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Method for knocking out NANS gene in iPSC (induced pluripotent stem cells) and application

A cell and gene technology, applied in the field of cell model construction, can solve problems such as brain organoid mental development disorder models that have not yet been seen, and achieve the effect of improving gene editing efficiency, complete and thorough effect, and thorough knockout effect

Active Publication Date: 2020-09-15
成都华西海圻医药科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] So far, there has been no report on the construction of a mental retardation model based on NANS-deficient brain organoids

Method used

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  • Method for knocking out NANS gene in iPSC (induced pluripotent stem cells) and application
  • Method for knocking out NANS gene in iPSC (induced pluripotent stem cells) and application
  • Method for knocking out NANS gene in iPSC (induced pluripotent stem cells) and application

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Experimental program
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Effect test

Embodiment 1

[0035] Embodiment 1: Utilize CRISPR-CAS9 technology to knock out the method for iPSC cell NANS gene

[0036] 1. sgRNA design

[0037] "sgRNA", that is, Single guide RNA, single guide RNA, can recognize specific genome fragments during CRISPR-Cas9 gene editing (including gene knockout knockout). sgRNA can be mainly divided into two parts. The first part is the reverse complementary sequence to the DNA sequence near the gene editing target (the DNA sequence is referred to as "recognition sequence"), which varies with different targets; the second part is the sequence that binds Cas 9 The sequence of the enzyme is a conserved sequence. In practical applications, the second part is built into the sgRNA expression vector (in this example, the commercial pCas-Guide vector), and when used, the double-stranded form of the first part sequence is inserted into the sgRNA expression vector to form a complete expressible sgRNA gene.

[0038] According to the human NANS gene sequence (Se...

Embodiment 2

[0087] Example 2: Construction Method and Application of NANS Knockout Brain Organoids

[0088] Using the NANS knockout iPSC cells in Example 1, with the help of STEMdiff TM ((Stem celltechnologies) Brain Organoid Kit to cultivate brain organoids, the operation is as follows:

[0089] (1) On Day 0, when the cell confluence is about 80% and the differentiation rate is less than 10%, the human pluripotent stem cells maintained in mTeSRTM1 are dissociated into a single cell suspension using TrypLE, and the density is 8000-10000 cells Each well was inoculated into a 96-well ultra-low adhesion culture plate, cultured with embryoid body (EB) formation medium (EB Seeding Medium) + 10 μM Rho-kinase inhibitor (ROCKi), 100 μL of medium per well.

[0090] (2) The cells were cultured in the EB formation medium for 5 days, and replaced with the EB formation medium without ROCKi every 2 days (Day2, Day4).

[0091] (3) After 5 days (Day5), cut off the tip of the prepared tip, and transfer ...

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Abstract

The invention discloses a method for knocking out an NANS gene in iPSC (induced pluripotent stem cells) and application, and belongs to the field of cell model construction. According to the invention, a CRIPSR-Cas9 gene editing system is transferred into the iPSC by adopting an electroporation method; Donor DNA containing a Puromycin resistance gene and a GFP gene is inserted into an NANS exon, and the stem cells subjected to gene editing are screened by Puromycin; and after transfection, CloneRTM culture cells are added, and the system can greatly improve gene editing efficiency of a CRISPR-Cas9 genome editing technology in the stem cells. The method is helpful for constructing an NANS defect type 3D brain organ model, and can be applied to in-vitro study and drug research and development on the basis of pathogenesis of mental development disorders caused by the NANS mutation.

Description

technical field [0001] The invention relates to the field of cell model construction, in particular to a method and application for knocking out the NANS gene of induced pluripotent stem cells based on CRISPR-CAS9 gene editing technology. Background technique [0002] Intellectual developmental disorders (Intellectual developmental disorders, IDD), also known as mental retardation, mental retardation C Mental Retardation, MR), is a group of onset before the age of 18, cognitive dysfunction (IQ value <70 points), social A disorder characterized by deficits in adaptive capacity. In preschool age (below 5 years old), it is manifested as language ability and motor development delay, which is also known as developmental delay (Developmental Delay, DD). Syndromic mental retardation is usually accompanied by multiple congenital anomalies (Multiple congenital anomalies, MCA), special face and so on. The etiology of ID / DD is complex, including environmental factors, perinatal hy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/85C12N15/90C12N5/10C12N5/079
CPCC12N15/1137C12N15/85C12N15/907C12N5/0696C12N5/0618C12N2310/20C12Y205/01057C12N2500/90C12N2510/00C12N2800/107C12N2506/45C12N2503/02Y02A50/30
Inventor 卜迁岑小波张华琴代艳萍
Owner 成都华西海圻医药科技有限公司
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