Construction and application of novel RNA cyclization expression vector

A technology of circularization and construction, applied in the biological field, can solve the problems of cumbersome construction process and limited types of circular RNA molecules

Active Publication Date: 2016-10-05
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Description
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Problems solved by technology

This method can generate circular RNA molecules in vivo, but the types of circular...

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  • Construction and application of novel RNA cyclization expression vector
  • Construction and application of novel RNA cyclization expression vector
  • Construction and application of novel RNA cyclization expression vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1 Construction and Detection of pZW1-Circ-Vector Circular RNA Molecular Expression Vector

[0083] According to the current molecular structure and production mechanism of circular RNA molecules derived from exons, the present invention constructs a circular RNA molecule expression vector named pZW1-Circ-Vector, and uses Northern Blot and other experimental methods to verify the expression of circular RNA molecules. In this example, the 9th and 10th exons of the POLR2A gene are taken as examples to describe its construction method and verification implementation process.

[0084] 1. Experimental materials

[0085] 1.1 Experimental reagents

[0086] Restriction endonucleases Nhel and MluI were purchased from NEB Company in the United States; T4 DNA ligase and PrimeSTAR high-fidelity PCR system were purchased from Takara Company in Japan; Reagent was purchased from Invitrogen, USA; AmpliScribe TM T7 Transcription Kits were purchased from Epicentre Company of ...

Embodiment 2

[0151] Example 2 Construction and detection of pZW1-Circ-Vector-High high-efficiency circular RNA molecule expression vector

[0152] On the basis of the pZW1-Circ-Vector expression vector, the original Alu element can be replaced by two completely reverse complementary sequences, which can express exon-derived circular RNA more efficiently and obtain more circular RNA molecules. The optimized vector is named pZW1-Circ-Vector-High efficient circular RNA molecule expression vector. For a schematic diagram of plasmid construction see image 3 . The specific implementation process is as follows:

[0153] 1. Experimental materials

[0154] 1.1 Experimental reagents

[0155] With reagent used in embodiment 1.

[0156] 1.2 Cell lines and vectors

[0157] In this example, the H9 cell line of human embryonic stem cells and the HeLa cell line of human cervical cancer cells are still used. For details, please refer to 1.2 Cell lines and vectors in Example 1. The vector used to co...

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Abstract

The invention provides construction and application of a novel RNA cyclization expression vector. Particularly, a nucleic acid constructor for constructing RNA cyclization molecules has the structure as shown in the formula I from 5' to 3'. In the formula I, A1 and A2 together form a reporter gene, B1 is a first 5' splicing site sequence, C1 is a first cyclization pairing sequence, D1 is a region rich in pyrimidine, E1 is a first 3' splicing site sequence, F is a linear sequence of exogenous RNA cyclization molecules, B2 is a second 5' splicing site sequence, C2 is a second cyclization pairing sequence, D2 is a region rich in pyrimidine, and E2 is a second 3' splicing site sequence, wherein the first cyclization pairing sequence C1 and the second cyclization pairing sequence C2 are complementary sequences. A method based on the constructor has the advantages of being easy to operate, high in expression efficiency, low in requirement on a target expression sequence, wide in application range, capable of achieving in vivo expression and the like. The formula I is A1-B1-C1-D1-E1-F-B2-C2-D2-E2-A2.

Description

technical field [0001] The invention relates to the biological field, in particular to the construction and application of an exon-derived circular RNA molecule high-efficiency expression vector. Background technique [0002] Circular RNA was first discovered in the 1970s. With the development of related technologies, people have gradually deepened the research on this type of molecule. [0003] In terms of species, whether in viruses, yeast or archaea, whether in humans, primates or mice, the presence of circular RNAs has been found. [0004] In terms of quantity, early circular RNAs were only sporadically discovered. Due to the advancement of next-generation high-throughput sequencing technology and molecular biology technology, the current calculation and analysis results of bioinformatics predict that the number of circular RNAs is equivalent to 1% of mRNA. Left, right, or even more, they are found in different cell lines, and there is species-specificity and tissue-spe...

Claims

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Application Information

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IPC IPC(8): C12N15/79C12N15/10
Inventor 杨力陈玲玲张晓鸥张杨
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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