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Method for constructing LQT disease model and application of LQT disease model in drug screening

A technology of disease model and culture, applied in the field of constructing LQT disease model, can solve the problem that LQTS disease model needs to be further developed, and achieve the effect of low cancer risk, high safety and high reprogramming efficiency

Inactive Publication Date: 2015-06-24
SHENZHEN SANQI BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, the current research on LQTS disease models still needs to be in-depth

Method used

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  • Method for constructing LQT disease model and application of LQT disease model in drug screening
  • Method for constructing LQT disease model and application of LQT disease model in drug screening
  • Method for constructing LQT disease model and application of LQT disease model in drug screening

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Retroviral reprogramming

[0050] (1) Donor cells LQTL1 and LQTL2 were cultured with urine cell expansion medium (CIB, Cat#Iso-1030). The day before virus infection, urine epithelial cells were divided into 1 well of 12-well plate according to 20,000 cells.

[0051] (2) Transfect pMXs virus plasmid into 293T cells by PEI transfection method, that is, use 293T cells to package virus pMXs-Oct4(O), pMXs-Sox2(S), pMXs-Klf4(K), pMXs-c-Myc respectively (M) and pMXs-eGFP, the retrovirus encoding pMXs-Sox2 / Klf4 / Oct4 / c-Myc was obtained, and the virus supernatant was filtered with a 0.45 μm filter membrane to obtain the infection solution.

[0052] (3) Add 8 μg / ml polybrene to the infection solution, and use the virus combination OSKM to infect the cells LQTL1 and LQTL2 obtained in step (1) twice, and eGFP-infected cells as a control for infection efficiency, after infection The cells were further cultured with human induced pluripotent stem cell (hiPSC) reprogrammin...

Embodiment 2

[0057] Example 2: Non-integrated iPSC reprogramming

[0058] 1) The donor cell LQTH1 was cultured with urine cell expansion medium (CIB, Cat#Iso-1030).

[0059] 2) Digest the donor cell LQTH1 into single cells with trypsin before transfection, stop the digestion with urine cell expansion medium (CIB, Cat#Iso-1030), centrifuge at 200g for 3mins, discard the supernatant, and use the culture medium The cells were resuspended, counted with trypan blue, and 1 million cells were taken for nucleofection.

[0060] 3) Combining Episome plasmids (purchased from Addgene, where the plasmid pCEP4-M2L uses CIB to replace c-Myc with L- Myc plasmids) were transferred into 1 million donor cells in total, and the structure diagram of the Episome plasmid combination is shown in figure 2 a.

[0061] 4) Spread the transfected cells into 2 ECM-coated T25 flasks, culture them with human induced pluripotent stem cell (hiPSC) reprogramming medium (CIB, Cat#Rep-1101), at 37°C, 5% CO 2 cultured und...

Embodiment 3

[0065] Example 3: iPSC identification

[0066] (1) Karyotype analysis

[0067] 1) The induced pluripotent stem cells obtained in Example 2 were treated with colchicine 3 hours before the termination of the culture, and the cells were collected by adding trypsin after the termination of the culture.

[0068] 2) Treat with 0.075M hypotonic solution (0.075M hypotonic solution KCl solution), then add fixative solution (glacial acetic acid:methanol=1:3) to fix the cells, and repeat the fixation 1-2 times.

[0069] 3) Depending on the amount of cells, add an appropriate amount of fixative to make a cell suspension, absorb the cell suspension and drop it on a glass slide, and put the slide in an oven to bake.

[0070] 4) Digest slides in trypsin with a pH of 7.0-7.2, and then wash them in NaCl solution at 37°C.

[0071] 5) Stain the slides with Giemsa staining solution, dry them and read them with OLYMPUS, BX41 / BX2.

[0072] (2) Methylation analysis

[0073] In this example, conv...

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Abstract

The invention provides a method for constructing an LQT disease model and application of the LQT disease model in drug screening. The method comprises the following steps: (1) introducing a reprogramming factor coding gene into somatocytes carrying LQTS related mutant gene by using an attachment vector to obtain induced pluripotent stem cells; and (2) under the conditions suitable for differentiation of the induced pluripotent stem cells, culturing the induced pluripotent stem cells to obtain myocardial cells, wherein the myocardial cells constitute the LQTS disease model. The method can be utilized to quickly and effectively prepare the LQTS disease model.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for constructing an LQT disease model and its application in drug screening. Background technique [0002] Congenital long QT syndrome (long QT syndrome, LQTS) is a hereditary arrhythmia, which is caused by the abnormality of the gene encoding the ion channel of myocardial cells, also known as "ion channel disease". LQTS patients have electrocardiographic features of prolonged QT interval (greater than 0.48 seconds) and abnormal T wave morphology, clinical manifestations of recurrent syncope and ventricular arrhythmias, especially Torsade de pointes (TdP) . According to statistics, the 10-year mortality rate of untreated LQTS patients is as high as 50%, and about 1 / 2500 patients have a lifetime risk of sudden death. According to recent data from the United States, the incidence of LQTS in the population is about 1:2000-1:5000, especially in children and adolescents. Based...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N5/10C12Q1/02
Inventor 李翠兰杨佳银王震刘雨晴周敏刘文玲胡大一杨波
Owner SHENZHEN SANQI BIOTECH
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