CN113969264B - COG5 gene knockout human embryonic stem cell line, construction method and application - Google Patents
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- C12N2510/00—Genetically modified cells
Abstract
The invention discloses a human embryonic stem cell line with a COG5 gene knocked out, a construction method and application thereof, wherein the CRISPR/Cas9 gene editing technology is used for knocking out the COG5 in a human embryonic stem cell line chHES-90 and inducing the COG5 into a midbrain neuron, thereby providing a convenient and reliable experimental model for researching the genetic disease; meanwhile, the multi-directional differentiation capability of hESC is utilized, and a platform is provided for researching multi-system lesions caused by COG5 defects. The invention constructs a COG 5-defective human embryonic stem cell line by CRISPR/Cas9 gene editing technology, and induces the cell line into a midbrain neuron as a cell model for research, thereby providing a convenient and reliable experimental model for the gene disease.
Description
Technical Field
The invention relates to construction of a human embryonic stem cell line, in particular to a human embryonic stem cell line with a COG5 gene knocked-out function, a construction method and application. Belongs to the technical field of genetic engineering.
Background
The conserved oligomeric golgi (conserved oligomeric golgi, COG) complex is the core of golgi retrograde transport and is responsible for repositioning the glycosyltransferase that performed the last time into the golgi apparatus' flat capsule. The COG complex consists of eight different subunits, COG1-4 constituting leaf A and COG5-8 constituting leaf B. Loss of leaf a poses a threat to cell survival, while failure of leaf B often causes abnormalities in the glycosylation process. The defect of COG5 and COG7 interactions does not disturb leaf B stability but has an impact on vesicle transport and post processing of glycosyl groups in the flat vesicles.
The glycosylation disease of the COG5 defect is an autosomal recessive genetic disease, and the complete deletion of proteins is caused by gene mutation. The first case of COG5 gene mutation was reported by Paesold-Burda in 2009. To date, a total of 7 articles have been reported. The clinical symptoms of patients mainly comprise hypoevolutism, mental disorder, brain atrophy, microcephaly, dystonia, etc. Currently, there is no effective treatment means for patients with the gene. Since human nerve cells are hardly available in vitro, it is also unclear what changes are caused by glycosylation abnormalities to the nerve cells. Research using animal models or cell models of COG5 mutations is a possible research tool. At present, no report of a COG5 mutant mouse model is found at home and abroad, and the difference between brain development and nervous system in mice and human is caused, so that the situation of human brain cells cannot be truly reflected by the mouse brain cells after all, and the research by using the mouse model has limitation.
In recent years, many genome editing technologies have emerged, including Zinc Finger Nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided CRISPR/Cas9 nuclease systems. The first two techniques use a tethering strategy of the endonuclease catalytic domain to modularize the DNA binding protein, thereby inducing targeted DNA Double Strand Breaks (DSBs) at specific genomic sites. In contrast, cas9 is a small RNA-guided nuclease that, by pairing with target DNA via PAM sequences, represents a more easily designed, highly specific, efficient system that is well suited for gene editing of various cell types.
CRISPR/Cas9 is a gene editing system. Cas9 endonucleases are guided by guide RNAs (sgrnas) to recognize PAM (NGG) sequences on foreign genetic elements and cleave several bases upstream thereof, thereby stimulating Double Strand Breaks (DSBs) at the genomic locus of interest to facilitate genome editing. Once cleaved by Cas9, the target site typically undergoes two major DNA damage repair pathways: non-homologous end joining (NHEJ) or homologous repair (HDR) pathways. Without repair templates, DSBs are religated by the NHEJ process, which leaves an imprint in the form of an insertion/deletion (INDEL) mutation. INDELs occurring within the coding exons may lead to frameshift mutations and premature termination of protein translation, thereby mediating gene knockout. The same CPRSPR/Cas9 plasmid has the advantages that the efficiency of successfully transfecting embryonic stem cells is far lower than that of tumor cells, the shearing efficiency of sites is low, and the cells with successful gene defects are few; drug screening, monoclonal culture, etc. all increase the difficulty of editing cell growth, and meanwhile, the influence of the processes on stem cell stem property, karyotype, etc. needs to be considered.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a COG5 gene knockout human embryonic stem cell line, a construction method and application aiming at a research model lack of COG defect.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
1. the construction method of the COG5 gene knockout human embryonic stem cell line comprises the following specific steps:
(1) Annealing two sgrnas shown in SEQ ID No.1 and SEQ ID No.2 into single strands, and cloning into an improved lunti-CRISPR plasmid containing puromycin resistance and BsmBI endonuclease to obtain a vector lunti-CRISPR-COG 5 plasmid containing the sgrnas;
(2) Then co-transfecting plasmid N75, plasmid VSV-G and Lenti-CRISPR-COG5 plasmid into 293T cells with 80% confluency, centrifuging to obtain supernatant, infecting chHES-90 cells with the supernatant, and screening to obtain surviving cells;
(3) And then carrying out monoclonal amplification and sequencing on the surviving cells, and reserving cells defective in the COG5 gene to obtain the COG5 gene knockout human embryonic stem cell line.
Preferably, in step (2), the supernatant is filtered through a 0.45 μm filter before cell infection.
Preferably, in the step (2), the specific method of cell infection is as follows: the supernatant was first allowed to act on chHES-90 cells for 24 hours, then fresh medium was changed and infection continued until the total duration of action was 48 hours.
Preferably, in step (2), cell selection is performed using 3. Mu.g/mL purine.
2. The COG5 gene knockout human embryo stem cell line obtained by the construction method is used.
3. Application of COG5 gene knockout human embryonic stem cell line in preparing COG5 defect disease model.
Preferably, the human embryonic stem cell line firstly forms an embryoid body, and then is subjected to suspension for 5 days and adherent culture for 35 days to form a midbrain neuron, so that the preparation of the COG5 defect disease cell model can be realized.
The invention has the beneficial effects that:
the invention knocks out COG5 in the human embryonic stem cell line chHES-90 by CRISPR/Cas9 gene editing technology and induces the COG5 into neurons, thereby providing a convenient and reliable experimental model for researching the gene disease; meanwhile, the multi-directional differentiation capability of hESC is utilized, and a platform is provided for researching multi-system lesions caused by COG5 defects.
The invention constructs a COG 5-defective human embryonic stem cell line by CRISPR/Cas9 gene editing technology, and induces the cell line into neurons as a cell model for research, thereby providing a convenient and reliable experimental model for the gene disease.
The invention has the main advantages that the prior research researches human nervous system diseases through experimental animals such as mice and the like, has species differences, and the invention provides human nerve cells by inducing human embryonic stem cells into midbrain neurons, spans species barriers, and has more comprehensive and real experimental results.
Drawings
FIG. 1 is a flow chart for mediating knock-out of the COG5 gene in a human embryonic stem cell line using lentiviral vectors.
FIG. 2 is a Sanger sequencing of COG5 deficient abnormal human embryonic stem cell DNA showing a 4bp deletion of one strand and a 21bp deletion of the other strand.
FIG. 3 is a protein expression pattern of Western Blot detection defective hESCs. Wild type was used as a normal control and beta-actin was used as an internal reference protein. Defective no COG5 protein expression.
FIG. 4 is a LAMP2 protein migration map of Western Blot detection of defective hESCs. Wild type was used as a normal control and beta-actin was used as an internal reference protein. Defective LAMP2 proteins migrate on SDS-PAGE, suggesting a defect in glycosylation.
FIG. 5 is a graph of Golgi apparatus for wild-type and defective hESCs before and after BFA treatment. BFA may cause collapse of the golgi apparatus and migration toward the endoplasmic reticulum, while loss of COG5 may retard this process. Wild type and defective were subjected to BFA (2 ug/ml) for 11 minutes, after which the Golgi apparatus was labeled with Giantin. Wild type Giantin fluorescent markers diffuse, indicating that the golgi body is fusing to the endoplasmic reticulum; whereas the defective fluorescent label is not significantly dispersed.
FIG. 6 is a Golgi apparatus morphology of detection of wild-type and defective hESCs by transmission electron microscopy.
FIG. 7 is a diagram of immunofluorescence detection of knock-down pluripotency markers NANOG, TRA-1-81, OCT4, SOX 2.
FIG. 8 is a diagram showing a multi-functional marker for detecting defects by flow cytometry, wherein A is NANOG and B is OCT4.
FIG. 9 is a graph showing the ability to differentiate defective three germ layers. And (3) performing suspension culture on a complete culture medium (DMEM, knockout serum substitute, nonessential amino acid, beta mercaptoethanol and L-glutamine) without bFGF for 7 days to form embryoid bodies, and then pasting the embryoid bodies on the wall for 14 days to perform free differentiation. Post immunofluorescence detects the internal (AFP), intermediate (α -SMA), and external (TUBB 3) germ layer markers, respectively.
FIG. 10 is a diagram of a defective karyotype analysis. The results showed no chromosomal abnormalities.
FIG. 11 is a graph showing the results of detection of Mycoplasma deficiency. The Mycoplasma detection kit of Lonza was used to detect the presence of Mycoplasma contamination in the defect. B/A <1 indicates no mycoplasma contamination. The results showed no contamination.
FIG. 12 is a flow chart for differentiation of human embryonic stem cell lines into dopaminergic neurons provided by STEMCELL company.
Fig. 13 is a bright field view of dopaminergic neurons in humans induced by a defect for 40 days.
FIG. 14 is a graph showing immunofluorescence detection of defective human dopaminergic neuron maturation markers induced for 40 days. MAP2, neuronal markers; TH, dopaminergic neuron markers.
FIG. 15 is a statistical analysis of the differences between wild-type and defective N-glycosylation sites.
FIG. 16 is a plot of the frequency of amino acid occurrences upstream and downstream of defective glycosylation sites. The defective N-glycosylation site follows the N-X-S/T amino acid sequence.
FIG. 17 is a functional cluster map of defective N-glycosylation decreasing proteins compared to wild-type.
FIG. 18 is a profile of differences in N-glycosylation sites between wild-type and defective.
FIG. 19 is an enrichment heatmap of wild-type versus defective N-glycosylation differential sites in the KEGG pathway. The marked decrease in defective N-glycosylation sites is concentrated in Huntington's disease, long-acting inhibition of the hippocampus, dopamine synapses, etc.
Detailed Description
The invention is further illustrated in the following figures and examples, which are provided for the purpose of illustration only and are not intended to be limiting.
Examples:
1. the flow chart of the preparation method of the human embryonic stem cell model of the COG5 defect disease is shown in figure 1, and the specific steps are as follows:
(1) Cas9 endonuclease is guided by guide RNA (sgRNA) to recognize PAM (NGG) sequence on the exogenous genetic element and cleave several bases upstream thereof, thereby stimulating double strand breaks at the genomic site of interest to facilitate COG5 genome editing. The PAM site of the targeted human COG5 was designed as follows:
TACAGTCCGGGAACTTCTGC(+)AGG
(2) And sgrnas were designed for appropriate restriction sites:
f5'-3': CACCGTACAGTCCGGGAACTTCTGC, as shown in SEQ ID NO. 1;
r5'-3': AAACGCAGAAGTTCCCGGACTGTAC, as shown in SEQ ID NO. 2;
(3) The two sgrnas were annealed to double strands and cloned into a modified Lenti-CRISPR plasmid containing puromycin resistance and BsmBI endonuclease to form a Lenti-CRISPR-COG5 plasmid.
(4) Two packaging plasmids (N75, VSV-G, helper plasmids to help better enter the host) and the Lenti-CRISPR-COG5 plasmid containing sgRNA were co-transfected into 293T cells at 80% confluency using lipofectamine 2000 (purchased by INVITROGEN).
(5) After 24 hours the viral particle supernatant containing sgrnas was collected and filtered through a 0.45 μm filter.
(6) Lentiviral supernatants were allowed to react with human embryonic stem cells (chHES-90) for 24h. Fresh medium mTESR was changed after 24h TM (purchased by STEMCELL Co.).
(7) 48 days after infection, cells were screened with 3. Mu.g/mL puromycin.
(8) Surviving cells were subjected to monoclonal expansion on Matrigel (CORNING).
(9) Sanger sequencing (FIG. 2) was performed, and cells defective in the COG5 gene (homozygous mutation, composite heterozygous mutation) were retained, one of which was designated chHES-90-Cog5 -/- -C2. Western Blot was used to detect the content of COG5 protein (FIG. 3), confirming that homozygous mutation of COG5 resulted in its protein deletion.
(10) And (5) carrying out cell characterization on the selected human embryonic stem cell monoclonal. Immunofluorescence detection of pluripotency markers NANOG, SOX-2, OCT4, TRA-1-81 (fig. 7) and pluripotency markers AFP, α -SMA, TUBB3 (fig. 9); the cells after gene editing are proved to still have the characteristics of hESC. Flow cytometry examined pluripotency markers NANOG (a in fig. 8) and OCT4 (B in fig. 8), and determined that COG5 gene-deficient monoclonal cells were still high purity human embryonic stem cells. Meanwhile, the genetic stability of the monoclonal was determined by performing a karyotype test on the monoclonal (FIG. 10). The presence or absence of mycoplasma was detected for the monoclonal, demonstrating that it was free of common microbial contamination (fig. 11).
2. The phenotypic results of human embryonic stem cells for COG 5-deficient disease and the induced dopaminergic neuron cell model are as follows:
(1) COG5 deficient hescs were induced to human midbrain dopaminergic neurons following the induction protocol determined in this laboratory (fig. 12). COG 5-deficient cells are in aggresell TM Embryoid bodies were formed in containers (purchased by STEMCELL company) and subjected to suspension for 5 days and adherent culture for 35 days to form midbrain neurons, and the induction process was sequentially carried out using a nerve induction kit/midbrain neuron differentiation kit (+SHH)/midbrain neuron maturation kit of STEMCELL company. The dopaminergic neuron morphology after induction is shown in figure 13. The mature human dopaminergic neuron markers induced by immunofluorescence detection were seen to be positive for MAP2 (markers of mature neuronal tubulin) and TH (markers of mature dopaminergic neuron neurotransmitter) (FIG. 14).
(2) The human embryonic stem cells show cell phenotype caused by COG5 deficiency, and as shown in FIG. 4, the human embryonic stem cells with COG5 deficiency show tail phenomenon of LAMP2 protein migration, which is consistent with sample protein electrophoresis of COG5 gene mutant patients; protein transport inhibitor BFA treatment may result in the collapse of the golgi apparatus and migration to the endoplasmic reticulum, while loss of COG5 may retard this process. Normal hESC and COG5 deficient hESC were treated at 2ug/ml BFA for 11 min and the Golgi apparatus labeled with Giantin. As shown in FIG. 5, migration of normal hESCs after BFA treatment occurred, whereas migration of COG 5-deficient human embryonic stem cells was not seen in the Golgi apparatus. COG5 deficient hESC electron microscopy showed swelling of golgi and increased vacuoles, which were also consistent with that of COG5 gene mutated patients (fig. 6); the above results demonstrate that knocked-out hescs have been initially associated with the same phenomenon as cog5 deficient patient cells.
(3) To demonstrate that hESC-induced mesodopaminergic neurons are a good cell model for studying nervous system lesions caused by COG 5-deficient glycosylation, applicants used LC-MS/MS to quantify N-glyco-proteomics, comparing the levels of protein and glycoprotein expression in normal human embryonic stem cells and COG 5-deficient human embryonic stem cells. The results of library search and mass spectrometry data analysis give the signal intensity for each peptide in the different samples. Based on this information, the relative amounts of proteins are calculated. The ratio of the average of all biological duplicate quantitative values for each modification site in the comparative sample pair was regarded as fold change in difference (FC). The change threshold for significant upregulation is greater than 1.5 and the change threshold for significant downregulation is less than 1/1.5. The results showed 426 up-regulation modification sites and 223 down-regulation modification sites. Since there may be multiple peptides corresponding to one protein, 289 up-regulated proteins and 155 down-regulated proteins were identified (fig. 15).
(4) Large-scale modification histology experiments can identify thousands of post-translational modification sites for proteins in one experiment, knowing the underlying biological processes that lead to these modifications is an important research aspect of proteomics. Since the partial biochemical preference of an enzyme for a given substrate may be determined by residues surrounding the modification site, a peptide sequence consisting of 10 amino acids upstream and downstream of all the identified modification sites is used as an analysis object. The N-glycosylation sequence motif heat map shows that the most significantly enriched motif is the N-glycosylation site: alanine (a), cysteine (C), histidine (H), glycine (G), serine (S), threonine (T), valine (V) and tyrosine Y. Notably, serine and threonine are both frequently present in the second position (fig. 16).
(5) Since defects in COG5 may lead mainly to aberrant glycosylase recovery and localization, meaning that the degree of glycosylation of synapses, neurotransmitters, channel proteins associated with possible neural cells is reduced, applicants' analytical strategies have focused mainly on glycoprotein analysis with reduced glycosylation. As shown in FIG. 17, it was also confirmed that the differential glycoproteins with reduced glycosylation levels were mainly concentrated in receptor activity, channel activity, synapses, membranes, etc. (P < 0.005).
(6) In addition, the differentially expressed glycoproteins were further divided into four parts, called Q1 to Q4, according to fold differential expression (fig. 18). Q1 and Q2 are glycosylated down-regulated differential proteins and Q3 and Q4 are up-regulated differential proteins. Applicant performed GO classification, KEGG, and protein domain enrichment for each Q group, and performed cluster analysis to find correlations of protein function with different fold differential expression. KEGG enrichment results indicate that interactions of neurotransmitters down-regulated by N-glycosylation modification with synapses and extracellular matrix accumulate in COG5 deficient human embryonic stem cell-derived dopaminergic neurons, possibly associated with mental and neurological related diseases (fig. 19). Of those significantly different proteins modified by glycosylation, AMPAR and NMDAR are most often down-regulated in these pathways. This finding may suggest that cleavage of glutamine and its receptors may be an important cause of neurological abnormalities.
(7) Among proteins with significantly reduced levels of COG5 deficient dopaminergic neuron glycosylation, applicants focused on α -L-fucosidase (FUCA 1). Fucose deposition is an autosomal recessive lysosomal storage disease, caused by alpha-L-fucosidase deficiency, and accumulates fucose in tissues. Clinical features include angiokeratomas, progressive mental retardation, neurological symptoms, facial features roughness and multiple bone dysplasia. These symptoms are highly coincident with those of COG5 deficiency cases, suggesting that COG5 deficiency may cause abnormal glycosylation of FUCA1 and dysfunction, and cause accumulation of fucose in the patient. In addition, applicants have found that glycosylation of several enzymes, receptors and neurotransmitters is significantly reduced and can be used as a target protein for subsequent studies.
(8) In summary, COG5 knockout of hescs was performed by the constitutive gene editing plasmid, and COG5 homozygous deleted hescs were obtained. And then inducing to neurons to obtain normal and defective cell sample pairs with completely consistent genetic background, overcoming the defect that nerve cells of patients are not advisable, also making up the defect that mice are different from people in brain development and neuron functions, establishing a cell model for research of COG defect diseases, and providing examples of a COG5 gene knockout human embryonic stem cell line, a construction method and application.
While the foregoing description of the embodiments of the present invention has been presented with reference to the drawings, it is not intended to limit the scope of the invention, but rather, various modifications or variations can be made by those skilled in the art without the need of inventive effort on the basis of the technical solutions of the present invention.
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Claims (3)
1. The COG5 gene knockout human embryonic stem cell line is constructed by the following method:
(1) Annealing two sgrnas shown in SEQ ID NO.1 and SEQ ID NO.2 into single strands, and cloning the single strands into an improved Lenti-CRISPR plasmid containing puromycin resistance and BsmBI endonuclease to obtain a vector Lenti-CRISPR-COG5 plasmid containing the sgrnas;
(2) Then co-transfecting plasmid N75, plasmid VSV-G and Lenti-CR ISPR-COG5 plasmid into 293T cells with 80% confluency, centrifuging to obtain supernatant, infecting chHES-90 cells with the supernatant, and screening to obtain surviving cells;
(3) And then carrying out monoclonal amplification and sequencing on the surviving cells, and retaining the cells with homozygous mutation of the COG5 gene to obtain the human embryonic stem cell line with the COG5 gene knocked out.
2. Use of the COG5 gene knockout human embryonic stem cell line of claim 1 for preparing a COG defect disease model.
3. The use according to claim 2, wherein the human embryonic stem cell line of claim 1 is subjected to 5 days suspension and 35 days adherent culture to form a mesobrain neuron, thereby realizing the preparation of the COG5 deficiency disease model.
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