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GPD1L-deleted human embryonic stem cell strain and construction method and application thereof

A technology of human embryonic stem cells and construction methods, applied in the field of GPD1L-deficient human embryonic stem cell lines and their construction, can solve the problems of large species and physiological differences, physiological activity cannot be maintained, and the pathological process of heart disease cannot be effectively simulated. Achieve good stability and stable cell karyotype

Pending Publication Date: 2021-12-17
QIQIHAR MEDICAL UNIVERSITY
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  • Description
  • Claims
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Problems solved by technology

[0003] However, the main problem facing the current research is the lack of effective human myocardial models, and it is impossible to analyze the pathophysiological process of the myocardium from a relatively normal state to a phenotypic change. Conventional pathological methods can only be used to compare normal myocardium and diseased myocardium. possible pathogenesis speculated
A few studies have isolated diseased cardiomyocytes from the operation and tested them. However, after long-term enzyme treatment and artificial culture during the isolation of primary cardiomyocytes, their physiological activity cannot be maintained for more than 48 hours, and the progression of the disease cannot be displayed.
In the past, heart disease research mainly used animal models carrying disease-causing genes. However, there are large species and physiological differences between animal cardiomyocytes and human cardiomyocytes. For example, the heart rate of mice can reach 600 beats per minute, while that of humans is 60. ~100 beats / min, species differences make the electrophysiological activities of cardiomyocytes also very different, so it cannot effectively simulate the pathological process of human heart disease

Method used

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  • GPD1L-deleted human embryonic stem cell strain and construction method and application thereof
  • GPD1L-deleted human embryonic stem cell strain and construction method and application thereof
  • GPD1L-deleted human embryonic stem cell strain and construction method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0036] A method for constructing a GPD1L-deleted human embryonic stem cell line, comprising the following:

[0037] 1. Human embryonic stem cell maintenance culture and passage,

[0038] (1). In the biological safety cabinet, discard the old culture medium, wash once with PBS buffer solution, and replace with new E8 culture medium.

[0039] (2). Cells can be subcultured when the growth confluence reaches 70% to 80%. Wash once with PBS buffer, add 0.05mM EDTA, digest at 37°C for 5 minutes, gently absorb the digestion solution, add new E8 culture medium, blow and beat to make the cells fall off, the passage ratio can be determined according to the experimental situation, the general ratio is 1:6.

[0040] 2. GPD1L gene knockout sgRNA design,

[0041] (1) Download and save the CDS sequence of GPD1L: run Snapgene software, create a new DNA file, paste the CDS region sequence of GPD1L into New DNAFile, name and save for later use;

[0042] (2) Download and save the GPD1L gene sequ...

Embodiment 2

[0082] GPD1L missing human embryonic stem cell line GPD1L protein loss identification method, the specific method is

[0083] 1. Differentiation of GPD1L-deficient embryonic stem cells into cardiomyocytes

[0084] (1). Add 4ml of myocardial differentiation medium 1 to each well of GPD1L-deficient embryonic stem cells in a 6-well plate, and then add 4ml of myocardial differentiation medium 2 after culturing for 48 hours. After culturing for 48 hours, add 4ml of myocardial differentiation medium 3, and thereafter Change the myocardial differentiation medium 3 every 2 days;

[0085] (2). Taking the first day of myocardial differentiation culture medium 1 as the calculation, generally on the 8th to 10th day of myocardial differentiation, beating cardiomyocyte clusters can be seen under the microscope, and GPD1L-deficient embryonic stem cells can differentiate into cardiomyocytes.

[0086] 2. Extraction of total protein from cardiomyocytes derived from GPD1L-deficient human embryo...

Embodiment 3

[0107] GPD1L deletion human embryonic stem cell karyotype identification test, the specific method is as follows:

[0108] (1). GPD1L-deficient human embryonic stem cells are passaged into 6-well plates, and the confluence reaches about 50% for karyotype analysis;

[0109] (2). Replace the E8 culture medium containing 100ng / ml Colcemid (Gibco#15210040) for 2h;

[0110] (3). Wash once with PBS, add 0.05mM EDTA, digest for 5min, and centrifuge the cell suspension at 1000rpm for 3min to retain the cell pellet;

[0111] (4). Add 3ml of 0.075mol / L potassium chloride at 37°C to the cell pellet, mix well, and treat with hypotonicity at 37°C for 20 minutes;

[0112] (5). Add 1ml of newly prepared fixative solution (methanol:glacial acetic acid=3:1, volume ratio) for pre-fixation, mix the cell suspension, centrifuge at 1000rpm for 3min, and discard the supernatant;

[0113] (6). Add 3ml of fixative in step (5), mix gently, and let stand at room temperature for 20 minutes;

[0114] (...

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Abstract

The invention provides a GPD1L-deleted human embryonic stem cell strain and a construction method and application thereof, and relates to the technical field of genetic engineering. According to a gene editing principle, sgRNA of a targeted GPD1L gene exon 4 is synthesized, a gene knockout plasmid is constructed, screening is carried out after human embryonic stem cells are transfected, a basic group A is inserted into the obtained GPD1L diallele exon 4, a termination codon TGA appears at the 157 amino acid position of protein after GPD1L expression, and protein translation is terminated in advance. The GPD1L-deleted human embryonic stem cell line has the advantages of GPD1L protein deletion, normal karyotype, normal stem cell pluripotent marker and good continuous passage stability, trigerm teratoma can be formed in an animal body, and mycoplasma pollution is avoided. The GPD1L-deleted human embryonic stem cell H9 line can be used for researching the influence of GPD1L deletion on the function of differentiated myocardial cells and the screening of therapeutic drugs.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a GPD1L-deficient human embryonic stem cell line and its construction method and application. Background technique [0002] With the introduction of the concept of Precision Medicine, the era of human post-genome medicine has been opened, and the Human Gene Mutation Database (HGMD) has been constructed to reveal the gene function and the pathogenic mechanism after mutation from the molecular biology level. Reasonable individualized treatment plan. The protein expressed by GPD1L is glycerol phosphatedehydrogenase 1-like, which is mainly distributed in the cytoplasm and under the cell membrane. It participates in the regulation of the function of the cardiomyocyte membrane protein Nav1.5 and is highly expressed in human cardiomyocytes. In HGMD, the diseases caused by point mutations of GPD1L are recorded as Brugada syndrome, sudden cardiac death, sudden neo...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85C12N15/53C12N15/113C12Q1/02
CPCC12N5/0606C12N15/85C12N9/0006C12N15/113G01N33/5008C12Y101/01008C12N2510/00C12N2800/107Y02A50/30
Inventor 董韬林岩岳丽玲沈雷潘洪明纪慧金海峰刘吉成
Owner QIQIHAR MEDICAL UNIVERSITY
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