Construction and application of transgenic mouse model for exosome tracing

A technology of transgenic mice and exosomes, applied in the construction of transgenic mouse models, can solve the problems of silencing, reading frame changes, loss of function of target genes, etc.

Pending Publication Date: 2022-07-01
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Under normal circumstances, cells will use efficient non-homologous end joining (NHEJ) to repair the broken DNA; however, base insertion or deletion mismatches usually occur during the repair process, resulting in frameshift mutations, (Frameshift mutation: refers to the change of the reading frame of the DNA molecule due to the deletion or insertion of a certain base, resulting in a series of downstream codon changes, so that the gene that originally coded for a certain peptide chain becomes a complete code for another Different peptide chain sequences;) to disable the target gene, so that the expression of the source DNA is silenced

Method used

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  • Construction and application of transgenic mouse model for exosome tracing
  • Construction and application of transgenic mouse model for exosome tracing
  • Construction and application of transgenic mouse model for exosome tracing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 Construction of a plasmid expressing stop-EGFP-Cas9-tracrRNA element (exosome receptor plasmid)

[0020] Construct a plasmid expressing the stop-EGFP-Cas9-tracrRNA element. The plasmid contains elements such as stop-EGFP-Cas9-tracrRNA for constructing exosome receptors; the plasmid vector contains the Cas9 protein sequence, and we need to insert it into the plasmid vector The stop-EGFP sequence and the tracrRNA sequence are sufficient. The specific operation method is as follows

[0021] Double-enzyme digestion of the plasmid vector, run 1% agarose gel and gel to recover larger gene fragments; complete gene synthesis of stop-EGFP DNA sequence and tracrRNA DNA sequence; double-enzyme cut double-stranded stop-EGFP sequence, pass through the column to recover the enzyme Cut the product, then connect the double-enzyme-cut vector and fragment, transform it into DH5α, select bacteria and sequence, and screen out the successfully constructed clone; cut the correctly ...

Embodiment 2

[0022] Example 2 Construction of a plasmid expressing crRNA or sgRNA (exosome donor plasmid)

[0023] Construct a plasmid expressing crRNA or sgRNA, the plasmid contains crRNA-1 / crRNA-2 or sgRNA-1 / sgRNA-2; used to construct an exosome donor; insert the crRNA / sgRNA sequence behind the U6 promoter of the plasmid vector Yes, the specific operations are as follows:

[0024] Double-enzyme digestion of the plasmid vector, run 1% agarose gel and gel to recover larger gene fragments, design crRNA-1 and crRNA-2 sequences targeting stop sequences and anneal them into oligo double-stranded in vitro. The crRNA-2 gene fragment was enzymatically linked with the double-enzyme-digested plasmid vector, and after transformation and sequencing, the successfully constructed clone was screened.

Embodiment 3

[0025] Example 3 Construction of Donor Cells and Verification of Exosomes

[0026] The construction of the donor cells is divided into two parts. The first part uses the tool cell HEK 293T cells to prepare the lentivirus expressing the donor element and obtains the virus suspension. The second part uses the obtained virus suspension to infect human non-small cell lung cancer. A549 cells, after screening, pick out the stable transfection monoclonal cell line, expand the culture to collect exosomes and detect; the specific operations are as follows

[0027] 1. Mix Opti-MEM, Lipo2000 / PEI, the target plasmid and the helper plasmid according to a certain ratio and let it stand for 20 minutes, then slowly drop it into a HEK 293T cell culture dish with a growth density of 70% to 80%, change it after 6 hours After 48 hours, the cell culture medium (containing lentivirus) was collected in a biological safety cabinet, and filtered with a 0.45 μm filter to obtain the virus supernatant, w...

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Abstract

The invention discloses a construction method and application of a transgenic mouse model for exosome tracing. The specific implementation process mainly comprises the following steps: constructing plasmids for expressing stop-EGFP-tracrRNA and expressing crRNA or sgRNA; construction of donor cells and verification of exosomes; constructing, breeding and identifying transgenic mice; the five parts are verified by the tracing exosome transgenic mouse model, and finally the transgenic mouse model for tracing the exosome is successfully obtained; the successful construction of the model lays a foundation for the research of the exosome as a gene drug carrier, and scientists can more easily and clearly observe the distribution of the exosome in vivo under pathological or physiological conditions.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular, to a construction method and application of a transgenic mouse model for exosome tracking. Background technique [0002] The CRISPR-Cas system is a natural immune system of prokaryotes. After some bacteria or archaea are invaded by viruses, they can store a small segment of the viral gene in a storage space called CRISPR in their own DNA; When a virus invades, bacteria can identify the virus according to the stored and written segments, and cut off the DNA of the virus to make it invalid; the mechanism of CRISPR-Cas9 is to drive immunity, and its mechanism of action can be divided into three stages to understand 1. Acquisition: Immunity is generated by ingesting exogenous DNA sequences and integrating them into new CRISPR spacers; 2. Expression: CRISPR sequences are transcribed under the control of the leader region to generate pre-crRNA (precursor of crRNA), which is compatible with p...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/66C12N15/65C12N15/55A01K67/027C12N15/867C12N5/10
CPCC12N15/8509C12N15/65C12N9/22A01K67/0275C12N15/86C12N5/0693C12N5/0688C12N2800/107C12N2840/102A01K2207/05A01K2227/105A01K2267/0393C12N2740/15043C12N2510/00
Inventor 张翔谢菲徐彬叶扬扬杨婷施倩张辰宇李菁
Owner NANJING UNIV
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