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RNA interference vector based on site-specific recombination, and construction method and application of same

An RNA interference and site-specific technology, applied in the biological field, can solve the problems of affecting the expression of the target gene and the inability to accurately locate the insertion position

Active Publication Date: 2014-05-07
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of these techniques is that the direct and accurate positioning of the insertion position cannot be performed, and random insertion may affect the expression of the target gene

Method used

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  • RNA interference vector based on site-specific recombination, and construction method and application of same
  • RNA interference vector based on site-specific recombination, and construction method and application of same
  • RNA interference vector based on site-specific recombination, and construction method and application of same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1 A method for constructing an RNA interference vector based on site-specific recombination

[0073] 1. Experimental materials

[0074] Pig liver tissue: purchased from an experimental animal supply base in Gansu

[0075] iαv-pENTR / U6 vector: preserved by our laboratory

[0076] PGT-V1: Sponsored by Wang Huayan, Northwest A&F University

[0077] Multiple cloning restriction sites, related primers used in the experiment: synthesized by Shanghai Bioengineering Co., Ltd.

[0078] Various kits used in the experiment were purchased from Bao Biological Engineering (Dalian) Co., Ltd., and XmaI and AgeI were purchased from NEB Company.

[0079] 2. Method

[0080] 2.1 The composition of the carrier

[0081] The composition of the carrier such as PSN-U6 structure map Figure 9 As shown, its functions are introduced as follows:

[0082] HA1, HA2: for homologous recombination with a single intron region of the porcine genome β3 gene

[0083] hU6 (human U6 promoter):...

Embodiment 2

[0117] Example 2 Application of the vector of the present invention in the construction of a pig-derived cell silencing expression vector

[0118] 1. Construct a pig-derived cell silencing expression vector for expressing shRNA and inserting it into a specific position of the pig-derived cell genome through homologous recombination

[0119] (1) Use ClaI and SfiI to double digest the vector to expose the cohesive end between the U6 promoter and the termination signal on PSN-U6. The reaction conditions are 1*M buffer solution, 2 kinds of enzymes are added at the same time, 30°C for 1.5h, and then the temperature is adjusted to 50°C for 1.5h, and the digested products are recovered.

[0120] (2) Synthesize the DNA sequence required to transcribe shRNA, such as Figure 12 Shown:

[0121] (3) Ligate the synthesized fragment with the digested PSN-U6, transform the ligated product, pick a positive clone for sequencing verification, and the sequencing primers are:

[0122] 5'-G GAC...

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Abstract

The invention discloses a RNA interference vector based on site-specific recombination, and construction method and application of the same, belonging to the field of biotechnology. The interference vector provided by the invention sequentially comprises the following connected parts: a homologous recombination arm 1 in homologous recombination with the upstream of a certain intron region of the integrin receptor beta 3 gene of pig, a human U6 promoter sequence, a multiple cloning enzyme digestion site, a human U6 terminator sequence, a new Neomycin resistant gene containing a PCMV (Porcine cytomegalovirus) promoter, a homologous recombination arm 2 in homologous recombination with the downstream of a certain intron region of the integrin receptor beta 3 gene of pig, a replication origin gene, an ampicillin resistant screened gene and a enzyme digestion site for vector linearization. The interference vector provided by the invention realizes accurate targeting to swine-origin cell genome, and facilitates screening, meanwhile, the influence of insertion of segment on the gene expression can be predicted, which is convenient for the following experiment.

Description

technical field [0001] The invention relates to an RNA interference carrier and its construction method and application, in particular to an RNA interference carrier based on site-specific recombination and its construction method and application, belonging to the field of biotechnology. Background technique [0002] RNA interference (RNA interference, RNAi) refers to the phenomenon of efficient and specific degradation of homologous mRNA induced by double-stranded RNA (double-stranded RNA, dsRNA), which is highly conserved during evolution. Since the use of RNAi technology can specifically knock out or shut down the expression of specific genes, this technology has been widely used in the field of gene therapy for exploring gene functions and infectious diseases and malignant tumors. [0003] In animals, RNAi can be achieved by expressing shRNA from the U6 promoter. The current U6 vector can be expressed in animal cells in two ways: transient expression and stable expressi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/64
Inventor 常惠芸马延滨丛国正高闪电独军政邵军军林彤郝春霞
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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