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

A technology of pluripotent stem cells and green fluorescence, applied in the field of VSX2 green fluorescence reporter human induced pluripotent stem cell lines and its construction, can solve the problems of unclear mechanism of occurrence and achieve highly specific effects

Active Publication Date: 2021-01-05
ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Mutations in the VSX2 gene are associated with anophthalmic syndrome and microphthalmia, but the exact mechanism is unclear

Method used

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

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Experimental program
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Embodiment 1

[0035] figure 1 A is the CRISPR / Cas9-mediated BC1-VSX2 of the present invention eGFP - Schematic diagram of the construction of hiPSC lines.

[0036] 1. Maintenance culture of hiPSC

[0037] 1.1 Materials and instruments:

[0038] 1.1.1 Cell: BC1-hiPSC, donated by Professor Cheng Linzhao (University of Science and Technology of China).

[0039] 1.1.2 Reagents and consumables:

[0040] 1) mTeSR1 medium: STEM CELL, #05851;

[0041] 2) EDTA: Invitrogen, 15575-038;

[0042] 3) PBS (1×): Gino Biomedical Technology Co., Ltd., 14111202;

[0043] 4) Matrigel: Corning, 354277;

[0044] 5) (-)-Blebbistatin: Sigma, B0560;

[0045] 6) Six-hole plate: FALCON, 353046;

[0046] 7) Centrifuge tube: FALCON, 352096.

[0047] 1.1.3 Instruments:

[0048] 1) CO 2 Incubator: SANYO, MCO-20A1C;

[0049] 2) Inverted microscope: Nikon, TS100.

[0050] 1.2 Specific steps:

[0051] hiPSCs were maintained in mTeSR1 medium and passaged when the confluence was about 80%-90%. Aspirate the mTe...

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Abstract

The invention discloses a VSX2 green fluorescence reporter gene human induced pluripotent stem cell line and a construction method thereof. The constructed VSX2 green fluorescence reporter gene hiPSCline is bright in reporter fluorescence expression, high in specificity and capable of being stably expressed for a long time, and strong fluorescence signals can still be detected during tissue fixation and end detection of frozen sections. According to the VSX2 green fluorescence reporter gene hiPSC line and the construction method thereof, a useful tool can be provided for retina related disease modeling, development mechanism and prevention and treatment research, and the VSX2 green fluorescence reporter gene hiPSC line is used for tracking of retinal cell fate and return, optimization ofa differentiation system, separation and purification of eye cups, even drug screening, cell transplantation and other downstream research.

Description

technical field [0001] The invention belongs to the technical field of cell biology, and in particular relates to a VSX2 green fluorescent reporter gene human induced pluripotent stem cell line and a construction method thereof. Background technique [0002] The normal formation and development of the eye is controlled by a network of genes, and any disruptive influence of genetic or environmental factors on this process can result in growth and structural defects of the eye, such as anophthalmia and microphthalmia. Anophthalmia and microphthalmia are currently considered to be the most serious developmental ocular abnormalities, which can cause severe visual impairment, accounting for about 3-12% of visual impairment in children, and seriously affect human's healthy life. The pathogenesis of anophthalmos and microphthalmia is still unclear. [0003] Visual system homeobox 2 (VSX2), also known as CHX10, encodes a transcription factor that plays an important role in mammalia...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85C12N15/65G01N21/64
CPCC12N5/0696C07K14/47C07K14/43595C12N15/85C12N15/65G01N21/6486C12N2800/107C12N2510/00Y02A50/30
Inventor 钟秀风向孟清郑丹丹王远张祖明晋康新
Owner ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV
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