CN112175909A

CN112175909A - VSX2 green fluorescent reporter gene human induced pluripotent stem cell line and construction method thereof

Info

Publication number: CN112175909A
Application number: CN202010953610.9A
Authority: CN
Inventors: 钟秀风; 向孟清; 郑丹丹; 王远; 张祖明; 晋康新
Original assignee: Zhongshan Ophthalmic Center
Current assignee: Zhongshan Ophthalmic Center
Priority date: 2020-09-11
Filing date: 2020-09-11
Publication date: 2021-01-05
Anticipated expiration: 2040-09-11
Also published as: CN112175909B

Abstract

The invention discloses a VSX2 green fluorescent reporter gene human induced pluripotent stem cell line and a construction method thereof. The VSX2 green fluorescence reporter gene hipSC constructed by the invention has bright reported fluorescence expression and strong specificity, can stably express for a long time, and can still detect strong fluorescence signals when tissue fixation and end detection of frozen sections are carried out. The VSX2 green fluorescence reporter gene hipSC line and the construction method thereof can provide useful tools for modeling, development mechanism and prevention and treatment research of retina-related diseases, and are used for tracking of retina cell fate return, optimizing of a differentiation system, sorting and purifying of eye cups, even downstream research such as drug screening and cell transplantation.

Description

VSX2 green fluorescent reporter gene human induced pluripotent stem cell line and construction method thereof

Technical Field

The invention belongs to the technical field of cell biology, and particularly relates to a VSX2 green fluorescent reporter gene human induced pluripotent stem cell line and a construction method thereof.

Background

The normal process of formation and development of the eye is controlled by a gene network, and any destructive influence of genetic or environmental factors on this process may lead to growth and structural defects of the eye, such as, for example, aphakia and microcuvetty. The ophthalmopathy and the microcephaly are considered as the most serious developmental eye abnormality at present, can cause serious visual disturbance which accounts for about 3 to 12 percent of the visual disturbance of children, and seriously affect the healthy life of human beings. The pathogenesis of the ophthalmopathy and the microphthalmia is not clear at present.

Visual system homeobox 2 (VSX 2), also known as CHX10, encodes a transcription factor that plays an important role in mammalian eye development (Liu et al, 1994). VSX2 was expressed early in retinal progenitor cells and finally abundantly in the inner nuclear layer of mature retina (Liu et al, 1994). In 2000, Ferda Percin et al found the first mutation in VSX2 in patients with small eye abnormalities. Later, the VSX2 mutation was also found in several other families with ocular abnormalities (Ammar et al, 2017; Burkitt Wright et al, 2010; Chassaing et al, 2014; Iseri et al, 2010; Ullah et al, 2016). The VSX2 gene mutation is associated with aphakia and microphthalmia, but the specific mechanism of occurrence is not clear at present.

Human induced pluripotent stem cells (hipscs) were first obtained by reprogramming in 2007 (Takahashi K et al, 2007). Since hipscs have unlimited proliferation ability and multipotentiality, various types of retinal cells can be theoretically obtained by induced differentiation in vitro. In recent years, related researches show that the hiPSCs can be differentiated to obtain three-dimensional (3D) retina organs under specific conditions, can simulate the development process of neural retina and the natural pathological change process of retina in vitro, provide a research platform for screening intervention drugs for retinal degenerative diseases and exploring other treatment methods, and also provide abundant seed cells for retinal transplantation treatment (Zhong et al, 2014; Li et al, 2018; Li et al, 2019). The rapid development of 3D retinal organoid technologies by induced differentiation of hiPSCs has made possible in vitro studies of human retinal development and retinal diseases. In recent years, the development of gene editing technologies such as clustered regularly spaced short palindromic repeats and related proteins 9 (CRISPR/Cas 9) and the like, and the fluorescent reporter cell lines constructed by using the gene editing technologies provide powerful tools for the live real-time observation of retinal organs, the mechanism research of retinal diseases, the screening of retinal disease intervention drugs, the retinal transplantation treatment research and the like (Phillips et al, 2018; Kaewkhaw et al, 2016).

Disclosure of Invention

Aiming at the current situations that living body research of VSX2 expressed in human retina is vacant and pathogenesis of VSX2 related diseases is not clear at present, the invention aims to construct an enhanced green fluorescent protein (eGFP) reporter gene hipSC system of VSX2 and a construction method thereof, and provides tools for real-time observation of retinal development, mechanism research of related diseases, screening of retinal disease intervention medicines and cell replacement therapy.

The first purpose of the invention is to provide a VSX2 green fluorescent reporter gene human induced pluripotent stem cell line, which is a human induced pluripotent stem cell line co-expressing a human VSX2 gene and a green fluorescent reporter gene eGFP.

The second purpose of the invention is to provide a construction method of a VSX2 green fluorescent reporter gene human induced pluripotent stem cell line, which comprises the following steps:

a. designing sgRNA according to a human VSX2 gene sequence, synthesizing an upstream primer and a downstream primer of the sgRNA, and inserting the sgRNA into a pSpCas9(BB) -2A-Puro (PX459) vector after denaturation and annealing to construct a PX459-VSX2_ sgRNA knockout plasmid;

b. inserting a LA-P2A-eGFP-RA fragment into a pBluescript SK (-) vector serving as a framework to construct a pBluescript-LA-P2A-eGFP-RA donor plasmid;

c. simultaneously electrotransfering a PX459-VSX2_ sgRNA knockout plasmid and a pBluescript-LA-P2A-eGFP-RA donor plasmid to hipSC; and screening and culturing the electroporated hipscs by using puromycin, selecting surviving monoclonals, carrying out subculture amplification, and identifying and confirming to obtain the VSX2 green fluorescent reporter gene human induced pluripotent stem cell line.

Preferably, the nucleotide sequence of the sgRNA upstream primer and the nucleotide sequence of the sgRNA downstream primer are shown in SEQ ID No.1 and SEQ ID No.2, respectively.

Preferably, the nucleotide sequence of the LA-P2A-eGFP-RA fragment is shown as SEQ ID NO. 3.

In the invention, an LA-P2A-eGFP-RA fragment (LA and RA are respectively a left homologous arm sequence and a right homologous arm sequence, P2A is a porcine teschovirus self-shearing peptide sequence, and eGFP is an enhanced green fluorescent protein sequence) is artificially synthesized and inserted into a pBluescript SK (-) vector, so that a pBluescript-LA-P2A-eGFP-RA gene editing donor plasmid is obtained.

Preferably, the step a comprises the following steps: designing sgRNA according to a human VSX2 gene sequence and synthesizing an upstream primer and a downstream primer of the sgRNA; digesting a pSpCas9(BB) -2A-Puro (PX459) vector by using BbS I enzyme for linearization, then connecting the linearized vector and denatured and annealed sgRNA upstream and downstream primers by using T4 DNA ligase, transforming a connection product into DH5 alpha competent escherichia coli, screening and culturing ampicillin, obtaining a plasmid with correct insertion through sequencing verification, and constructing to obtain a PX459-VSX2_ sgRNA knockout plasmid.

Preferably, the step b comprises the following steps:

b1. carrying out double digestion on pBluescript SK (-) vector linearization by using EcoR I enzyme and Xho I enzyme;

b2. amplifying a LA-P2A-eGFP-RA fragment by using an upstream primer and a downstream primer of LA-P2A-eGFP-RA, and performing double enzyme digestion on an amplification product by using EcoRI enzyme and XhoI enzyme; the nucleotide sequence of the upstream primer of the LA-P2A-eGFP-RA is shown as SEQ ID NO.4, and the nucleotide sequence of the downstream primer of the LA-P2A-eGFP-RA is shown as SEQ ID NO. 5;

b3. the linearized pBluescript SK (-) vector and the double-restriction enzyme-digested LA-P2A-eGFP-RA fragment are connected by using T4 DNA ligase, the connection product is transformed into DH5 alpha competent escherichia coli, ampicillin is screened and cultured, a plasmid with correct insertion is obtained through sequencing verification, and the pBluescript-LA-P2A-eGFP-RA donor plasmid is constructed.

Preferably, the step c of performing electrotransformation specifically comprises: culturing hiPSC with mTeSR1 culture solution before electrotransformation, removing the culture solution, washing with PBS, adding digestive juice to digest cells, centrifuging, and collecting cells; resuspend cells with electrotransfer buffer containing PX459-VSX2_ sgRNA knockout plasmid and pBluescript-LA-P2A-eGFP-RA donor plasmid, with the electrotransfer parameters set to: 1100V, 10ms/pulse, 3pulse, for electrical switching.

The puromycin screening culture in the step c specifically comprises the following steps: the electroporated hipscs were cultured until they were fused, and cultured and selected in mTeSR1 medium containing puromycin until the number of cell clones was not decreased.

The passage amplification culture of the step c specifically comprises the following steps: hiPSC cultures were passaged on plates with mTeSR1 medium and Matrigel coatings at confluency reaching 80% -90%.

The identification confirmation in the step c specifically comprises the following steps: the cell clones obtained were identified as follows: PCR identification of the insertion of the report fluorescent fragment, Sanger sequencing identification of the base sequence of the clone of the inserted report fluorescent fragment, and karyotype analysis identification of the change of the karyotype of the cell; STR detection to eliminate cell contamination; detecting the expression of the molecular marker of the pluripotent stem cell of the report cell line by an immunofluorescence method and RT-PCR; identifying the ability of the reporter cell line to differentiate into the three germ layers by an in vitro three germ layer formation assay; the retinal organoids are obtained by inducing the 3D retinal organoid technology, and the co-localization condition of the VSX2 protein and the autofluorescence protein eGFP is evaluated by an immunofluorescence method.

The invention has the following advantages:

by utilizing the VSX2 green fluorescence reporter gene hipSC system constructed by the invention, the expression condition of the VSX2 protein can be monitored in real time in vivo by observing the eGFP fluorescence intensity under a fluorescence microscope without fussy end detection;

the VSX2 green fluorescent reporter gene hipSC system constructed by the invention can be used for obtaining 3D retina organs in a differentiated manner, and the VSX2-eGFP reporter system has strong specificity, the expression time and cell specificity of eGFP green fluorescent protein are completely matched with VSX2 protein, precursor cells and bipolar cells which are expressed in a neural retina layer are enriched, and the cells are obviously distinguished from other types of cells such as retinal pigment epithelial cells and photoreceptor cells;

by utilizing the VSX2 fluorescent reporter gene hipSC system constructed by the invention, the VSX2-eGFP fluorescent reporter protein can still detect strong fluorescent signals when tissue fixation and end detection is carried out on frozen sections;

the VSX2 green fluorescence reporter gene hipSC system can provide a useful tool for modeling, development mechanism and prevention and treatment research of relevant diseases of retina, and is used for tracking of fate of retinal cells, optimizing a differentiation system, sorting and purifying eye cups, even performing downstream research such as drug screening and cell transplantation.

Drawings

Fig. 1 shows PCR and Sanger sequencing indicating correct insertion of eGFP reporter (100 μm scale), where: 1A: CRISPR/Cas9 mediated BC1-VSX2^eGFPSchematic representation of the construction of the hiPSC line; 1B: a hiPSC clone bright field map after puromycin screening; 1C: PCR identification results of 20 hiPSC clones randomly picked after puromycin screening are shown: WT is wild type control BC 1-iPSC; m is 100bp DNA Ladder; C1-C20 corresponded to 20 hipSC clones, respectively, and red indicated that P2A-eGFP knock-in succeeded clones; 1D: sanger sequencing results of clone C5.

FIG. 2 is BC1-VSX2^eGFP-karyotyping analysis of ipscs (scale 100 μm), wherein: 2A: BC1-VSX2 before colchicine treatment^eGFP-a brightfield map of ipscs; 2B: BC1-VSX2 after 2.5h colchicine treatment^eGFP-a brightfield map of ipscs; 2C: BC1-VSX2^eGFPiPSC karyotype analysis, showing normal G-band karyotype.

FIG. 3 is BC1-VSX2^eGFP-hiPSC-expressing pluripotent stem cell molecular tags(scale 100 μm), wherein: 3A: RT-PCR detection shows BC1-VSX2^eGFPThe pluripotency markers NANOG, OCT4, SOX2, DNMT3B and GDF3 of the hipSC (denoted VSX2 in the figure) and BC1-hipSC (denoted BC1 in the figure) were expressed at mRNA levels that were similar; 3B-E: immunofluorescence detection shows BC1-VSX2^eGFPNANOG, SSEA4, TRA-1-60 and OCT4 were all positively expressed in hipscs in red fluorescence (Alexa Fluor-555) and blue fluorescence (DAPI) in nuclei.

FIG. 4 is BC1-VSX2^eGFP-hiPSC possesses a capacity for tripdermic differentiation (scale 100 μm), wherein: 4A-F: immunofluorescence shows that the three-germ layer tissues obtained after induced differentiation express endoderm markers AFP (liver cells), mesoderm markers alpha-SMA (smooth muscle) and ectoderm markers TUJ1 (nerve cells), the markers (AFP, alpha-SMA and TUJ1) are red fluorescence (Alexa Fluor-555), and the cell nucleus is blue fluorescence (DAPI).

Fig. 5 is a graph of eGFP co-expressed with VSX2 protein in 3D retinal organoids (scale 100 μm) where: 5A-C: BC1-VSX2^eGFPThe iPSC is differentiated to obtain retina organoids and express a reporter fluorescent protein eGFP; 5A: a bright field map; 5B: eGFP expression in the neural retina layer; 5C: brightfield and eGFP overlay; 5D-I: immunofluorescence assay VSX2 protein co-localized with eGFP in retinal organoids, VSX2 was red fluorescent (Alexa Fluor-555), eGFP was green fluorescent (autofluorescence), and nuclei were blue fluorescent (DAPI).

Detailed Description

The following examples are further illustrative of the present invention and are not intended to be limiting thereof.

Example 1

FIG. 1A is the CRISPR/Cas9 mediated BC1-VSX2 of the invention^eGFPSchematic representation of the construction of the hiPSC line.

Maintenance culture of hipscs

1.1 materials and instruments:

1.1.1 cells: BC1-hiPSC, which is a gift given by Chilobrachys professor (university of science and technology of China).

1.1.2 reagents and consumables:

1) mTeSR1 medium: STEM CELL, # 05851;

2)EDTA：Invitrogen，15575-038；

3) PBS (1 ×): jinuo biomedical technologies, Inc., 14111202;

4)Matrigel：Corning，354277；

5)(-)-Blebbistatin：Sigma，B0560；

6) a six-hole plate: FALCON, 353046;

7) centrifuging the tube: FALCON, 352096.

1.1.3 Instrument:

1)CO₂an incubator: SANYO, MCO-20A 1C;

2) and (3) inverting the microscope: nikon, TS 100.

1.2 concrete steps:

hipscs were maintained in mTeSR1 medium with a confluency of approximately 80% to 90% for passaging. mTeSR1 medium was aspirated, washed with sterile PBS, added 0.5mM EDTA, and digested at 37 ℃ for 4-6 min. The EDTA was aspirated, the cells were blown down with a small amount of mTeSR1 medium, inoculated onto a Matrigel-coated plate, cultured with mTeSR1 containing 10. mu.M (-) -Blebbistatin at 37 ℃ with 5% CO₂Culturing in incubator, and changing the culture medium to mTeSR1 the next day.

Construction of PX459-VSX2_ sgRNA knockout plasmid

2.1 materials and instruments:

2.1.1 reagents and consumables:

1) BbS I endonuclease: NEB, # R3539S;

2) t4 DNA ligase: NEB, # M0202S;

3) agar powder: guangdong Huanji microbial science and technology, Inc., 028990A;

4) peptone: guangdong Huanji microbial science and technology Co., Ltd., 050170B;

5) yeast powder: kyoto Tokay microbial science, Inc., 050090;

6) sodium chloride: aladdin, C111533;

7) DH 5. alpha. competent E.coli: beijing Quanji Biotech Ltd, CD 201-01;

8) plasmid extraction kit: guangzhou Meiji Biotech Co., Ltd., P1231-03;

9) DNA gel recovery kit: guangzhou meji biotechnology limited, D2110;

10) agarose: biofroxx, 0002A;

11) ampicillin sodium: Sigma-Aldrich, A9518;

12) a bacterial culture dish: MCD100150, cantonese, filth bio-filtration gmbh;

13) pSpCas9(BB) -2A-Puro (PX459) plasmid (hereinafter referred to as PX459 plasmid): addgene, # 48139.

2.1.2 Instrument:

1) a constant-temperature incubator: Shanghai-Hengchang scientific instruments Inc., LH 150;

2) horizontal constant temperature shaking table: shanghai-chang scientific instruments ltd, THZ 300;

3) nucleic acid concentration detector: thermo, Nanodrop 2000;

4) nucleic acid level electrophoresis apparatus: beijing, six Biotech Ltd, DYCP-31 CN.

2.2 concrete steps:

2.2.1 design of synthetic VSX2_ sgRNA primers

The genome sequence information of a human VSX2 gene (GeneID:338917) is downloaded in an NCBI GeneBank database, the fact that the human VSX2 gene contains 5 exons in total is determined, sgRNAs are designed on a website http:// crispor.tefor.net/aiming at 100bp sequences (200 bp in total) before and after a No.5 exon reading frame terminator, the sgRNAs with the highest comprehensive scores (high cutting efficiency and low miss rate) are selected and sent to Shanghai biological engineering limited company to synthesize upstream and downstream primers, and the primers are subjected to denaturation and annealing (reaction condition parameters are: 95 ℃ for 5min, 4 ℃ and infinity) and then are reserved (namely VSX2_ sgRNAs). The sequences of the upstream and downstream primers of the sgRNA are shown in Table 1 (corresponding to SEQ ID NO.1 and SEQ ID NO.2 in the sequence table), and the PCR reaction system is shown in Table 2.

Table 1 VSX2_ sgRNA primer synthesis sequences

Table 2 VSX2_ sgRNA primer denaturation annealing system

2.2.2 amplification of PX459 plasmid

Absorbing 1 mu L of PX459 plasmid bacterial liquid, adding the bacterial liquid into 100mL of LB liquid culture medium (the LB culture medium configuration method is shown in Table 3), placing the bacterial liquid in a constant-temperature horizontal shaking table at 37 ℃ for 18h, then taking out the bacterial liquid, extracting to obtain PX459 plasmid, dissolving the plasmid by using TE Buffer, and detecting the concentration of the obtained plasmid.

TABLE 3 LB Medium protocol

2.2.3 enzyme digestion linearization of PX459 vector

The amount of PX459 plasmid was calculated according to the concentration, and 1. mu.g of the plasmid was used for the digestion reaction in a PCR apparatus (reaction conditions: 1h at 37 ℃ C., 4. degreeC.), and the digestion system was configured as shown in Table 4.

TABLE 4PX459 vector restriction enzyme linearization System

Preparing 1% agarose gel in advance, and after the enzyme digestion reaction is finished, carrying out electrophoretic separation to obtain a linearized PX459 vector for later use.

2.2.4 ligation of PX459 linearization vector to VSX2_ sgRNA

The VSX2_ sgRNA reaction solution prepared for use after annealing in 2.2.1 was treated with ddH₂The reaction mixture was diluted 200-fold with O, 2. mu.L of the diluted mixture was used for ligation reaction, and the ligation reaction was carried out in a PCR apparatus (25 ℃ C. for 1 hour, 4 ℃ C. for. infinity), and the ligation system was prepared as shown in Table 5.

TABLE 5 PX459 vector and VSX2_ sgRNA ligation System

2.2.5 transformation and selection of recombinants

Taking the ligation product in 2.2.4 and transforming the ligation product into DH5 alpha competent Escherichia coli, wherein the specific reaction conditions are as follows: after 2 mul of the ligation product was mixed with 50 mul of DH5 alpha, the mixture was ice-washed for 30min, heat-shocked for 1min at 42 ℃ in a PCR instrument, ice-washed for 3min, spread on LB solid medium plate containing ampicillin, incubated in a constant temperature incubator at 37 ℃ for 16h, and then single colonies were picked up and sent to Shanghai Biotechnology engineering Co., Ltd for Sanger sequencing.

2.2.6 extraction of PX459-VSX2_ sgRNA recombinant plasmid

And amplifying a single colony with the VSX2_ sgRNA inserted correctly by sequencing by using an LB liquid culture medium containing ampicillin, extracting a plasmid, and determining the concentration to obtain a PX459-VSX2_ sgRNA knockout plasmid.

Construction of pBluescript-LA-P2A-eGFP-RA Donor plasmid

3.1 materials and instruments:

3.1.1 reagents and consumables:

1) t4 DNA ligase: NEB, # M0202S;

2) agar powder: guangdong Huanji microbial science and technology, Inc., 028990A;

3) peptone: guangdong Huanji microbial science and technology Co., Ltd., 050170B;

4) yeast powder: kyoto Tokay microbial science, Inc., 050090;

5) sodium chloride: aladdin, C111533;

6) DH5 α competent e.coli: beijing Quanji Biotech Ltd, CD 201-01;

7) low endotoxin plasmid extraction kit: guangzhou Meiji Biotech Co., Ltd., P1231-03;

8) DNA gel recovery kit: guangzhou meji biotechnology limited, D2110;

9) agarose: biofroxx, 0002A;

10) ampicillin sodium: Sigma-Aldrich, A9518;

11) a bacterial culture dish: MCD100150, cantonese, filth bio-filtration gmbh;

12) pBluescript SK (-) vector: shanghai Lianmai bioengineering, Inc., LM 1893.

3.1.2 Instrument:

3) nucleic acid concentration detector: thermo, Nanodrop 2000;

3.2 concrete steps:

3.2.1 Synthesis of LA-P2A-eGFP-RA fragment and addition of enzymatic cleavage sites

Synthesizing a LA-P2A-eGFP-RA sequence (the nucleotide sequence of which is shown as SEQ ID NO. 3) by Shanghai Biotechnology engineering Co., Ltd, designing and synthesizing primers for amplifying LA-P2A-eGFP-RA by Primer 5 software (the Primer sequences are shown in Table 6 and correspond to SEQ ID NO.4 and SEQ ID NO.5 in the sequence table), and configuring a PCR reaction system: 2 XTaqMasterMix 25. mu.L, upstream and downstream primers 1.5. mu.L each, LA-P2A-eGFP-RA fragment 1. mu.L, sterile ultrapure H₂O21. mu.L, 50. mu.L total. Preparing 1.5% agarose, carrying out electrophoresis, purifying according to the specification of a gel recovery kit for later use, and thus obtaining the LA-P2A-eGFP-RA fragment with EcoRI and XhoI enzyme cutting sites added to two ends respectively. The reaction is carried out in a PCR instrument, and the reaction conditions are as follows: 95 ℃ for 3min, (95 ℃ for 15s, 55 ℃ for 15s, 72 ℃ for 90s) x 35 cycles, 72 ℃ for 5min, 4 ℃ infinity.

TABLE 6 amplification of LA-P2A-eGFP-RA fragment primer synthetic sequences

Note: the EcoRI cleavage sites are in italics and the Xho I cleavage sites are underlined.

3.2.2 amplification of pBluescript SK (-) plasmid

The pBluescript SK (-) plasmid bacterial liquid is sucked and added into 100mL LB liquid medium (the LB medium configuration method is shown in Table 3), and then placed into a constant temperature horizontal shaking table at 37 ℃ for 18h, and then the bacterial liquid is taken out, pBluescript SK (-) plasmid is obtained by extraction, and the concentration of the obtained plasmid is detected by dissolving the plasmid with TE Buffer.

3.2.3 double digestion linearization of pBluescript SK (-) vector and double digestion of LA-P2A-eGFP-RA fragment

A pBluescript SK (-) vector and a LA-P2A-eGFP-RA fragment double-enzyme digestion system with EcoRI and XhoI enzyme digestion sites added at two ends are respectively configured according to the table 7 and the table 8, reacted in a PCR instrument (1 h at 37 ℃ and infinity at 4 ℃), 1.5% agarose gel is configured, and after electrophoretic separation and purification according to the instructions of a gel recovery kit, a linearized pBluescript SK (-) vector and a double-enzyme digested LA-P2A-eGFP-RA fragment are obtained for later use.

TABLE 7 double-restriction linearization of the pBluescript SK (-) vector

TABLE 8 double cleavage of LA-P2A-eGFP-RA fragment

3.2.4 ligation of the linearized pBluescript SK (-) vector and the digested LA-P2A-eGFP-RA fragment

Adding the double-enzyme-digested LA-P2A-eGFP-RA fragment in 3.2.3 into ddH according to a certain proportion₂O dilution, ligation reaction was performed in 1. mu.L (100ng) and the ligation reaction was performed in a PCR apparatus (25 ℃ C. for 1h, 4 ℃ C. for. infinity), and the ligation system was prepared as shown in Table 9.

TABLE 9 ligation of the cleaved LA-P2A-eGFP-RA fragment to the linearized pBluescript SK (-) vector

3.2.5 transformation and screening of recombinants

Taking the ligation product in 3.2.4 and transforming the ligation product into DH5 alpha competent Escherichia coli, wherein the specific reaction conditions are as follows: after 2 mul of the ligation product was mixed with 50 mul of DH5 alpha, the mixture was ice-washed for 30min, heat-shocked for 1min at 42 ℃ in a PCR instrument, ice-washed for 3min, spread on LB solid medium plate containing ampicillin, incubated in a constant temperature incubator at 37 ℃ for 16h, and then single colonies were picked up and sent to Shanghai Biotechnology engineering Co., Ltd for Sanger sequencing.

3.2.6 extraction of pBluescript-LA-P2A-eGFP-RA plasmid

A single colony, which showed correct insertion of the LA-P2A-eGFP-RA fragment by sequencing, was amplified in LB liquid medium containing ampicillin, and plasmids were extracted and the concentration was determined to obtain pBluescript-LA-P2A-eGFP-RA donor plasmid.

4. Electric converter

4.1 materials and instruments:

4.1.1 reagents and consumables:

1) mTeSR1 medium: STEM CELL, # 05851;

2)Accutase：Gibco，A1110501；

3) PBS (1 ×): jinuo biomedical technologies, Inc., 14111202;

4)Matrigel：Corning，354277；

5)(-)-Blebbistatin：Sigma，B0560；

6) a six-hole plate: FALCON, 353046;

7) centrifuging the tube: FALCON, 352096;

8) electrotransfer buffer reagent: invitrogen, Neon MPK 10096.

4.1.2 Instrument:

1)CO₂an incubator: SANYO, MCO-20A 1C;

2) and (3) inverting the microscope: nikon, TS 100;

3) electrotransformation appearance: invitrogen, Neon MPK 5000;

4) a centrifuge: flying pigeon brand, TDL-40B.

4.2 concrete steps: BC1-hipSC was cultured until the confluency reached about 90%, mTeSR1 medium was aspirated. Washing with PBS, adding Accutase, digesting at 37 deg.C for 5min, removing Accutase, adding mTeSR1 culture medium, blowing off all cells, centrifuging at 1500r/min for 5min, and collecting cell precipitate. Cells were resuspended in electrotransfer buffer (total 100. mu.L) containing 10. mu.g of PX459-VSX2_ sgRNA knockout plasmid and 10. mu.g of pBluescript-LA-P2A-eGFP-RA donor plasmid, with the electrotransfer parameters set to: 1100V, 10ms/pulse, 3pulse, for electrical switching. The cell suspension after the electric conversion is inoculated in a six-well plate coated with Matrigel in advance according to different inoculation density gradients, and mTeSR1 culture medium is added for continuous culture.

5. Puromycin screening

5.1 materials and instruments:

5.1.1 reagents and consumables:

1) mTeSR1 medium: STEM CELL, # 05851;

2)EDTA：Invitrogen，15575-038；

3) PBS (1 ×): jinuo biomedical technologies, Inc., 14111202;

4)Matrigel：Corning，354277；

5)(-)-Blebbistatin：Sigma，B0560；

6) a six-hole plate: FALCON, 353046;

7) centrifuging the tube: BD FALCON, 352096;

8) puromycin: solarbio, P8230.

5.1.2 Instrument:

1)CO₂an incubator: SANYO, MCO-20A 1C;

2) and (3) inverting the microscope: nikon, TS 100.

5.2 concrete steps:

the cells after electrotransformation are firstly cultured by mTeSR1 culture solution until cell clones are about to fuse, then cultured and screened by mTeSR1 culture solution containing puromycin with the final concentration of 300ng/mL, fresh culture solution is replaced every day until the cell clone number is not reduced any more, and surviving monoclonals are picked under an optical microscope and respectively subjected to amplification culture.

PCR identification and Sanger sequencing

6.1 materials and instruments:

6.1.1 reagents and consumables:

1)EDTA：Invitrogen，15575-038；

2) the genome extraction kit comprises: shanghai Biyuntian Biotech Co., Ltd, D0065M;

3) PBS (1 ×): jinuo biomedical technologies, Inc., 14111202;

4) PCR enzyme: takara, RR 902;

5)DNA Marker：100bp DNA Ladder，BM301；

6) agarose; brofrox, 0002A;

7) centrifuging the tube: FALCON, 352096.

6.1.2 Instrument:

1) a PCR instrument: Bio-Rad, T100;

2) nucleic acid level electrophoresis apparatus: beijing, six Biotech Ltd, DYCP-31 CN.

3) A centrifuge: flying pigeon brand, TDL-40B.

6.2 concrete steps:

collecting sample by digesting and centrifuging the cell clone after amplification culture, preparing DNA mixed liquor by using a Biyuntian animal genome rapid identification kit, wherein the DNA mixed liquor adopts Takara Premix Taq^TM(Ex Taq^TMVersion 2.0plus dye) premixed enzyme, wherein PCR primers are verification upstream Primer 1 (VF 1) and verification downstream Primer 1 (VF Reverse Primer 1, VR1) (the Primer information is shown in Table 10), wherein VF1 is positioned at the upstream of the 5 'end of the genome corresponding to the left homology arm of the donor plasmid, and VR1 is positioned at the downstream of the 3' end of the genome corresponding to the right homology arm of the donor plasmid. The size of the insert fragment is 786bp, the size of a band obtained by cloning the insert fragment through PCR is 1468bp, and the size of a band of a wild type control BC1-iPSC clone is 682 bp. And (3) PCR system: premix Taq^TM(Ex Taq^TMVersion 2.0plus dye) 25. mu.L, upstream and downstream primers 1.5. mu.L each, DNA mixture 1. mu.L, sterile ultrapure H₂O21. mu.L, 50. mu.L total. The PCR program was set to: 95 ℃ for 3 min: (95 ℃ 15s, 55 ℃ 15s, 72 ℃ 1 min). times.35 cycles, 72 ℃ 5min, 4 ℃ infinity. 1.5% agarose gel electrophoresis, the band of interest was cut and sent to Sanger sequencing by Shanghai Bioengineering Co., Ltd.

TABLE 10 PCR identification primer information Table

STR detection and karyotyping

7.1 materials and instruments:

7.1.1 cells: BC1-hiPSC, which is a gift given by Chilobrachys professor (university of science and technology of China).

7.1.2 reagents and consumables:

1) mTeSR1 medium: STEM CELL, # 05851;

2)EDTA：Invitrogen，15575-038；

3) PBS (1 ×): jinuo biomedical technologies, Inc., 14111202;

4)Matrigel：Corning，354277；

5) a six-hole plate: FALCON, 353046;

6) culture dish: FALCON, 353003;

7) centrifuging the tube: FALCON, 352096.

7.1.2 Instrument:

1)CO₂an incubator: SANYO, MCO-20A 1C;

2) and (3) inverting the microscope: nikon, TS 100;

3) a centrifuge: flying pigeon brand, TDL-40B.

7.2 concrete steps:

BC1-VSX2^eGFPand (3) when the-iPSC is cultured until the fusion degree reaches about 80-90%, sucking out mTeSR1 culture medium, washing with PBS, adding 0.5mM EDTA, digesting at 37 ℃ for 5min, removing EDTA, adding mTeSR1 culture medium, blowing down all cells, centrifuging at 1500r/min for 5min, collecting cell precipitates, and sending the cell precipitates to Cobioer Biosciences company for STR detection. BC1-VSX2 with a degree of fusibility of about 50%^eGFP2.5 treatment of mTeSR1 medium with a final concentration of 0.8. mu.g/ml colchicine to iPSCh, absorbing the culture medium, washing by PBS, adding 0.25% trypsin, digesting for 1min at 37 ℃, adding mTeSR1 culture medium to blow down the cells, centrifuging for 10min at 1500r/min, collecting the cells, and sending to Guangzhou Daan clinical inspection center to finish the slide preparation and karyotype analysis.

8. Reverse transcription-polymerase chain reaction (RT-PCR)

8.1.1 cells: BC1-hiPSC, which is a gift given by Chilobrachys professor (university of science and technology of China).

8.1.2 reagents and consumables:

1) mTeSR1 medium: STEM CELL, # 05851;

2)EDTA：Invitrogen，15575-038；

3) PBS (1 ×): jinuo biomedical technologies, Inc., 14111202;

4)Matrigel：Corning，354277；

5) a six-hole plate: FALCON, 353046;

6) centrifuging the tube: FALCON, 352096.

8.1.2 Instrument:

1)CO₂an incubator: SANYO, MCO-20A 1C;

2) and (3) inverting the microscope: nikon, TS 100;

3) a centrifuge: flying pigeon brand, TDL-40B.

8.2 concrete steps:

BC1-hipSC and BC1-VSX2^eGFPCulturing iPSC until the fusion degree reaches about 80% -90%, sucking out mTeSR1 culture medium, washing with PBS, adding 0.5mM EDTA, digesting at 37 deg.C for 4-6min, removing EDTA, adding mTeSR1 culture medium, blowing down all cells, centrifuging at 1500r/min for 5min, collecting cell precipitate, sending to Guangzhou Hongxiang biological medicine science and technology limited, and detecting BC1-VSX2^eGFPmRNA expression levels of pluripotent stem cell molecular markers in-iPSC and BC1-iPSC, and GAPDH was used as an internal reference gene. The primer information is shown in Table 11.

TABLE 11 RT-PCR detection primer information Table

9. Formation of three germ layers

9.1 materials and instruments:

9.1.1 reagents and consumables:

1) mTeSR1 medium: STEM CELL, # 05851;

2)EDTA：Invitrogen，15575-038；

3) PBS (1 ×): jinuo biomedical technologies, Inc., 14111202;

4)Matrigel：Corning，354277；

5)(-)-Blebbistatin：Sigma，B0560；

6) DMEM/F12 basal medium: gibco, C11330500 BT;

7) non-essential amino acids (MEM-NEAA): gibco, 11140-050;

8)GlutaMAX：Gibco，35050-061；

9) fetal Bovine Serum (FBS): natocor, 10099-141;

10) beta-mercaptoethanol: invitrogen, 21985123;

11) culture dish: BIOFIL, TCD 000100;

12) centrifuging the tube: FALCON, 352096.

9.1.2 Instrument:

1)CO₂an incubator: SANYO, MCO-20A 1C;

2) and (3) inverting the microscope: nikon, TS 100;

9.2 concrete steps:

BC1-VSX2^eGFPwhen the fusion degree of-iPSC is about 80-90%, the mTeSR1 medium is aspirated, washed with PBS, and digested with 0.5mM EDTA at 37 ℃ for 4-6 min. Removing EDTA, blowing down all cells with mTeSR1 medium, transferring into a low adsorption dish, adding mTeSR1 medium containing 10 μ M (-) -Blebbistatin, shaking up, 37 deg.C, 5% CO₂The culture was carried out overnight. The day of embryoid body construction was regarded as day 0 of induced differentiation, day 1, the medium was replaced with mTeSR1 medium, day 12, the medium was replaced with MEFG medium (MEFG component: DMEM/F12 basal medium supplemented with 1% by volume MEM-NEAA, 10% by volume FBS, 1% by volume GlutaMAX, and 100. mu.M. beta. -mercaptoethanol), culturing was continued for 14 days, and collection was performedAnd (3) carrying out immunofluorescence detection on the sample to obtain a three-germ-layer marker.

10. Immunofluorescence detection

10.1 materials and instruments:

10.1.1 reagents and consumables:

1) PBS powder: boster immunolader, AR 0030;

2) PFA powder: sigma, P6148-1 KG;

3) sucrose powder: biofrox, 1245GR 500;

4) DAPI dye: NY809, 1:1000, Dong ren chemical technology (Shanghai) Co., Ltd;

5)TritonX100：MP Biomedicals,LLC；194854；

6)Donkey serum：Hyclone，XT-100；

7) rabbit anti-human NANOG primary antibody (1:100, ab21624, abbam, uk), mouse anti-human SSEA4 primary antibody (1:100, ab16287, abbam, uk), mouse anti-human TRA-1-60 primary antibody (1:100, ab16288, abbam, uk), mouse anti-human TUJ1 primary antibody (1:500, ab7751, abbam, uk), mouse anti-human alpha-SMA primary antibody (1:500, ab119952, abbam, uk), mouse anti-human AFP primary antibody (1:200, ab3980, abbam, uk), rabbit anti-human OCT4 primary antibody (1:200, a7920, bocam bio-technologies, ltd, martian, aitheny), goat anti-human VSX2 primary antibody (1:200, ab9016, Millipore);

8) donkey anti-rabbit Alexa Fluor-555 labeled secondary antibody (1:500, A31572, Thermo Fisher Scientific, USA), donkey anti-mouse Alexa Fluor-555 labeled secondary antibody (1:500, A31570, Thermo Fisher Scientific, USA), and donkey anti-sheep Alexa Fluor-555 labeled secondary antibody (1:500, A21436, Thermo Fisher Scientific, USA);

9) o.c.t embedding agent: oriental cherry, 4583;

10) anti-fluorescence quenching encapsulated tablet: biyunyan, P0128M-2.

10.1.2 Instrument:

1) inverted fluorescence microscopy: ZEISS, HAL 100;

2) solid microscope: LEICA, M26;

3) freezing microtome: LEICA, CM 1950;

4) a slide scanner: ZEISS, Axio Scan.21.

10.2 concrete steps:

BC1-VSX2^eGFPimmunofluorescence detection was performed after-iPSC and BC1-iPSC were immobilized with 4% PFA. Fixing the three-germ layer tissue and retina organs in 4% PFA, transferring to phosphate buffer solution containing sucrose with different mass fractions and gradients, performing gradient dehydration, O.C.T embedding, freezing and slicing, and performing immunofluorescence detection. The immunofluorescence detection method comprises the following steps: sealing and permeating 0.25% X-Triton100 containing 10% donkey serum at room temperature for 1h, dripping corresponding primary antibody, incubating at 4 ℃ overnight, washing out the primary antibody, dripping corresponding secondary antibody, and incubating at room temperature in a dark place for 1 h. Washing the secondary antibody, adding a DAPI solution, dyeing the nucleus for 5min in a dark place at room temperature, washing the DAPI, sealing the chip by an anti-fluorescence quenching sealing agent, and observing and recording the fluorescence expression condition of the corresponding channel under a fluorescence microscope.

11. Inducing differentiation to obtain 3D retina organoids

11.1 materials and instruments:

11.1.1 reagents and consumables:

1) mTeSR1 medium: STEM CELL, # 05851;

2)Matrigel：Corning，354277；

3) DMEM/F12 basal medium: gibco, C11330500 BT;

4) DMEM Basic culture medium: gibco, C11995500 BT;

5) n2 additive: gibco, 17502-048;

6) non-essential amino acids (MEM-NEAA): gibco, 11140-050;

7)GlutaMAX：Gibco，35050-061；

8) b27 (without vitamin a): gibco, 17504044;

9) heparin: sigma, 2mg/mL in PBS;

10) taurine: sigma, # T-0625;

11) fetal Bovine Serum (FBS): natocor, 10099-141;

12) antibacterial fungal agent (AA): gibco, 15240;

13) culture dish: BIOFIL, TCD 000100;

14) a tungsten needle;

11.1.2 Instrument:

1)CO₂an incubator: SANYO, MCO-20A 1C;

2) and (3) inverting the microscope: nikon, TS 100;

3) inverted fluorescence microscopy: ZEISS, HAL 100;

11.2 the concrete steps are as follows:

BC1-VSX2^eGFPwhen the fusion degree of-iPSC is about 70% -80%, the mTeSR1 medium is aspirated, washed with PBS, and digested with 0.5mM EDTA at 37 ℃ for 4-6 min. Removing EDTA, blowing down the cells with mTeSR1 culture medium to obtain small cell mass, transferring the cell mass into a low adsorption dish, adding mTeSR1 culture medium containing 10 μ M (-) -Blebbistatin, shaking up, 37 deg.C, 5% CO₂The culture was carried out overnight. The day of construction of embryoid bodies was taken as day 0 of induced differentiation. On days 1-3, the medium was gradually changed to NIM medium (NIM component: DMEM/F12 basal medium supplemented with 1% by volume of N2 additive, 1% by volume of MEM-NEAA, heparin to a final concentration of 2. mu.g/mL). The embryoid bodies were inoculated on days 4-7 onto Matrigel-coated plates for further culture. On day 16, the medium was changed to RDM medium (RDM component: DMEM/F12 basal medium supplemented with 35% by volume of DMEM Basic medium, 2% by volume of B27 (containing no vitamin A), 1% by volume of AA, and 1% by volume of MEM-NEAA). And on the 28 th day, the 3D retina with a good structure can protrude out of the bottom surface of the culture hole, the 3D retina tissue is picked up by a tungsten needle and is cultured in a low-adsorption culture dish in a suspension manner, and the 3D retina cup is obtained. The medium was changed to RC2 medium on day 42 (RC2 component: DMEM/F12 basal medium supplemented with 40% volume fraction DMEM Basic medium, 2% volume fraction B27 (without vitamin A), 1% volume fraction AA, 1% volume fraction MEM-NEAA, 10% volume fraction FBS, 1% volume fraction GlutaMAX, final concentration 100. mu.M taurine). And taking a picture to record the green fluorescence expression change, and collecting a sample to perform immunofluorescence detection.

Specific results are shown in FIGS. 1-5.

In puromycin screening, surviving monoclonals are picked under an optical microscope after puromycin screening, and the bright field diagram of one hiPSC clone is shown in fig. 1B.

FIG. 1C is the PCR-identified agarose gel electrophoresis results of 20 random hipSC clones picked after puromycin screening: WT is wild type control BC 1-iPSC; m is 1500bp DNA Ladder; C1-C20 correspond to 20 hipSC clones respectively, wherein C5, C11, C14 and C15 are clones with successful P2A-eGFP knock-in. The Sanger sequencing result of clone C5 is shown in FIG. 1D. The results of the above PCR and Sanger sequencing indicate that the eGFP reporter gene is correctly inserted.

BC1-VSX2 in STR detection and karyotype analysis^eGFPThe karyotyping results of the-ipscs are shown in fig. 2, wherein: 2A: BC1-VSX2 before colchicine treatment^eGFP-a brightfield map of ipscs; 2B: BC1-VSX2 after 2.5h colchicine treatment^eGFP-a brightfield map of ipscs; 2C: BC1-VSX2^eGFPiPSC karyotype analysis, showing normal G-band karyotype. The comparison of the STR detection result with an authoritative database such as ATCC shows that BC1-VSX2^eGFPThe-ipscs were not cross-contaminated with other cells.

Detection of BC1-VSX2 in reverse transcription-polymerase chain reaction (RT-PCR)^eGFP-mRNA expression level of pluripotent stem cell molecular markers in ipscs and BC 1-ipscs with GAPDH as reference gene; the results are shown in FIG. 3A, and the RT-PCR assay showed BC1-VSX2^eGFPThe pluripotency markers NANOG, OCT4, SOX2, DNMT3B and GDF3 of-hipSC (VSX 2 in the figure) and BC1-hipSC (BC 1 in the figure) were expressed at the same level of mRNA level.

The results of FIGS. 3B-E show that immunofluorescence assays revealed BC1-VSX2^eGFPThe pluripotent stem cell molecular markers NANOG, SSEA4, TRA-1-60 and OCT4 in hipSC are positively expressed and show red fluorescence (Alexa Fluor-555) and blue fluorescence (DAPI) in cell nucleus.

The results in FIGS. 4A-F show that immunofluorescence shows that the resulting trimodal tissue after induced differentiation expresses the endodermal marker AFP (liver cells), the mesodermal marker α -SMA (smooth muscle) and the ectodermal marker TUJ1 (nerve cells), with the markers (AFP, α -SMA, TUJ1) all fluorescent in red (Alexa Fluor-555) and the nucleus fluorescent in blue (DAPI). Description of BC1-VSX2^eGFP-hipscs possess a tripdermal differentiation capacity.

FIGS. 5A-C: BC1-VSX2^eGFPThe iPSC is differentiated to obtain retina organoids and express a reporter fluorescent protein eGFP; 5A: a bright field map; 5B: eGFP expression in the neural retina layer; 5C: brightfield and eGFP overlay; FIGS. 5D-I: immunofluorescence assay VSX2 protein co-localized with eGFP in retinal organoids, VSX2 was red fluorescent (Alexa Fluor-555), eGFP was green fluorescent (autofluorescence), and nuclei were blue fluorescent (DAPI). Indicating that eGFP is specifically expressed in early neural retinal layers and co-expressed with VSX2 protein.

Sequence listing

<110> Zhongshan ophthalmic center of Zhongshan university

<120> VSX2 green fluorescent reporter gene human induced pluripotent stem cell line and construction method thereof

<160> 5

<170> SIPOSequenceListing 1.0

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<211> 25

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<213> Artificial Sequence (Artificial Sequence)

<400> 1

caccgggagg acatggctta ggtca 25

<210> 2

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

aaactgacct aagccatgtc ctccc 25

<210> 3

<211> 1289

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ccaagctcga caagatggag caggacgagc ggggccccga cgctcaggcg gccatctccc 60

aggaggaact gagggagaac agcattgcgg tgctccgggc caaagctcag gagcacagca 120

ccaaagtgct ggggactgtg tctgggccgg acagcctggc ccggagtacc gagaagccag 180

aggaggagga ggccatggat gaagacaggc cggcggagag gctcagtcca ccgcagctgg 240

aggacatggc tggaagcgga gctactaact tcagcctgct gaagcaggct ggagacgtgg 300

aggagaaccc tggacctatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca 360

tcctggtcga gctggacggc gacgtgaacg gccacaagtt cagcgtgtcc ggcgagggcg 420

agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc 480

ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct 540

accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc 600

aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt 660

tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg 720

gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg 780

ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg 840

gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc 900

tgctgcccga caaccactac ctgagcaccc agtccgccct gagcaaagac cccaacgaga 960

agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg 1020

acgagctgta caagtaagtc aaggcgcgct cagatgccgg agccccaaga ctctgctctc 1080

ctcgggccct gtggtgctgg gagatgctct ctgaggcaag gcccagacct ggcctctgcc 1140

atcctccctg ttccccacag gtcctccatc acccctggtg gctgcaggca ccgctgggtt 1200

ctgactctgg accatgctga gacatccctc atctagtctt gacctctcca gcatcccagc 1260

ctcagaagcc ttcttgctgc ccacaacgt 1289

<210> 4

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atagaattcc caagctcgac aagatggagc a 31

<210> 5

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atactcgaga cgttgtgggc agcaagaagg cttctga 37

Claims

1. A VSX2 green fluorescence reporter gene human induced pluripotent stem cell line is characterized by being a human induced pluripotent stem cell line co-expressing a human VSX2 gene and a green fluorescence reporter gene eGFP.

2. A construction method of a VSX2 green fluorescent reporter gene human induced pluripotent stem cell line is characterized by comprising the following steps:

3. The method according to claim 2, characterized in that the sgRNA upstream and downstream primers have nucleotide sequences shown in SEQ ID No.1 and the sgRNA downstream primer has nucleotide sequences shown in SEQ ID No. 2.

4. The method of claim 2, wherein the nucleotide sequence of the fragment LA-P2A-eGFP-RA is shown in SEQ ID No. 3.

5. The method according to claim 3, wherein the step a comprises the steps of: designing sgRNA according to a human VSX2 gene sequence and synthesizing an upstream primer and a downstream primer of the sgRNA; digesting a pSpCas9(BB) -2A-Puro (PX459) vector by using BbS I enzyme for linearization, then connecting the linearized vector and denatured and annealed sgRNA upstream and downstream primers by using T4 DNA ligase, transforming a connection product into DH5 alpha competent escherichia coli, screening and culturing ampicillin, obtaining a plasmid with correct insertion through sequencing verification, and constructing to obtain a PX459-VSX2_ sgRNA knockout plasmid.

6. The method of claim 4, wherein said step b comprises the steps of:

7. The method according to claim 2, wherein the step c of electrically switching comprises: culturing hiPSC with mTeSR1 culture solution before electrotransformation, removing the culture solution, washing with PBS, adding digestive juice to digest cells, centrifuging, and collecting cells; resuspend cells with electrotransfer buffer containing PX459-VSX2_ sgRNA knockout plasmid and pBluescript-LA-P2A-eGFP-RA donor plasmid, with the electrotransfer parameters set to: 1100V, 10ms/pulse, 3pulse, for electrical switching.

8. The method according to claim 2, wherein the puromycin screening culture of step c comprises: the electrotransferred hipscs were screened by incubation with a puromycin-containing mTeSR1 medium until the number of cell clones no longer decreased.

9. The method according to claim 2, wherein the subculture of step c comprises: hiPSC cultures were passaged on plates with mTeSR1 medium and Matrigel coatings at confluency reaching 80% -90%.

10. The method according to claim 2, wherein the identification confirmation of step c is specifically: the cell clones obtained were identified as follows: PCR identification of the insertion of the report fluorescent fragment, Sanger sequencing identification of the base sequence of the clone of the inserted report fluorescent fragment, and karyotype analysis identification of the change of the karyotype of the cell; STR detection to eliminate cell contamination; detecting the expression of the molecular marker of the pluripotent stem cell of the report cell line by an immunofluorescence method and RT-PCR; identifying the ability of the reporter cell line to differentiate into the three germ layers by an in vitro three germ layer formation assay; the retinal organoids are obtained by inducing the 3D retinal organoid technology, and the co-localization condition of the VSX2 protein and the autofluorescence protein eGFP is evaluated by an immunofluorescence method.