO1 according to claim 1, characterized in that: It includes: transcriptome sequencing analysis of pluripotent stem cells and MSC cells, screening of MSC-specific highly expressed genes, prediction and synthesis of promoter fragments, construction of fluorescence verification vectors to verify promoter function, optimization of promoter sequences, and finally screening out promoters that are specifically expressed only in MSCs.

5. The use of the MSC-specific promoter FMO1 according to claim 1 in preparing a product in which an exogenous gene is specifically expressed in MSC cells derived from pluripotent stem cells, characterized in that: Using this promoter, foreign genes are carried into pluripotent stem cells, but are not expressed in the pluripotent stem cell stage, and transcriptional activity is initiated only after the pluripotent stem cells differentiate into MSCs.

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