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Ribonucleic acid (RNA) interference vector for bovine genome locus specificity recombination, vector construction method and application

An RNA interference and site-specific technology, applied in the field of RNA interference vectors and their construction, can solve the problems of direct and accurate positioning of the inability to insert the position, affecting the expression of the target gene, and affecting the research of transgenic animals.

Active Publication Date: 2013-06-05
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 technologies is that it is impossible to directly and accurately locate the insertion position, and random insertion may affect the expression of the target gene, causing inestimable impact on gene function research or transgenic animal research.

Method used

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  • Ribonucleic acid (RNA) interference vector for bovine genome locus specificity recombination, vector construction method and application
  • Ribonucleic acid (RNA) interference vector for bovine genome locus specificity recombination, vector construction method and application
  • Ribonucleic acid (RNA) interference vector for bovine genome locus specificity recombination, vector construction method and application

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Experimental program
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Effect test

Embodiment 1

[0074] Example 1 A method for constructing an RNA interference vector for site-specific recombination of the bovine genome

[0075] 1. Experimental materials

[0076] Bovine liver tissue: purchased from an experimental animal supply base in Gansu

[0077] iαv-pENTR / U6: preserved by our laboratory (refer to the literature "Lentviral-mediated RNAi to inhibit target gene expression of the porcine integrin av subunit, the FMDV receptor, and against FMDV infection in PK-15cells")

[0078] PGT-V1: Sponsored by Teacher Wang Huayan from Northwest A&F University (refer to the literature "Construction and Functional Identification of Double Screening Marker Targeting Vector")

[0079] pORF9-HSV1tk: purchased from Invitrogene

[0080] Multiple cloning restriction sites, related primers used in the experiment: synthesized by Shanghai Bioengineering Co., Ltd.

[0081] Various kits used in the experiment were purchased from Bao Biological Engineering (Dalian) Co., Ltd., and restriction e...

Embodiment 2

[0133] Embodiment 2 Application of the carrier of the present invention in the construction of transgenic bovine-derived cells

[0134] 1. Construct a bovine cell silencing expression vector for expressing shRNA (for bovine ITGB3NM_001206490) and inserting it into a specific position in the bovine cell genome through homologous recombination

[0135] (1) Use ClaI and SfiI to double digest the vector to expose the cohesive end between the U6 promoter and the termination signal on PBKN-U6. The reaction conditions were 1*NEB Buffer4, 100 μg / ml BSA, ClaI and SfiI enzymes were added at the same time, 30°C for 1.5h, and then the temperature was adjusted to 50°C for 1.5h, and the digested product was recovered.

[0136] (2) Synthesize the DNA sequence required to transcribe shRNA, such as Figure 10 Shown:

[0137] (3) Ligate the synthesized fragments with the digested PBKN-U6, transform the ligated products, pick positive clones for sequencing verification, and the sequencing prim...

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Abstract

The invention discloses a ribonucleic acid (RNA) interference vector for bovine genome locus specificity recombination, a vector construction method and application and belongs to the field of biotechnology. The interference vector comprises the following sequentially connected parts: an HSV-TK expression element, A digestion locus for vector linearization, a homologous recombination arm 1 in homologous recombination with a bovine scaffold sequence upstream, a human U6 promoter sequence, a polyclonal digestion locus, a human U6 stop subsequence, a neomycin resistance gene containing a PCMV promoter, a homologous recombination arm 2 in homologous recombination with a bovine scaffold sequence downstream, a replication origin gene and an ampicillin resistance screening gene. The RNA interference vector achieves accurate target shooting on bovine derived cell bovine, and brings convenience to screening, insertion locus and insertion element do not affect the integral structure of chromosome and cause no non-research gene expression effect on genome, and RNA interference vector provides convenience for research.

Description

technical field [0001] The invention relates to an RNA interference vector and its construction method and application, in particular to an RNA interference vector for bovine genome site-specific recombination, its construction method and application, and belongs 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 ex...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N5/071
Inventor 常惠芸马延滨邵军军高闪电赵付荣丛国正林彤独军政
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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