Mouse model with miRNA-125a knocked in based on CRISPR/Cas9 technology and construction method

A mouse and technology technology, applied in the field of genetic engineering, can solve problems such as the lack of C57BL/6J mouse construction method

Inactive Publication Date: 2021-06-22
国家卫生健康委科学技术研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Generally speaking, compared with coding genes, there are few studies on miRNAs knock-in based on CRISPR/Cas9 technology; in particul

Method used

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  • Mouse model with miRNA-125a knocked in based on CRISPR/Cas9 technology and construction method
  • Mouse model with miRNA-125a knocked in based on CRISPR/Cas9 technology and construction method
  • Mouse model with miRNA-125a knocked in based on CRISPR/Cas9 technology and construction method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1 Design sgRNA

[0056] Compare the human and mouse sequences in this region in ensemble, and select the sequence difference site as the region to be knocked in (design sgRNA in this region) to ensure that the splicing information is not destroyed. Available sequences in the mouse genome were analyzed using the website (http: / / crispr.mit.edu). We selected one sgRNA (SEQ ID NO:1) as the site to be knocked in, as follows:

[0057] The sequence of SEQ ID NO:1 is (sgRNA1): CTCACAGGTTAAAGGGTCTC(5'→3')

[0058] According to the characteristics of the sticky end produced by the restriction endonuclease digestion of the vector, in order to make the sgRNA fragment connect to the linearized vector exactly, add GTTT before the 5' end of the forward strand, and add AAAC before the 5' end of the reverse strand. . The target sequences with adapters and their complementary sequences designed for the above sgRNAs are as follows:

[0059] The sequence of SEQ ID NO:2 is (sgRN...

Embodiment 2

[0061] Example 2: Construction of expression vectors

[0062] a) Plasmid linearization

[0063] Digest the lentiCRISPR V2 vector in the system shown in the table below:

[0064] Table 1 enzyme digestion system

[0065]

[0066] Digest overnight at 37°C, add loading buffer to a final concentration of not less than 1×, purify the digested product by 0.8% agarose gel electrophoresis, and recover the linear DNA band by cutting the gel. The steps for gel recovery are as follows (commercialized by Omega Corporation) Reagent test kit):

[0067] 1. Cut out the target DNA band in the agarose gel, add an equal volume of gel solution GSB, and put it in a metal bath at 65°C for 5-10 minutes to completely dissolve it.

[0068] 2. Add all the glue solution to the spin column in batches and let it stand for 1min, centrifuge at 12000rpm for 1min, and discard the effluent.

[0069] 3. Add 300 μL GSB to wash the spin column, centrifuge at 12000 rpm for 1 min, and discard the effluent.

...

Embodiment 3

[0106] Example 3: Verification of sgRNA activity

[0107] 1) Routinely culture mouse liver AML12 cells, spread 12-well plates, and transfect the constructed LentiCRISPR V2 vector when the confluence is about 80%, transfect 1.5 μg of plasmid per well.

[0108] 2) Prepare a complete medium containing 2 μg / mL puromycin (puro). After 24 hours of transfection, change to the selection medium containing puro, and continue to culture normally for 48 hours; at this time, it can be observed that the cells in the control wells without transfection plasmid gradually die, and continue to culture with the normal complete medium until the cells are basically confluent. The genomic DNA of the cells can be extracted, and the target fragment containing the sgRNA target region is amplified by PCR, and the PCR primers are designed as F: 5'-GATAGCGAAGTGGTGGTGGT-3' (SEQ ID NO: 4), R: 5'-TTCTGAAGGAGGAGGGGATT-3' ( SEQ ID NO:5), the full length is 735bp. The PCR amplification system is as follows: ...

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Abstract

The invention discloses a mouse model with miRNA-125a knocked in based on a CRISPR/Cas9 technology and a construction method. According to the mouse model with the miRNA-125a knocked in based on the CRISPR/Cas9 technology, the CRISPR/Cas9 technology is utilized, fixed-point knock-in of a section of exogenous DNA fragment including pre-miRNA-125a is realized in a C57BL/6J mouse genome, and the expression quantity of the miRNA-125a in a homozygous knock-in mouse is obviously increased. The invention provides an accurate, efficient, simple and convenient method for knocking in the miRNA-125a at a fixed point, the construction method can be used for the mouse model for miRNA-125a function research, and also has a reference value for realizing the fixed-point knock-in of other miRNAs.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and more specifically relates to a mouse model and a construction method for knocking in miRNA-125a based on CRISPR / Cas9 technology. Background technique [0002] Gene site-specific modification technology is one of the important means to study gene function. Following the early gene targeting, researchers have discovered three generations of artificial endonucleases. The third-generation artificial endonuclease Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) / CRISPR-associated (Cas) has been widely used because of its simple operation, low cost and high efficiency[1]. Among them, CRISPR / Cas9 is the most widely used gene editing tool so far. It consists of an RNA chain chimerized together with crRNA and tracrRNA——sgRNA (singleguide RNA) and Cas9 protein. Cas9 protein is an endonuclease, including two active centers of RuvC and HNH, which can cut one strand of DNA un...

Claims

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Application Information

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IPC IPC(8): A01K67/027C12N15/90C12N9/22C12N15/113C12Q1/6888
CPCA01K67/0275C12N15/907C12N9/22C12N15/113C12Q1/6888A01K2217/072A01K2227/105A01K2217/15A01K2267/03C12N2310/141C12Q2600/124
Inventor 王瑚夏红飞马旭管纯一张璐
Owner 国家卫生健康委科学技术研究所
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