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Alternative splicing reporter vector and preparation method thereof

A vector and reporting technology, applied in the introduction of foreign genetic material using vectors, recombinant DNA technology, etc., can solve the problems of limited alternative splicing, lack of detection tools for alternative splicing, no alternative splicing research report vector, etc. The effect of stitching

Pending Publication Date: 2019-04-26
NORTHWEST A & F UNIV
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Problems solved by technology

[0004] However, the current research on the functional impact of alternative splicing and its mechanism is still relatively limited, and there is still a lack of intuitive detection tools for the study of alternative splicing in vitro, and most of the current research is on the analysis of alternative splicing. Reporter vectors for targeted regulation of regulatory factors, and no reporter vectors available for the study of alternative splicing at the cellular and in vivo levels

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  • Alternative splicing reporter vector and preparation method thereof
  • Alternative splicing reporter vector and preparation method thereof
  • Alternative splicing reporter vector and preparation method thereof

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Embodiment 1

[0078] Such as figure 1 As shown, the present invention constructs the vector pEGFP-C1-ASR containing special sequences intron1 and intron2, and transfects the vector pEGFP-C1-ASR into cells by electroporation, and observes the expression of the marker gene EGFP by fluorescence microscope and flow cytometry In this case, after PCR identification, it was confirmed that the pre-mRNA after gene transcription could be spliced ​​correctly.

[0079] The reporter vector pEGFP-C1-ASR was co-transfected with the general-purpose transcriptome editing vector pCas13d-gEGFP, and the expression of the marker gene EGFP was observed under a fluorescence microscope. After PCR identification, it was confirmed that the vector pCas13d-gEGFP can interfere with the splicing event, making the reporter vector Alternative splicing occurs.

[0080] The specific method of alternative splicing reporter vector construction is as follows:

[0081] (1) Synthesize the sequence intron1 containing 5' splice...

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Abstract

The invention discloses an alternative splicing reporter vector. Two sections of intron sequences intron1 and intron2 are inserted into a reporter gene sequence of a reporter gene expression vector pEGFP-C1; each of the intron1 and the intron2 has a 5' splicing site, a branch site and a 3' splicing site. The invention also discloses a preparation method of the alternative splicing reporter vector.The intron1 and the intron2 are designed and synthesized according to a splicing mechanism of RNA (Ribonucleic Acid); the intron1 and the intron2 are inserted into a reporter sequence of the reportergene expression vector respectively and are used for detecting an alternative splicing event on a cell level and a living body level; the introns containing the splicing sites are inserted so that correct splicing of post-transcriptional mRNA (messenger Ribonucleic Acid) is facilitated.

Description

technical field [0001] The invention relates to the technical field of gene cloning, in particular to an alternative splicing reporter carrier and a preparation method thereof. Background technique [0002] Alternative splicing is a general regulatory mechanism of gene expression that enables a single gene to produce multiple different kinds of mRNA. Alternative splicing includes exon skipping, selection between mutually exclusive exons, splice site utilization, and intron retention, among others. Through the alternative splicing mechanism, mRNAs with differences in the untranslated regions or coding sequences can be produced, and these differences may affect the stability, localization or translation of mRNAs. Furthermore, some spliced ​​mRNA isoforms can alter the reading frame, generating differential protein isoforms with different roles or localizations. Genome-wide studies predict that 90-95% of human genes undergo some degree of alternative splicing. High-resolutio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N15/66
CPCC12N15/63C12N15/66
Inventor 刘旭郑小曼权富生张涌
Owner NORTHWEST A & F UNIV
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