Dlc1 gene knockout embryonic stem cell line as well as construction method and application thereof

Abstract

The invention provides a Dlc1 gene knockout embryonic stem cell line as well as a construction method and application thereof. A gRNA sequence of a targeted Dlc1 gene is designed by adopting a CRISPR / Cas9 gene editing technology, so that the Dlc1 gene is efficiently knocked out. Besides, the PX459-gRNA vector is electrically transferred to embryonic stem cells, and the Dlc1 diallele knockout cell line is obtained through puromycin screening and sequencing identification. The multidirectional differentiation capability of embryonic stem cells is utilized to construct a three-germ-layer embryoid body, and Dlc1 deletion is found to promote mid-germ and endogerm differentiation; the embryoid body is further induced and differentiated into myocardial cells, and Dlc1 deletion damages myocardial development, myofilament assembly and contraction functions. The invention provides a good experimental platform for researching multisystem diseases caused by Dlc1 defects, can be used for analyzing the action mechanism of Dlc1 in cardiovascular diseases, and provides an important tool for myocardial regeneration, drug screening, disease diagnosis and gene intervention.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to a Dlc1 gene knockout embryonic stem cell line and a construction method and application thereof. BACKGROUND

[0002] DLC1 (Deleted in liver cancer-1) gene encodes a RhoGTPase activating protein, which can promote the conversion of active RhoGTP to inactive RhoGDP, and is involved in cytoskeleton formation, proliferation, apoptosis, adhesion and migration. The Dlc1 protein includes an N-terminal sterile alpha motif (SAM) domain, an intermediate Rho GTPase activating protein (RhoGAP) domain and a C-terminal steroidogenic acute regulatory-related lipid-transfer (START) domain.

[0003] DLC1 is widely expressed in mouse and human embryonic development tissues and adult tissues, including brain, heart, kidney, liver, lung, skin, ovary, spleen, thyroid, intestinal tissue, etc., and is involved in embryonic growth, tissue and organ formation and differentiation. Dlc1 knockout mice homozygous embryos develop slowly, and the neural tube, heart and placental vascular system have serious defects, and E10.5 embryos die due to multiple organ system failure.

[0004] Genome sequencing analysis shows that DLC1 is a susceptible gene for various diseases, including nervous system diseases (such as dyslexia, neural tube defects such as anencephaly and myelomeningocele, and congenital megacolon); cardiovascular system diseases (such as congenital heart disease, myocardial infarction, and coronary artery spasm angina); kidney diseases (such as nephrotic syndrome) and lung diseases (such as chronic obstructive pulmonary disease and sleep breathing disorders), etc., but the specific molecular pathogenesis has not been elucidated.

[0005] Dlc1 knockout mice die at E10.5 due to multiple organ failure, which makes it impossible to obtain tissue and organ samples at later developmental stages, severely limiting the study of the mechanism of action of Dlc1 in embryonic development stages and adult organ systems, so it is urgent to construct a suitable Dlc1 knockout model.

[0006] Embryonic stem cells (ESC) are derived from the inner cell mass of early blastocysts, which are a type of pluripotent stem cells with the dual characteristics of self-renewal and pluripotency, and can differentiate into all embryonic germ layer cells, i.e. endoderm, mesoderm and ectoderm, and develop into all types of cells, tissues, organs and / or body parts. Embryoid bodies (EB) are three-dimensional spherical structures formed by ES cells under specific culture conditions, which include endoderm, mesoderm and ectoderm, and exhibit similar morphological and molecular characteristics to early embryos, simulating the early embryonic development process. EBs can be further differentiated into functional cells (such as endodermal cells such as intestinal epithelial cells, liver cells and lung epithelial cells; mesodermal cells such as cardiomyocytes, osteoblasts, kidney cells and smooth muscle cells; ectodermal cells such as neural cells, epidermal cells and retinal cells) and organoids (such as brain organoids, heart organoids and liver organoids) through specific induction conditions and signal pathway regulation.

[0007] In summary, constructing Dlc1 knockout embryonic stem cells is of great significance for studying the mechanism of Dlc1 in germ layer differentiation, tissue and organ development, and related diseases. SUMMARY

[0008] In order to overcome the shortcomings of the prior art, the purpose of the present application is to provide a Dlc1 gene knockout ES cell line and its construction method and application, so as to realize efficient and stable gene knockout and promote the development of related disease research and targeted therapy.

[0009] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0010] In a first aspect, the present application provides a gRNA specifically targeting the Dlc1 gene, wherein the sequence of the gRNA specifically targeting the Dlc1 gene is a combination of gRNA1, gRNA2 and gRNA3, wherein the sequence of gRNA1 is shown in SEQ ID NO. 1, the sequence of gRNA2 is shown in SEQ ID NO. 2, and the sequence of gRNA3 is shown in SEQ ID NO. 3.

[0011] In a second aspect, the present application provides a gene knockout vector, which is constructed by connecting the DNA sequence corresponding to the above-mentioned gRNA to the vector, and the gene knockout vector expresses the gRNA as claimed in claim 1.

[0012] In a third aspect, the present application provides a CRISPR / Cas9 system for specifically targeting and knocking out the Dlc1 gene, which comprises the combination of gRNAs targeting the Dlc1 gene and Cas9 protein.

[0013] In a fourth aspect, the present application provides a method for constructing a Dlc1 gene knockout embryonic stem cell line, comprising the following steps:

[0014] Step S1: constructing a gene knockout vector;

[0015] Step S2: electroporating the gene knockout vector into the embryonic stem cells;

[0016] Step S3: obtaining the Dlc1 gene knockout embryonic stem cell line through puromycin screening and genotyping.

[0017] Preferably, in the step S1, the construction of the Dlc1 gene knockout vector adopts the CRISPR-Cas9 gene editing technology.

[0018] Preferably, in the step S1, the method comprises the following steps:

[0019] designing gRNA primers and obtaining a combination of gRNA1, gRNA2 and gRNA3, wherein the sequence of gRNA1 is shown in SEQ ID NO. 1, the sequence of gRNA2 is shown in SEQ ID NO. 2, and the sequence of gRNA3 is shown in SEQ ID NO. 3;

[0020] annealing and phosphorylating the gRNA primers to obtain a gRNA double-stranded fragment;

[0021] cloning the gRNA double-stranded fragment into a puromycin-resistant vector PX459 (i.e. pSpCas9(BB)-2A-Puro) to obtain a gene knockout vector, wherein the gene knockout vector is named as PX459-Dlc1-gRNA1, PX459-Dlc1-gRNA2 and PX459-Dlc1-gRNA3.

[0022] Preferably, in the step S2, the amount of each Dlc1 gene knockout vector is 1.5 μg, and the electroporation transfection adopts a Neon electroporator with the following specific parameters: pulse voltage 1300 V, pulse width 20, and pulse number 2.

[0023] Preferably, in the step S3, the amount of puromycin added is 1.5 μg / mL, and the screening time is 3 days.

[0024] In a fifth aspect, the present application provides a Dlc1 gene knockout embryonic stem cell line, which is obtained by the method described above.

[0025] In a sixth aspect, the present application provides a method for constructing a hanging drop-embryoid body differentiation model, comprising the following steps:

[0026] Culture of the embryoid body: the embryoid body is formed by culturing the Dlc1 gene knockout embryonic stem cell line in a manner of hanging drop culture for 2 days and three-dimensional suspension culture for 3 days, thereby constructing a hanging drop-embryoid body differentiation model;

[0027] RNA-seq analysis: the influence of Dlc1 gene knockout on germ layer differentiation is analyzed by RNA-seq technology, and the differential genes related to mesoderm and endoderm development are screened by GO (Gene ontology) analysis.

[0028] In a seventh aspect, the present application provides a method for constructing an embryoid body-cardiomyocyte differentiation model, the method comprising the following steps:

[0029] Cardiomyocyte differentiation: the embryoid body cultured for 5 days is subjected to adherent culture to induce differentiation into cardiomyocytes, the induction and differentiation culture time is 4 weeks, thereby constructing an embryoid body-cardiomyocyte differentiation model;

[0030] RNA-seq analysis: the influence of Dlc1 gene knockout on cardiomyocyte differentiation is analyzed by RNA-seq technology, and the differential genes related to heart development, heart morphogenesis and cardiomyocyte function are screened by GO analysis.

[0031] In an eighth aspect, the present application provides the application of the above-mentioned Dlc1 gene knockout embryonic stem cell line or the hanging drop-embryoid body model constructed by the above-mentioned method in germ layer differentiation, Dlc1 deficiency related disease research model and targeted Dlc1 related disease drug screening and / or preparation.

[0032] In a ninth aspect, the present application provides the application of the above-mentioned Dlc1 gene knockout embryonic stem cell line or the embryoid body-cardiomyocyte differentiation model constructed by the above-mentioned method in Dlc1 related cardiovascular system differentiation research and disease modeling.

[0033] It should be noted that, due to the low transfection efficiency and low cell survival rate of the CRISPR / Cas9 gene editing technology in the prior art in the application of embryonic stem cells. The present application successfully constructs the Dlc1 gene knockout embryonic stem cell line by optimizing the transfection method (such as improving the pulse voltage to 1300V, the pulse width to 20, and the pulse number to 2), improving the shearing efficiency (such as multi-site gRNA design), and optimizing the screening conditions (such as using 1.5 μg / mL puromycin for 3 days). The cell line provides an important tool for gene function research and disease mechanism analysis, and provides technical support for the development of targeted Dlc1 related disease treatment strategies.

[0034] Compared with the prior art, the above-mentioned at least one technical solution adopted by the embodiments of the present application can achieve at least the following beneficial effects:

[0035] (1) Highly efficient targeting: This invention provides a combination of gRNAs that specifically target the Dlc1 gene, which can achieve precise and efficient gene knockout through the CRISPR / Cas9 system, and has the advantages of simple operation and low cost.

[0036] (2) Thorough knockout effect: The Dlc1 gene knockout ES cell line constructed based on the CRISPR / Cas9 system has been confirmed by gene and protein level detection to have successfully knocked out Dlc1. Compared with gene silencing, interference or knockdown techniques, knockout is more thorough and helps to conduct in-depth research on the function of Dlc1 protein.

[0037] (3) Stable ES cell line: The Dlc1 gene knockout ES cells constructed in this invention have no significant differences from the normal group cells in terms of proliferation, apoptosis and pluripotency. They can be stably passaged for a long time, providing an ideal gene knockout model for the study of Dlc1 gene and protein function.

[0038] (4) Multi-system research platform related to Dlc1 deficiency: By utilizing the multi-directional differentiation characteristics of Dlc1 gene knockout ES cells, a hanging drop-embryomorph model was successfully constructed, providing a research platform for studying germ layer differentiation, multi-system lesions caused by Dlc1 deficiency, and drug screening and preparation for Dlc1-related diseases.

[0039] (5) Cardiovascular system research tools related to Dlc1 deficiency: The embryonic body-myocardial differentiation model established based on Dlc1 gene knockout ES cells can be used to analyze the role mechanism of Dlc1 in cardiovascular diseases and provide important tools for cardiomyocyte regeneration, drug screening, diagnosis of heart-related diseases and development of gene therapy strategies. Attached Figure Description

[0040] Figure 1 The flowchart illustrates the construction of a Dlc1 knockout ES cell line using CRISPR-Cas9 technology. Based on the common coding region of the Dlc1 transcript, gRNAs 1, 2, and 3 were designed and synthesized. These three gRNAs were then cloned into the PX459 vector to construct the PX459-Dlc1-gRNA1 / 2 / 3 expression vector (i.e.,...). Figure 1 The green plasmid structure loop in the PX459-Dlc1-gRNA plasmid was transfected into ES cells, and single-clonal cell lines were isolated after screening with puromycin and the genotype was determined by sequencing.

[0041] Figure 2 The image shows the initial screening of Dlc1 knockout ES cell clones using PCR with identification primers P-sg1 / 2. The figure shows a representative agarose gel electrophoresis image of the PCR products; the first column is the DNA marker, control1-3 are the wild-type control cell numbers, and A4-F12 are the ES cell clone numbers.

[0042] Figure 3 Figure 1 shows the PCR primary screening of Dlc1 knockout ES cell clones using identification primer P-sg3. The figure is a representative agarose gel electrophoresis map of PCR products, the first column is DNA marker, and A1-F12 is the ES cell clone number.

[0043] Figure 4 Figure 2 shows the DNA level identification of Dlc1 knockout ES cell lines. Wherein Figure 4 Figure 2A is the DNA sequencing map of Dlc1 knockout cell strain D2, Figure 2B is the DNA sequencing map of Dlc1 knockout cell strain A5, and Figure 2C is the DNA sequencing map of Dlc1 knockout cell strain F9.

[0044] Figure 5 Figure 3 shows the Western blot detection of protein level of Dlc1 knockout ES cell lines D2, A5, and F9, and β-tublin is the internal reference protein.

[0045] Figure 6 Figure 4 shows the flow cytometry analysis of Dlc1 + / + and Dlc1 - / - ES cells. Wherein Figure 6 Figure 4A is the representative flow cytometry detection map of EdU-labeled Dlc1 + / + and Dlc1 - / - ES cells; Figure 6 Figure 4B is the statistical map of the proliferation activity of Dlc1 + / + and Dlc1 - / - ES cells. The data is expressed as mean ± SD, n = 3 independent repeated experiments, ns (not significant).

[0046] Figure 7 Figure 5 shows the flow cytometry analysis of Dlc1 + / + and Dlc1 - / - ES cells. Wherein Figure 7 Figure 5A is the representative flow cytometry detection map of Annexin V and Propidium iodide (PI) double staining of Dlc1 + / + and Dlc1 - / - ES cells; Figure 7 Figure 5B is the statistical map of early apoptosis of Dlc1 + / + and Dlc1 - / - ES cells. The data is expressed as mean ± SD, n = 5 independent repeated experiments, ns (not significant).

[0047] Figure 8 Figure 6 shows the Dlc1 + / + and Dlc1 - / -Pluripotency analysis of ES cells. Among them Figure 8 A in Fig. 1 is Real-time PCR detection of the expression level of pluripotency marker gene Oct4 in wild type and Dlc1 knockout cell lines. The results were normalized by Gapdh as the internal reference gene. Data are expressed as mean ± SD, n = 6 independent repeated experiments, ns (not significant); Figure 8 B in Fig. 1 is the expression heat map of pluripotency genes selected based on RNA-seq data analysis in wild type and Dlc1 knockout cell lines; Figure 8 C in Fig. 1 is the GO enrichment analysis based on the differential genes between undifferentiated wild type and Dlc1 knockout cell lines.

[0048] Figure 9 Fig. 2 shows the schematic diagram of EB differentiation scheme of ES cells.

[0049] Figure 10 Fig. 3 shows the imaging record of Dlc1 + / + and Dlc1 - / - EB morphology and analysis. Among them Figure 10 A in Fig. 3 is the representative bright field image of Dlc1 + / + and Dlc1 - / - EB at day2-day5, scale bar 200 μm. Figure 10 B in Fig. 3 is the histogram of the long axis diameter of EB. Data are expressed as mean ± SD, N > 3 independent repeated experiments, n ≥ 91 in each group. *P < 0.05, ***P < 0.001.

[0050] Figure 11 Fig. 4 shows the flow cytometry analysis of Dlc1 + / + and Dlc1 - / - EB proliferation capacity. Among them Figure 11 A in Fig. 4 is the representative flow detection diagram of EdU labeled Dlc1 + / + and Dlc1 - / - EB at day3 and day5; Figure 11 B in Fig. 4 is the proliferation activity histogram of Dlc1 + / + and Dlc1 - / - EB cells. Data are expressed as mean ± SD, n = 3 independent repeated experiments, *P < 0.05, ns (not significant).

[0051] Figure 12 Fig. 5 shows the flow cytometry analysis of Dlc1 + / + and Dlc1 - / - EB apoptosis level. Among them Figure 12 A in Fig. 5 is the representative flow detection diagram of AnnexinV and PI double staining of Dlc1 + / + and Dlc1- / - Representative flow cytometry apoptosis detection plot of EBs; Figure 12 B in Fig. 1 is Dlcl + / + Dlcl - / - Statistical plot of early apoptosis of EB cells. Data are presented as mean ± SD, n = 6 independent repeats, ns (not significant).

[0052] Figure 13 Dlcl + / + Dlcl - / - Differential expression gene enrichment analysis of day3 EBs. Among them Figure 13 A in Fig. 2 is day3 Dlcl + / + Dlcl - / - EB GO enrichment analysis; Figure 13 B-C in Fig. 2 is based on day3 Dlcl + / + Dlcl - / - GO entries related to mesoderm and endoderm development between Dlcl Figure 13 D-E in Fig. 2 is GSEA (Gene set enrichment analysis) analysis of day3 Dlcl + / + Dlcl - / - Differential expression genes related to mesoderm and endoderm development in EBs. NES (Normalized enrichment score), normalized enrichment score.

[0053] Figure 14 Real-time PCR detection of Dlcl + / + Dlcl - / - EBs in the mesoderm and endoderm (Mesp1, Eomes, Gata4, Gata6) and ectoderm (Pax6, Nes) marker levels during germ layer differentiation period. The results were normalized by Gapdh as an internal control. Data are presented as mean ± SD, n≥6, *P<0.05, **P<0.01, ***P<0.001.

[0054] Figure 15 Teratoma germ layer differentiation. Among them Figure 15 A in Fig. 4 is Dlcl + / + Dlcl - / - HE staining of teratoma formed by ES cells, showing endoderm (small intestinal mucosal epithelial tissue), mesoderm (muscle and cartilage tissue), ectoderm (squamous epithelial tissue), scale bar = 20 μm; Figure 15 B in Fig. 4 is Real-time PCR quantitative analysis of Dlcl + / + Dlcl- / - Expression levels of endoderm, mesoderm and ectoderm markers in teratoma. Data are presented as mean ± SD, n = 4, *P < 0.05, **P < 0.01.

[0055] Figure 16 Schematic diagram of ES cell cardiomyocyte differentiation protocol.

[0056] Figure 17 Dlc1 + / + Dlc1 - / - The proportion of EBs containing beating clusters during ES cell cardiomyocyte differentiation. Data are presented as mean ± SD, n = 5, *P < 0.05, **P < 0.01.

[0057] Figure 18 Dlc1 + / + Dlc1 - / - Expression levels of cardiac marker genes (Tnnt2, Myl2, Myl7 and Myh6) during ES cell cardiomyocyte differentiation. Results were normalized to Gapdh. Data are presented as mean ± SD, n = 6, *P < 0.05, **P < 0.01, ***P < 0.001.

[0058] Figure 19 Dlc1 + / + Dlc1 - / - day9 EB differential expression gene function enrichment analysis. Wherein Figure 19 A in day9 Dlc1 + / + Dlc1 - / - EB GO enrichment analysis; Figure 19 B in day9 Dlc1 + / + Dlc1 - / - EB between the GO annotation items related to cardiac development, the volcano plot of differential genes is drawn; Figure 19 C in day9 Dlc1 + / + Dlc1 - / - EB differential expression genes related to cardiac development and cardiomyocyte differentiation. NES (Normalized enrichment score), normalized enrichment score.

[0059] Figure 20 Dlc1 + / + Dlc1 - / - day9 EB differential expression gene KEGG (Kyoto encyclopedia of genes and genomes) signal pathway enrichment analysis.

[0060] Figure 21 Dlc1 in cardiomyocyte differentiation process + / + Dlc1 - / - day23 EB differential expression gene function enrichment analysis. Among them Figure 21 Dlc1 + / + Dlc1 - / - EB GO enrichment analysis; Figure 21 Dlc1 + / + Dlc1 - / - GO annotation entries related to heart development between day23 Dlc1 Figure 21 Dlc1 + / + Dlc1 - / - Genes related to myocardial contraction in day23 Dlc1 DETAILED DESCRIPTION

[0061] The embodiments of the present application will be described in detail below with reference to the drawings.

[0062] The above examples are merely illustrative of the embodiments of the present application. Numerous modifications and adaptations thereof will be apparent to those skilled in the art without departing from the spirit and scope of the present application. Therefore, the scope of the present application is not limited to the specific embodiments described herein, but only defined by the claims appended hereto.

[0063] It is to be understood that the foregoing description is that of certain examples of the application and that numerous changes in the details of construction and the combination and arrangement of parts can be made by those skilled in the art without departing from the scope of the application. It is intended that all such changes be within the scope of the following claims.

[0064] The experimental methods in the following examples are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0065] The data of the specific examples are all expressed as mean ± standard deviation (SD), and at least three independent repeated experiments are performed. The data analysis uses Two-tailed unpaired t test to identify the differences between two groups. One-way ANOVA is used to analyze the differences between more than two groups. P < 0.05 is considered statistically significant. Specific examples:

[0067] Example 1: Construction of Dlc1 knockout ES cell line based on CRISPR-Cas9 technology

[0068] The process of constructing Dlc1 knockout ES cell line by CRISPR-Cas9 technology: design gRNA, construct PX459-gRNA expression vector, ES cell electroporation, puromycin screening, sequencing identification, etc. Figure 1 The main principle is that gRNA guides Cas9 nuclease to target and recognize PAM sequence (Protospacer adjacent motif, usually NGG) on Dlc1 DNA sequence and cut 3-4 nucleotides upstream thereof, thereby causing double-strand break, which can induce cells to repair Dlc1 DNA by non-homologous end joining, forming mutant alleles characterized by insertion / deletion, and if it is a frameshift mutation and premature termination of protein translation, it can form Dlc1 gene knockout.

[0069] 1. Experimental materials

[0070] Eukaryotic expression vector PX459 (pSpCas9(BB)-2A-Puro, puromycin selection marker).

[0071] Mouse ES cell line E14.

[0072] 2. Construction of PX459-Dlc1-gRNA expression vector

[0073] (1) Design Dlc1 gRNA

[0074] a.Dlc1 gene has three transcripts (https: / / www.ncbi.nlm.nih.gov / gene / 50768) in common, transcript 1 (NM_001194940.2) is 7.6 kb in length and is a heart-specific transcript; transcript 2 (NM_015802.3) is 6.1 kb in length and is widely expressed in different tissues; transcript 3 (NM_001194941.1) is 6.2 kb in length. The three are different in the 5' end, and the protein encoded by transcripts 2 / 3 differs from transcript 1 only in the first 13 / 45 amino acid residues at the 5' end. The common coding region was selected, and the exon structure in the common coding region was determined. Three pairs of gRNA sequences gRNA1, gRNA2, and gRNA3 were designed based on exon 9, exon 10, and exon 11 of transcript 1 (Table 1, shown in bold).

[0075] b.Add enzyme cutting sites at both ends of the gRNA, add 5'-CACC-3' at the 5' end of the forward primer, and add 5'-AAAC-3' at the 5' end of the reverse primer (Table 1, shown underlined).

[0076] c.The U6 RNA polymerase III promoter in the PX459 vector for expressing gRNA is more inclined to guanine (G) nucleotides as the first base of transcription, so when the gRNA does not start with G, an extra G is added at the 5' of the forward primer, and the complementary base C is added in the corresponding reverse primer (Table 1, shown in italics).

[0077] Table 1 gRNA primer

[0078]

[0079] (2) gRNA primer annealing and phosphorylation to obtain gRNA double-stranded fragments

[0080] Reaction system

[0081]

[0082] Reaction procedure: 37°C for 30 min; 95°C for 5 min; decrease to 25°C at a rate of 5°C / min.

[0083] (3) Cloning of gRNA double-stranded fragments into PX459 vector

[0084] a.The gRNA double-stranded fragments obtained in the previous step were diluted with ddH2O at a ratio of 1:200.

[0085] b.Ligation

[0086] Reaction system

[0087]

[0088] Reaction procedure: 37°C for 5 min, 21°C for 5 min, 6 cycles for 1 h.

[0089] (4) Transformation of competent cells and culture amplification

[0090] a. Take 50 μL Stbl3 competent cells on ice bath, add the ligation product of the previous step, and place on ice for 30 min.

[0091] b. Heat shock at 42°C water bath for 90 s.

[0092] c. Quickly transfer to ice, and ice bath for 2 min.

[0093] d. Add 500 μL LB medium to each tube, and incubate on a shaker at 37°C, 200 rpm for 1 h to recover the bacteria.

[0094] e. Take the bacterial culture solution to the LB culture plate containing 100 μg / mL ampicillin, and evenly spread the liquid. Place the plate upright in a 37°C incubator for 30 min to absorb the liquid, and then invert and incubate overnight.

[0095] (5) Small-scale plasmid extraction and identification

[0096] a. Select positive clones for culture: select 5-6 colonies from each plate, and aseptically inoculate a single colony into 3 mL of LB liquid medium (containing 100 μg / mL ampicillin) using a sterile 10 μL gun tip, and incubate on a shaker at 37°C, 200 rpm for 16 h for culture amplification.

[0097] b. Small-scale plasmid extraction: use the kit to perform small-scale plasmid extraction according to the manufacturer's instructions.

[0098] c. Sequencing identification: sequence the obtained plasmid using the U6 promoter forward primer U6-F, and select the correct PX459 vector with Dlc1-gRNA for the next experiment. Sequencing primer U6-F: 5'-GAGGGCCTATTTCCCATGATTCC-3' (as shown in SEQ ID NO. 10).

[0099] (6) Large-scale plasmid extraction: after sequencing verification, expand the bacterial culture. Use the large-scale extraction kit to prepare the endotoxin-free plasmid PX459-Dlc1-gRNA according to the manufacturer's instructions, and determine its concentration and purity.

[0100] 3. ES cell electroporation of PX459-Dlc1-gRNA expression vector

[0101] (1) Prepare cells

[0102] a. Culture the required amount of ES cells. The culture medium contains Knock-out DMEM, 15% fetal bovine serum, 1% penicillin-streptomycin, 1% L-glutamine, 1% non-essential amino acids, 0.1 mM β-mercaptoethanol, 10 3 U / mL leukemia inhibitory factor (LIF).

[0103] b. Centrifuge the cell suspension after digestion, and discard the supernatant. Then resuspend the cells by adding DPBS.

[0104] c. Perform cell counting, centrifuge again, and discard the supernatant. Resuspend the cells using buffer R in the Neon Transfection System 100 μL kit, so that the number of cells per 100 μL of buffer is 2 x 10 6 cells.

[0105] (2) Electroporation

[0106] a. According to the manufacturer's instructions, use the Neon electroporator and the matching transfection kit to perform electroporation.

[0107] b. According to the amount of cells obtained, the required amount of each plasmid is 1.5 μg. Take the corresponding volume of PX459-Dlc1-gRNA1 / 2 / 3 plasmid into a 1.5 mL sterile centrifuge tube.

[0108] c. Add the single-cell suspension to the above-mentioned tube containing plasmid DNA, and mix gently.

[0109] d. Choose the appropriate electroporation program determined in the pre-experiment to perform electroporation, pulse voltage 1300 V, pulse width 20, and pulse number 2. e. Transfer the cell suspension after completing electroporation to a culture dish containing preheated culture medium. Mix the cells and culture them in a 37°C, 5% CO2 incubator.

[0110] 4. Puromycin drug screening Dlc1 knockout ES cell clones

[0111] (1) After 24 h of electroporation, use culture medium containing 1.5 μg / mL puromycin for screening for 3 days, and change the medium every day.

[0112] (2) After digestion, centrifugation, and resuspension of the ES cells, count the cells, and add cells at 200-400 cells / 10 cm dish, and change the medium every day.

[0113] (3) After one week of culture, select the single clones formed by the above-mentioned cells into a 96-well plate (1 clone corresponds to 1 well), and change the medium every day. (4) After 2 days of culture of the ES cells in the 96-well plate, digest them into a single-cell suspension, take out half of the cells in each well to a 0.2% gelatin pretreated 96-well plate for continued culture, and freeze the remaining half of the cells.

[0114] 5. Extraction of ES cell clone DNA

[0115] (1) The ES cells on the 96-well gelatin plate were cultured for 2 days before DNA extraction.

[0116] (2) Lysis Buffer was prepared in advance: 10 mM Tris pH 8.0, 10 mM EDTA, 10 mM NaCl, 0.2% SDS. Proteinase K was added to the Lysis buffer at a ratio of 1:20.

[0117] (3) The culture medium in the 96-well plate was aspirated, and DPBS was added to wash twice. 50 μL of Lysis buffer with proteinase K was added to each well, sealed with sealing film, and incubated in a wet box at 60°C overnight.

[0118] (4) A mixture of 75 μM NaCl prepared with cold ethanol was prepared in advance. 100 μL of the mixture was added to each well, and incubated at room temperature for 30 min.

[0119] (6) 150 μL of 70% ethanol was added to each well and washed for 3 times.

[0120] (7) The plate was inverted and air-dried at room temperature for 40 min. 50 μL of ddH2O was added to each well to dissolve the DNA, and the plate was stored at 4°C.

[0121] 6. PCR amplification for screening Dlc1 knockout ES cell clones

[0122] (1) Two sets of corresponding identification primers P-sg1 / 2 and P-sg3 (Table 2) were designed for Dlc1 gRNA1 / 2 / 3 predicted cleavage sites, respectively, for detecting the cleavage caused by gRNA1 / 2 and gRNA3. Each ES cell clone DNA was amplified by PCR using two sets of primers, and the wild-type products were 2080 bp and 392 bp, respectively.

[0123] Table 2 gRNA verification primers

[0124]

[0125] Reaction system

[0126]

[0127] Reaction profile: 98°C 5 min; 30 cycles (98°C 10 s, 60°C 30 s, 72°C 1 kb / min); 72°C 10 min; 4°C hold. (2) PCR amplification using P-sg1 / 2 as primers with extracted ES cell clone DNA as template, and agarose gel electrophoresis detection, the PCR product size is about 800-2000 bp Figure 2 ) PCR amplification using P-sg3 as primers, the product size is about 200-400 bp Figure 3 ) The knockout ES cell clone number is recorded after the preliminary screening by this step.

[0128] 7. Identification of Dlc1 knockout ES cell clone genotype by T-A clone (blue-white spot)

[0129] (1) PCR amplification using P-sg1 / 2 and P-sg3 as primers with the DNA of selected Dlc1 knockout ES cell clone as template by the foregoing method.

[0130] (2) Agarose gel electrophoresis of the PCR product, and gel recovery.

[0131] (3) 4°C overnight ligation of the gel-purified PCR product with pGEM-T Easy vector.

[0132] 10 μL ligation system is as follows:

[0133]

[0134] (4) Transformation: 5 μL ligation product is added to 50 μL DH5α competent cells, mixed gently, and placed in ice bath for 30 min. 42°C heat shock for 90 s, and quickly transferred to ice for 3 min. 900 μL LB medium is added, and incubated at 37°C for 1 h.

[0135] (5) Preparation of ampicillin / IPTG / X-Gal culture plate: IPTG (0.5 mM) and X-Gal (80 μg / mL) are added to the LB culture plate containing 100 μg / mL ampicillin, and evenly coated, and the plate is placed at 37°C for 30 min to allow the liquid to be absorbed.

[0136] (6) Blue-white spot screening by plating: after centrifugation of the bacterial liquid after recovery, 700 μL supernatant is discarded from each tube, and the remaining bacterial liquid is plated on the ampicillin / IPTG / X-Gal culture plate, and the liquid is evenly coated. The plate is placed upright at 37°C until the liquid is absorbed, and incubated at 37°C overnight. The vector colonies with successful ligation appear white, and the colonies with unsuccessful ligation appear blue.

[0137] (7) Selection and expansion of white colonies: 2 mL LB medium containing 100 pg / mL ampicillin was dispensed into a 15 mL sterile centrifuge tube. A white colony was picked with a sterile 10 pL tip and directly placed into the centrifuge tube. The culture was incubated at 37 °C, 200 rpm, overnight.

[0138] (8) Sequencing identification: The overnight culture was subjected to small-scale plasmid extraction and sent to a sequencing company for genotype identification. Sequence alignment analysis of the sequencing data confirmed the type of allelic mutation, and ultimately identified three homozygous frameshift mutant cell clones: D2, A5 and F9 (Table 3, Figure 4 ). DNA sequencing results showed that the Dlc1 gene in the D2 cell strain had a 7 bp deletion, causing a frameshift mutation, which resulted in the 1101st amino acid of the encoded protein being replaced by Asp (Aspartic acid) and an early stop codon being generated at the subsequent 41st amino acid; the Dlc1 gene in the A5 cell strain had a 44 bp deletion, causing a frameshift mutation, which resulted in the 1097th amino acid of the encoded protein being replaced by Ala (Alanine) and an early stop codon being generated at the subsequent 8th amino acid; the Dlc1 gene in the F9 cell strain had a 212 bp deletion, causing a frameshift mutation, which resulted in the 833rd amino acid of the encoded protein being replaced by Phe (Phenylalanine) and an early stop codon being generated at the subsequent 16th amino acid (Table 3, Figure 4 ).

[0139] (9) gRNA knockout efficiency and mutation analysis: Three gRNAs (gRNA1: c.2673-2692, gRNA2: c.3072-3091, gRNA3: c.3285-3304) were designed, all of which could effectively mediate Dlc1 gene knockout. The deletion fragments of the D2 and A5 cell strains were mainly concentrated around the gRNA3 target site; the deletion fragments of the F9 cell strain were generated by the combined action of gRNA1, gRNA2 and gRNA3, resulting in multiple site deletions in the gene (Table 3).

[0140] Table 3 Genotype identification of Dlc1 knockout ES cell clones and corresponding mutant protein types

[0141]

[0142] (10) The above table of gene mutation naming rules mainly refers to the HGVS (Human genome variation society) naming rules. "del" = deletion, deletion mutation; "fs" = frame shift, frameshift mutation; "*" = termination codon, stop codon.

[0143] 8. Verification of protein expression level of Dlcl knock-out ES cell lines

[0144] Western blot detection of Dlcl protein level: Dlcl protein level was detected by Western blot. Dlcl - / - D2, Dlcl - / - A5, Dlcl - / - F9 and Dlcl + / + Cell lines protein. Western blot results showed that compared with Dlcl + / + Cell lines, Dlcl - / - Dlcl protein expression was not detected in D2, A5 and F9 cell lines. Figure 5

[0145] Based on the above DNA level and protein level verification, it is proved that the Dlcl gene knock-out ES cell line is successfully constructed. Based on the above results, A5 cell line is selected for subsequent functional study.

[0146] Example 2: Dlcl + / + and Dlcl - / - Phenotype observation and functional study of ES cells

[0147] ES cells have the characteristics of unlimited proliferation, self-renewal and pluripotent differentiation in vitro culture. In order to verify whether Dlcl gene is involved in the physiological process of ES cell growth and maintenance, the proliferation, apoptosis and pluripotency of Dlcl + / + and Dlcl - / - ES cells were detected respectively.

[0148] 1. Dlcl + / + and Dlcl - / - ES cell proliferation ability analysis

[0149] Flow cytometry was used to detect Dlcl + / + and Dlcl - / - ES cell proliferation activity:

[0150] (1) According to the manufacturer's instructions for EdU cell proliferation detection, 2x EdU working solution (20 μM) diluted with ES cell culture solution was prepared in advance. After preheating the working solution at 37°C water bath, the original culture medium was added into 6-well plate with the same volume, and cultured at 37°C, 5% CO2 incubator for 1.2 h.

[0151] (2) Remove the culture medium, digest the ES cells, centrifuge in the centrifuge tube, and remove the supernatant.

[0152] (3) Each tube was fixed with 1 mL 4% PFA for 15 min. Centrifuge to remove the supernatant.

[0153] ​(4) Resuspend and wash with PBS containing 3% BSA for 5 min. Repeat 3 times.

[0154] (5) Permeabilize with PBS containing 0.3% Triton X-100 for 10 min. Centrifuge to remove supernatant.

[0155] (6) Resuspend and wash with PBS containing 3% BSA for 5 min. Repeat 2 times.

[0156] (7) Prepare Click reaction solution in advance. The reaction system is 500 μL per well of 6-well plate:

[0157]

[0158]

[0159] Reaction procedure: 30 min at room temperature in the dark.

[0160] (8) Remove the reaction solution and resuspend and wash with PBS containing 3% BSA for 5 min. Repeat 3 times.

[0161] (9) Use flow cytometry to detect Aizde-Alexa Fluor 647 fluorescence intensity. It is found that Dlc1 knockout does not affect the proliferation activity of ES cells Figure 6 ).

[0162] 2, Dlc1 + / + and Dlc1 - / - Analysis of the apoptosis level of ES cells

[0163] Use flow cytometry to detect Dlc1 + / + and Dlc1 - / - Apoptosis of ES cells:

[0164] (1) According to the instructions of the apoptosis kit, prepare 1 x Binding Buffer

[0165] (2) Digest ES cells, collect cells, wash once with PBS, and then wash once with 1 x Binding Buffer.

[0166] (3) Resuspend the cells with 100 μL of 1 x Binding Buffer, and the density is 1-5 x 10 5 cells / 100 μL.

[0167] (4) Add 5 μL of APC-Annexin V flow cytometry antibody to the resuspended cells, mix well, and incubate at room temperature in the dark for 15 min.

[0168] (5) Wash cells once with 1x Binding Buffer and resuspend with 200 μL 1x Binding Buffer.

[0169] (6) Add 5 μL of PI staining solution to the resuspended cells and incubate at room temperature for 5 min.

[0170] (7) Flow cytometry analysis of apoptosis. It was found that Dlc1 + / + Dlc1 - / - ES cells showed no significant difference in apoptosis level Figure 7 ).

[0171] 3、Dlc1 + / + Dlc1 - / - Analysis of pluripotency of ES cells

[0172] The pluripotency of ES cells is controlled by a set of key transcription factors, including Oct4, Nanog and Sox2. Real-time PCR detection showed that knocking out Dlc1 did not affect the RNA level of the core pluripotency marker Oct4 Figure 8 A). To better understand the molecular function of Dlc1 in ES cells, undifferentiated Dlc1 + / + Dlc1 - / - ES cells were subjected to RNA sequencing (RNA-seq) analysis, and it was found that the knockout of the Dlc1 gene did not affect the expression of key pluripotency genes Figure 8 B). According to the screening criteria of |Fold Change|>2 and corrected p.value<0.05, a total of 93 differentially expressed genes were identified between undifferentiated Dlc1 + / + Dlc1 - / - ES cells, of which 55 were up-regulated and 38 were down-regulated, and the overall gap between the two sets of data was relatively small. GO enrichment analysis of differentially expressed genes was performed, and only 24 significantly enriched GO entries were obtained, which were mainly related to DNA-specific binding in transcriptional regulation, and there was no GO function closely related to the maintenance of stem cell pluripotency Figure 8 C).

[0173] Based on the above analysis, the Dlc1 gene may not play a major role in the regulation of proliferation, apoptosis and pluripotency of ES cells.

[0174] Example 3: Dlc1 + / + Dlc1 - / - Phenotypic observation and functional study of ES cell germ layer differentiation

[0175] ES cells have the ability to differentiate into cells of all three germ layers (endoderm, mesoderm and ectoderm). To assess whether the Dlcl gene is involved in the physiological process of regulating germ layer differentiation of ES cells, we selected the hanging drop-EB differentiation model and teratoma formation experiment to explore.

[0176] 1. Hanging drop-EB differentiation

[0177] (1) Prepare ES cell differentiation medium: 15% fetal bovine serum, 1% penicillin-streptomycin, 1% L-glutamine, 0.1 mM β-mercaptoethanol, 1% non-essential amino acids, Knock-out DMEM.

[0178] (2) Prepare a cell suspension of 4 x 10 4 cells / mL using the differentiation medium.

[0179] (3) Prepare a square bacterial culture dish and add DPBS to cover the bottom of the dish to prevent the cell suspension from drying.

[0180] (4) Drop 20 μl / drop (about 800 cells) of the mixed and evenly distributed cell suspension onto the inner surface of the bacterial culture dish cover, and carefully cover the dish cover. Incubate in a 37°C, 5% CO2 incubator, and mark day 0. Incubate for 2 days to allow the ES cells to aggregate and form EBs.

[0181] (5) On day 2, rinse the droplets on the dish cover with DPBS, carefully collect the EBs that have formed, transfer the EBs to a centrifuge tube and allow them to settle for a few minutes, discard the supernatant, resuspend the EBs in the ES cell differentiation medium, and transfer them to a bacterial culture dish for suspension expansion culture for 3 days. Figure 9 )。

[0182] 2. Dlcl + / + and Dlcl - / - EB morphology detection

[0183] During the EB differentiation process, from day 2 to day 5, a certain number of EBs were collected each day to image and record their growth changes, and the EB size was measured and counted. Figure 10 ) Dlcl + / + and Dlcl - / - EBs can form relatively uniform spherical EBs at day 2-day 4, and the shape of the EBs tends to be irregular at day 5 Figure 10 A). Compared with wild type, day 2 Dlcl - / - EBs are larger in diameter, while day 5 Dlcl - / - EBs are smaller in diameter; during day 3 and day 4, Dlcl - / - EBs are slightly smaller than wild type but have no statistical difference Figure 10B). The results showed that Dlc1 gene knockout might affect the growth of EBs.

[0184] 3、Dlc1 + / + With Dlc1 - / - Analysis of the proliferative capacity of EBs

[0185] Flow cytometry analysis of day3 and day5 Dlc1 + / + With Dlc1 - / - The proliferative activity of EBs, day3 Dlc1 - / - EBs had a lower proliferation rate than wild type, while day5 showed no significant difference, which was consistent with the results of Dlc1 - / - EB size change Figure 11 , suggesting that Dlc1 might be involved in regulating the proliferation of EB cells during differentiation.

[0186] 4、Dlc1 + / + With Dlc1 - / - Analysis of the apoptosis level of EBs

[0187] Flow cytometry analysis of day3 and day5 Dlc1 + / + With Dlc1 - / - The apoptosis level of EBs, both showed no significant difference in apoptosis level Figure 12 ).

[0188] 5、RNA-seq analysis of the effect of Dlc1 deletion on EB embryonic differentiation

[0189] (1) Dlc1 + / + With Dlc1 - / - EB cells at the day3 stage of embryonic differentiation were selected for RNA-seq and bioinformatics analysis.

[0190] (2) According to |Fold Change|>2 and corrected P<0.05, 1006 different genes were identified between day3 Dlc1 + / + With Dlc1 - / - EBs.

[0191] (3) The top 30 annotation terms of GO analysis showed that Dlc1 gene knockout at this stage significantly enriched genes related to mesoderm development / fate determination and endoderm fate determination Figure 13 A).

[0192] (4) According to GO annotation, a total of 24 different genes were related to mesoderm development and most were up-regulated genes, among which multiple mesoderm key markers (such as Mixl1, Dkk1, Eomes, T, Mesp1) were significantly increased in expression Figure 13 B).

[0193] (5) According to GO annotations, there are 18 differentially expressed genes related to endoderm development, and their expression is generally upregulated, including some important endoderm markers (such as Foxa2, Cxcr4, Sox17, and Gata6). Figure 13 C).

[0194] (6) Similar to the GO analysis results, the GSEA analysis showed that, compared with the wild type, Dlc1 - / - In EB, the expression of genes related to endoderm differentiation was significantly upregulated. Figure 13 DE).

[0195] (7) RNA-seq results were further validated by Real-time PCR. Compared with the control group, Dlc1 - / - In EB, on day 3, the expression of mesodermal and endoderm markers (Mesp1, Eomes, and Gata6) increased, while the expression of ectoderm marker (Pax6) decreased. Figure 14 ).

[0196] (8) In summary, the knockout of the Dlc1 gene promotes the formation of the mesoderm and endoderm.

[0197] 6. Experimental analysis of teratoma formation: the effect of Dlc1 deletion on in vivo germ layer differentiation.

[0198] In vitro EB differentiation experiments using ES cells demonstrated that Dlc1 gene knockout can affect trigerm layer differentiation to some extent. To further investigate whether this effect exists in the in vivo environment, we conducted a teratoma formation experiment.

[0199] (1) A certain number of Dlc1 + / + With Dlc1 - / - ES cells were mixed with matrix gel and subcutaneously injected into SCID-Beige mice to form teratomas. Four weeks later, the mice were sacrificed and teratoma samples were obtained.

[0200] (2) Teratoma samples were fixed overnight in neutral formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE). Dlc1 was found. + / + With Dlc1 - / - ES cells all have the ability to differentiate into the three germ layers (ectoderm, mesoderm, and endoderm), such as endoderm (intestinal mucosa), mesoderm (cartilage and muscle), and ectoderm (epidermis). Figure 15 A).

[0201] (3) RNA was extracted from teratoma samples, and germ layer differentiation markers were analyzed by real-time PCR. The results showed that Dlc1... - / -The expression levels of mesoderm and endoderm genes Mesp1 and Eomes in teratoma increased, and the expression level of ectoderm gene Pax6 decreased Figure 15 B), which is basically consistent with the detection results of EB differentiation.

[0202] (4) In summary, these results show that Dlc1 deletion still has the ability to form three germ layers, but promotes mesoderm and endoderm formation and inhibits ectoderm formation, further confirming the RNA-seq analysis.

[0203] The above results show that Dlc1 plays a key role in early embryonic development, and Dlc1 deletion promotes mesoderm and endoderm differentiation while inhibiting ectoderm differentiation at the three-germ layer stage. Abnormal germ layer development can lead to defects in important organs, such as cardiovascular hypoplasia, or absence or malformation of endoderm-derived organs (lungs, liver, digestive tract), ultimately leading to embryonic lethality. This mechanism can partially explain the phenomenon that Dlc1 knockout mice die at E10.5 due to multiple organ failure.

[0204] The construction of Dlc1 knockout ES cell models can further induce the formation of functional cells based on germ layer differentiation, such as liver cells and lung epithelial cells derived from endoderm, cardiomyocytes and kidney cells derived from mesoderm, and neurons and epidermal cells derived from ectoderm. This model overcomes the limitations of Dlc1 knockout mice, which cannot obtain tissue and organ samples at later developmental stages due to early embryonic lethality, providing an ideal experimental platform for the study of multiple system diseases caused by Dlc1 deficiency.

[0205] Example 4: Dlc1 + / + Dlc1 - / - Phenotypic observation and functional study of ES cell cardiomyocyte differentiation

[0206] DLC1 is a susceptibility gene for various diseases, including neurological diseases, cardiovascular diseases, kidney diseases, and lung diseases, but the specific molecular pathogenesis still needs further study. This embodiment constructs a cardiomyocyte differentiation model based on EB differentiation to explore the effect of Dlc1 deletion on cardiomyocyte differentiation, providing a theoretical basis for elucidating the mechanism of Dlc1 in the cardiovascular system.

[0207] 1. ES cell cardiomyocyte differentiation model

[0208] (1) The treatment of ES cells from day 0 to day 5 is the same as the hanging drop-EB differentiation model in Example 3.

[0209] (2) day5: 0.2% gelatin was used to treat 96-well plates for 15 min in advance, and then 100ul of ES cell differentiation medium was added to each well.

[0210] (3) Use a pipette to aspirate droplets and transfer EB one by one to a 96-well plate for adherent culture to induce cardiomyocyte differentiation. Differentiate to day 28 according to experimental requirements. Figure 16 ).

[0211] 2. Dlc1 + / + With Dlc1 - / - Analysis of the proportion of pulsations during the differentiation of ES-cell cardiomyocytes

[0212] During cardiomyocyte differentiation, the number of blast cells (EBs) containing pulsatile clusters was observed and recorded from day 6 to day 28. EBs began to show pulsation on day 7, and most EBs contained pulsatile clusters on days 8 and 9, until day 18 (Dlc1). + / + With Dlc1 - / - The EB pulsation phenotypes were basically the same, and the pulsation ratio remained at around 90%, indicating successful cardiomyocyte differentiation. From day 20 onwards, Dlc1... - / - The proportion of EB pulsation began to decline, and the rate of decline was significantly faster than that of wild-type ( Figure 17 The results suggest that Dlc1 gene knockout may affect the maintenance of cardiomyocyte phenotype.

[0213] 3. Dlc1 + / + With Dlc1 - / - Analysis of cardiac marker genes during ES cell myocardial differentiation

[0214] During the 28-day differentiation process of embryonic endocarditis (EB), real-time PCR was used to detect the expression of four genes: Tnnt2 (related to myocardial contraction), Myl2 (regulating early embryonic heart development and contraction), and Myl7 and Myh6 (important structural genes for atrial and ventricular myocardium). Overall, all four genes reached peak expression on day 8. Except for Tnnt2, which remained highly expressed until day 28, the expression levels of Myl2, Myl7, and Myh6 began to decline after day 16. This overall expression trend was largely consistent with the pulsational changes in EB. Figure 18 In addition, Dlc1 - / - ES cell-derived EB showed significant alterations in the expression levels of four genes compared to wild-type. Dlc1 - / - In EB, Tnnt2 expression levels remained consistently lower than in the wild-type population from day 12 until day 28. (Dlc1) - / - In EB, the expression levels of three other genes, Myl2, Myl7, and Myh6, were lower than those of wild-type from day 8 onwards, and this low expression of Myl2 and Myh6 persisted after day 20. Figure 18 This result is consistent with Dlc1. - / - EB gene knockout fails to maintain a consistent phenotype with myocardial pulsation. In conclusion, Dlc1 gene knockout affects cardiomyocyte differentiation and phenotype maintenance.

[0215] 4. RNA-seq analysis of the effect of Dlc1 deletion on EB differentiation into mature cardiomyocytes

[0216] Day 9, mid-stage of cardiomyocyte differentiation, and Day 23, late stage of cardiomyocyte differentiation, were selected for RNA-seq and bioinformatics analysis.

[0217] (1) Analysis of RNA-seq data of day 9, mid-stage of cardiomyocyte differentiation

[0218] a. According to the criteria of |Fold Change| > 2 and corrected P < 0.05, 29 differentially expressed genes were screened in day 9 Dlc1 + / + compared with Dlc1 - / - EBs.

[0219] b. GO analysis showed that genes related to heart morphogenesis were enriched at this stage Figure 19 A).

[0220] c. According to GO annotation, a total of 29 differentially expressed genes related to heart development and morphogenesis were screened, including multiple important cardiomyocyte structure genes such as Myh7, Myl2, Tnnc1, Actn2, Cacna1c Figure 19 B).

[0221] d. GSEA analysis showed that compared with wild type, Dlc1 - / - EBs were enriched in genes related to heart development, cardiomyocyte differentiation, and contraction, and their expression was significantly down-regulated Figure 19 C).

[0222] e. KEGG analysis was similar to GSEA analysis, and the top 10 enriched pathways were mainly related to cardiomyopathy and myocardial contraction Figure 20 ).

[0223] f. The above results were similar to the expression trend of mature cardiomyocyte markers detected by Real-time PCR, confirming that the transition of Dlc1 - / - EBs to mature cardiomyocytes was inhibited to a certain extent.

[0224] (2) Analysis of RNA-seq data of day 23, late stage of cardiomyocyte differentiation

[0225] a. According to the criteria of |Fold Change| > 2 and corrected P < 0.05, 351 differentially expressed genes were screened in day 23 Dlc1 + / + compared with Dlc1 - / - EBs.

[0226] b.The top 30 enriched terms before GO analysis were mainly related to muscle fiber assembly and proliferation and differentiation of myoblasts Figure 21 A), and most of the differential genes were down-regulated.

[0227] c.According to the GO annotation, 41 differential genes related to heart development and myocardial cell function were screened out, including multiple important myocardial cell structure genes such as Actc1, Actn2, Tnnc1 Figure 21 B).

[0228] d.Among the GSEA analysis results, multiple enrichment terms were related to myocardial contraction and myofilament structure and assembly, and most of them were down-regulated Figure 21 C).

[0229] e.These results are consistent with the changes in the EB beating phenotype and the expression trend of mature myocardial cell markers, proving that Dlc1 gene knockout affects the maintenance of myocardial cell phenotype in the late differentiation stage.

[0230] The above experimental results show that Dlc1 deletion leads to a decrease in the beating ability of myocardial cells, accompanied by abnormal expression of genes related to myocardial structure and contraction function (Tnnt2, Myl2, Myl7, Myh6). RNA-seq analysis further reveals that Dlc1 deletion significantly affects myocardial cell development, myofilament assembly and myocardial contraction-related pathways, and is enriched in gene sets related to cardiomyopathy and cardiovascular abnormalities, revealing the core role of Dlc1 in maintaining the function of mature myocardial cells.

[0231] The present application provides a Dlc1 knockout ES cell myocardial differentiation model for studying the key role of Dlc1 in myocardial cell differentiation, maturation and function maintenance, and providing experimental evidence for the pathogenesis of congenital heart disease and related cardiovascular diseases. The model overcomes the limitation of Dlc1 knockout mice that cannot be used to study myocardial development due to early embryonic lethality, and can be used as an important tool for studying the molecular pathological mechanism of cardiovascular diseases, and can be further used for myocardial cell regeneration, drug screening and exploration of gene intervention strategies.

[0232] The RT-PCR primers are as follows:

[0233]

[0234] The above examples are only for the purpose of illustrating the technical concept and characteristics of the present application, and the purpose is to enable those skilled in the art to understand the content of the present application and to implement it, and it cannot limit the protection scope of the present application. Any equivalent changes or modifications made in accordance with the essence of the present application should be covered within the protection scope of the present application.

Claims

1. A gRNA that specifically targets the Dlc1 gene, characterized in that, The gRNA specific to the Dlc1 gene is a combination of gRNA1, gRNA2 and gRNA3, wherein the sequence of gRNA1 is shown as SEQ ID NO. 1, the sequence of gRNA2 is shown as SEQ ID NO. 2, and the sequence of gRNA3 is shown as SEQ ID NO.

3.

2. A gene knockout vector, characterized by, The gene knockout vector is constructed by connecting the DNA sequence corresponding to the gRNA of claim 1 to a vector, and the gene knockout vector expresses the gRNA of claim 1.

3. A CRISPR / Cas9 system specifically targeting knock-out of a Dlc1 gene, characterized in that, The CRISPR / Cas9 system comprises the gRNA of claim 1 and the Cas9 protein.

4. The gRNA of claim 1, the gene knockout vector of claim 2, or the CRISPR / Cas9 system of claim 3 is used in any of the following: (1) specifically recognizing the Dlc1 gene; (2) knocking out the Dlc1 gene; (3) preparing a reagent or kit for knocking out the Dlc1 gene; (4) constructing a Dlc1 gene knockout cell line.

5. A method for constructing a Dlc1 gene knockout embryonic stem cell line, characterized by, The construction method comprises the following steps: Step S1: constructing a gene knockout vector; Step S2: electroporating the gene knockout vector into embryonic stem cells; Step S3: obtaining a Dlc1 gene knockout embryonic stem cell line by screening with puromycin and genotyping.

6. The construction method of claim 5, wherein, In the step S1, the following steps are specifically included: designing gRNA primers and obtaining a combination of gRNA1, gRNA2 and gRNA3, wherein the sequence of gRNA1 is shown as SEQ ID NO. 1, the sequence of gRNA2 is shown as SEQ ID NO. 2, and the sequence of gRNA3 is shown as SEQ ID NO. 3; annealing and phosphorylating the gRNA primers to obtain a gRNA double-stranded fragment; cloning the gRNA double-stranded fragment into a puromycin-resistant vector PX459 to obtain a gene knockout vector, wherein the gene knockout vector is named as PX459-Dlc1-gRNA1, PX459-Dlc1-gRNA2 and PX459-Dlc1-gRNA3.

7. The construction method of claim 5, wherein, In the step S2, each Dlc1 gene knockout vector is used in an amount of 1.5 μg, and electroporation transfection is performed using a Neon electroporator with the following parameters: pulse voltage 1300 V, pulse width 20, and pulse number 2.

8. The construction method of claim 5, wherein, In the step S3, the puromycin is added in an amount of 1.5 μg / mL, and the screening time is 3 days.

9. A Dlcl gene-knockout embryonic stem cell line, which is characterized by, The Dlc1 gene knockout embryonic stem cell line is constructed by the construction method of any one of claims 5-8.

10. A method for constructing a hanging drop-embryoid body differentiation model, characterized by, The construction method comprises the following steps: suspension drop culture for 2 days and three-dimensional suspension culture for 3 days to culture the Dlc1 gene knockout embryonic stem cell line to form a blastoid, thereby constructing a suspension drop-blastoid differentiation model; RNA-seq analysis: analyzing the effect of Dlc1 gene knockout on germ layer differentiation by RNA-seq technology, and screening differential genes related to mesoderm and endoderm development by GO analysis.

11. A method for constructing a model of embryoid body-cardiomyocyte differentiation, characterized by, The method comprises the following steps: Cardiomyocyte differentiation: the 5-day-old embryoid bodies were subjected to adherent culture to induce differentiation into cardiomyocytes, and the induction differentiation culture lasted for 4 weeks, thereby constructing an embryoid body-cardiomyocyte differentiation model; RNA-seq analysis: the effect of Dlc1 gene knockout on cardiomyocyte differentiation was analyzed by RNA-seq technology, and the differential genes related to heart development, heart morphogenesis and cardiomyocyte function were screened by GO analysis.

12. The use of the Dlc1 gene knockout embryonic stem cell line of claim 9 or the hanging drop-embryoid body model constructed by the method of claim 10 in germ layer differentiation, Dlc1 deficiency related disease research model, and in targeted Dlc1 related disease drug screening and / or preparation.

13. The use of the Dlc1 gene knockout embryonic stem cell line of claim 9 or the embryoid body-cardiomyocyte differentiation model constructed by the method of claim 11 in Dlc1 related cardiovascular system differentiation research and disease modeling.

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