Method for constructing chromosome translocation stem cell and animal model by CRISPR-Cas9 technology

A technology of chromosomal translocation and animal model, applied in the field of animal model

Active Publication Date: 2015-06-24
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] At present, there are no relevant literature reports on the use of CRISPR-Cas9 technology to artificially mediate chromosomal translocation in stem cells at home and abroad, and there is no report on the use of CRISPR-Cas9 technology to obtain stem cells carrying artificially mediated chromosomal translocation to further produce chromosomal translocation animals model coverage

Method used

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  • Method for constructing chromosome translocation stem cell and animal model by CRISPR-Cas9 technology
  • Method for constructing chromosome translocation stem cell and animal model by CRISPR-Cas9 technology
  • Method for constructing chromosome translocation stem cell and animal model by CRISPR-Cas9 technology

Examples

Experimental program
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Effect test

Embodiment 1

[0071] Example 1: Isolation, culture and passage of mouse embryonic stem cells (mESCs).

[0072] According to the method reported in the literature, the specific method can be referred to: Czechanski, A., et al., Derivation and characterization of mouse embryonic stem cells from permissive and nonpermissive strains. Nat Protoc, 2014.9(3): p. 559-74. After the mouse embryonic stem cells are isolated, they are cultured as follows:

[0073] In 0.1% gelatin-coated petri dishes or plates, the medium is mES medium and placed in 5% CO 2 , 37 ℃ cell culture incubator; the mES medium contains 1% non-essential amino acids (Invitrogen), 10% fetal bovine serum (HyClone), 0.1mM β-mercaptoethanol (Invitrogen), 2mM GlutaMax (Invitrogen), And 100 units / ml of GMEM medium (Sigma, G5414) of leukemia inhibitory factor.

[0074] When the cultured mESCs clones and clones are about to fuse, they need to be passaged, usually every 2-3 days. The specific method is:

[0075] 1. Remove the medium, and fine it ...

Embodiment 2

[0081] Example 2: Design and construct gRNA expression vectors targeting mouse chromosomes 7 and 5.

[0082] This example aims to construct a translocation between mouse chromosome 7 and chromosome 5 in embryonic stem cells.

[0083] The designed translocation site of chromosome 7 is located at the site of GSK3a gene, specifically: chr7:25235652-25235624, and its site sequence is: 25235652- GATTGGTAATGGCTCATT CGG-25235632, the design recognition site of its gRNA is: GATTGGTAATGGCTCATT (SEQ ID NO:1)

[0084] The designed translocation site of chromosome 5 is located at the site of CDX2 gene, specifically: chr5:

[0085] 147306976-147306947, its locus sequence is: 147306976- GTGAGCTACCTTCTGGACA AGG-147306955, the design recognition site of its gRNA is: GTGAGCTACCTTCTGGACA (SEQ ID NO: 2)

[0086] After the design is completed, a DNA synthesis company is entrusted to synthesize the expressed DNA sequence of gRNA-1 for chromosome 7 according to sequence 5, and the expression DNA sequence ...

Embodiment 3

[0087] Example 3: Inducing a specific site translocation between chromosome 7 and chromosome 5 in mouse embryonic stem cells by CRISPR / Cas9 technology.

[0088] 1. Cell culture and transfection

[0089] Mouse embryonic stem cells (mESCs) are divided into two groups, one group is an experimental group and the other is a control group. They are cultured in mES medium and placed in a 37°C, 5% carbon dioxide incubator. When the cell density of the two groups reached about 50%, Lipofectamine2000 reagent (Invitrogene, USA) was used for transfection according to the steps and reagent ratios in the transfection reagent instructions. The experimental group was transfected with gRNA-1, gRNA-2 expression vector, and Cas9 expression vector (purchased from Addgene Plasmid ID: 41815), and the control group was only transfected with Cas9 expression vector.

[0090] 2. Chromosome specific site translocation identification

[0091] (1) PCR to identify chromosomal translocations at specific sites

[00...

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Abstract

The invention relates to a method for preparing a chromosome translocation stem cell model and an animal model. The method mainly comprises the steps of inducing occurrence of specific site chromosome translocation in stem cells by means of CRISPR / Cas9 technology, preparing a cell model and further constructing an animal model carrying the specific site chromosome translocation by utilizing embryonic stem cell technology. The method particularly comprises the steps of simultaneously introducing Cas9 and gRNA aiming at two target chromosome sites into the stem cells to artificially inducing chromosome dislocation of specific sites, and screening to obtain the cell model carrying the chromosome translocation. The animal model carrying chromosome translation of the specific sites can be further prepared from the chromosome translocation animal embryonic stem cells carrying chromosome translation by utilizing a chimera technology. The method has relatively large application prospect in studies on chromosome translocation and functions of fusion genes, researches on chromosome interaction and medicine screening evaluation.

Description

Technical field [0001] The present invention relates to the field of biotechnology, in particular to a method for constructing a specific site chromosomal translocation in stem cells through CRISPR-Cas9 technology, and using stem cells to construct an animal model of a specific site chromosome translocation. Background technique [0002] Chromosome Translocation, the change in the position of chromosome fragments, is the most common chromosomal abnormality (see literature: Braude, P., Pickering, S., Flinter, F., and Ogilvie, CM (2002).Preimplantation genetic diagnosis.Nat Rev Genet 3,941-953). The production process is often DNA double-strand breaks (DSBs) at different chromosomal sites, and then the different chromosomal ends are repaired through the mechanism of non-homologous end joining (NHEJ), resulting in chromosome Changes in fragment location (see literature: Roukos, V., and Misteli, T. (2014). The biogenesis of chromosome translocations. Nat Cell Biol 16, 293-300.). Ch...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/55C12N15/11C12N5/10A01K67/027C12Q1/02A61K49/00
Inventor 刘厚奇蒋俊锋应其龙王越张莉
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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