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VSX2 green fluorescence reporter gene vector system and construction method thereof

A reporter gene and carrier system technology, applied in the field of cell biology, can solve the problem of unclear mechanism and achieve strong specificity

Active Publication Date: 2021-01-05
ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV
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  • Summary
  • 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 vector system and construction method thereof
  • VSX2 green fluorescence reporter gene vector system and construction method thereof
  • VSX2 green fluorescence reporter gene vector system and construction method thereof

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Experimental program
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Effect test

Embodiment 1

[0030]figure 1 A is CRISPR / Cas9 mediated BC1-VSX2 of the present inventioneGFP -Schematic diagram of the construction of the hiPSC system.

[0031]1. Maintenance and cultivation of hiPSC

[0032]1.1 Materials and instruments:

[0033]1.1.1 Cell: BC1-hiPSC, gifted by Professor Cheng Linzhao (University of Science and Technology of China).

[0034]1.1.2 Reagents and consumables:

[0035]1) mTeSR1 medium: STEM CELL, #05851;

[0036]2) EDTA: Invitrogen, 15575-038;

[0037]3) PBS(1×): Jinuo Biomedical Technology Co., Ltd., 14111202;

[0038]4) Matrigel: Corning, 354277;

[0039]5) (-)-Blebbistatin: Sigma, B0560;

[0040]6) Six-hole plate: FALCON, 353046;

[0041]7) Centrifuge tube: FALCON, 352096.

[0042]1.1.3 Apparatus:

[0043]1) CO2Incubator: SANYO, MCO-20A1C;

[0044]2) Inverted microscope: Nikon, TS100.

[0045]1.2 Specific steps:

[0046]hiPSCs are maintained in mTeSR1 medium and subcultured when the confluency is about 80%-90%. Aspirate the mTeSR1 medium, wash with sterile PBS, add 0.5mM EDTA, and digest at 37°C for 4-6min. As...

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Abstract

The invention discloses a VSX2 green fluorescence reporter gene vector system and a construction method thereof. The VSX2 green fluorescence reporter gene vector system comprises a PX459-VSX2_sgRNA knockout plasmid and a pBluescript-LA-P2A-eGFP-RA donor plasmid; and a VSX2 green fluorescence reporter gene hiPSC system constructed by transformation of the vector system is bright in reporter fluorescence expression, high in specificity and capable of being stably expressed for a long time, and a strong fluorescence signal can still be detected when a tissue is fixed and frozen sections are subjected to end detection. The VSX2 green fluorescence reporter gene vector system and the VSX2 green fluorescence reporter gene hiPSC system can provide useful tools for retina-related disease modeling,development mechanism and prevention and treatment research, and are used for downstream research such as tracking of retinal cell fate transfer and optimization of a differentiation system, separation and purification of eye cups and even drug screening and cell transplantation.

Description

Technical field[0001]The invention belongs to the technical field of cell biology, and specifically relates to a VSX2 green fluorescent reporter gene carrier system and a construction method thereof.Background technique[0002]The normal formation and development process of the eye is controlled by a gene network. Any destructive influence of genetic or environmental factors on this process may lead to the growth and structural defects of the eye, such as anphthalmia and microphthalmia. Aphthalmia and microphthalmia are currently considered to be the most serious developmental eye abnormalities, which can cause severe visual disturbances, accounting for about 3-12% of children’s visual disturbances, and seriously affecting human health. At present, the pathogenesis of asthenophthalmia and microphthalmia is not clear.[0003]Visual system homeobox 2 (VSX2), also known as CHX10, encodes a transcription factor that plays an important role in mammalian eye development (Liu et al., 1994). VS...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/65C12N15/66C12N5/10G01N21/64
CPCC12N15/85C12N15/65C12N15/66C12N5/0696C07K14/47C07K14/43595G01N21/6486C12N2800/107C12N2510/00Y02A50/30
Inventor 向孟清钟秀风晋康新张祖明王远郑丹丹
Owner ZHONGSHAN OPHTHALMIC CENT SUN YAT SEN UNIV
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