RNA (Ribonucleic Acid) interference carrier for inhibiting swine source p53 gene expression and application thereof

A p53 gene and RNA interference technology, applied in the field of genetic engineering, can solve the problems of short duration of RNAi effect and easy degradation of siRNA

Active Publication Date: 2013-08-28
INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Among them, chemical synthesis and in vitro transcription methods are to obtain siRNA in vitro and then import it into cells, but these two methods mainly have two insurmountable shortcomings: siRNA is easily degraded after entering cells; short duration of effect

Method used

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  • RNA (Ribonucleic Acid) interference carrier for inhibiting swine source p53 gene expression and application thereof
  • RNA (Ribonucleic Acid) interference carrier for inhibiting swine source p53 gene expression and application thereof
  • RNA (Ribonucleic Acid) interference carrier for inhibiting swine source p53 gene expression and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Preparation of RNA Interference Vector for Inhibiting the Expression of Porcine p53 Gene

[0065] (1) Mutate the BamH I and Hind III restriction sites in the pEGFP-C1 vector MCS:

[0066] Using the 1386-1646bp fragment on the pEGFP-C1 vector (that is, between Sma Ⅰ and Mlu Ⅰ restriction sites) as the target fragment, design primers B1 and B2 to mutate the BamH Ⅰ restriction site, and use the pEGFP-C1 vector as a template, Through PCR amplification (94°C 3min; 94°C 30s, 60°C 30s, 72°C 30s, 30 cycles; 72°C final extension 8min.) to obtain the target fragment B (such as image 3 As shown, M: DL2000, 1: target fragment B, 2: negative control), the size is 265bp.

[0067] The target fragment B and the pEGFP-C1 vector were digested with Sma Ⅰ and Mlu Ⅰ, respectively, and the digested fragments were ligated with T4 DNA ligase, and the ligated product was transformed into E. coli Top 10 to construct a recombinant vector, which was confirmed to be correct by PCR and sequencing ...

Embodiment 2

[0078] Relative Fluorescence Quantitative-PCR Detection of Interference Efficiency of RNA Interference Vectors Inhibiting Porcine p53 Gene Expression

[0079] (1) Lipofectamine with Invitrogen TM2000 Under the same conditions, pGenesilencer-GFP (control group) and the RNA interference vector pGenesilencer-GFP-p53 (experimental group) in Example 1 were transfected into PK-15 cells respectively.

[0080] The shRNA template in the pGenesilencer-GFP vector is obtained by annealing hybridization of the sense strand of the control shRNA and the antisense strand of the control shRNA, and the rest of the sequence is the same as that of pGenesilencer-GFP-p53.

[0081](2) After continuing to culture PK-15 cells for 48 hours, the cells were collected, and the total RNA of PK-15 cells was extracted with the MiniBEST Viral RNA / DNA Extraction Kit Ver4.0 kit from Takara Company.

[0082] (3) The extracted total RNA was reverse-transcribed with Takara’s PrimeScript RT Master Mix Perfect Real...

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Abstract

The invention discloses an RNA (Ribonucleic Acid) interference carrier for inhibiting swine source p53 gene expression and an application thereof, belonging to the field of gene engineering. The RNA interference carrier takes pEGFP-C1 as a framework carrier and comprises shRNA transcription templates of a swine U6RNA polymerase III promoter and an interference swine source p53 gene. The RNA interference carrier is obtained by that pEGFP-C1BamHI and HindIII digestion sites are mutated; and the shRNA transcription templates of the swine U6RNA polymerase III promoter and the interference swine source p53 gene are led into a pEGFP-C1 carrier through a digestion and connection manner. The RNA interference carrier is used for carrying out RNA interference research and / or identification gene functions through continuously inhibiting the expression of a target gene so as to lay the foundation on disease-resistant transgenosis breeding.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to an RNA interference carrier, in particular to an RNA interference carrier for inhibiting the expression of pig-derived p53 gene and its preparation and application. Background technique [0002] RNA interference (RNA interference, RNAi) is a phenomenon that inhibits the expression of specific genes in normal organisms. It refers to the introduction of double-stranded RNA (double stranded RNA, dsRNA) homologous to the endogenous mRNA coding region in cells, The degradation of the mRNA leads to the silencing of gene expression, which occurs at the post-transcriptional level, also known as post-transcriptional gene silencing (PTGS). The antisense strand of small interfering RNA (siRNA) produced after exogenous dsRNA enters the cell forms a silencing complex (RNA-induced silencing complex, RISC) with a variety of nucleases. RISC has the ability to bind and cut mRNA The process of med...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/85C12N15/66
Inventor 沈海燕张建峰郭鹏举张春红周恒朱余军
Owner INST OF ANIMAL HEALTH GUANGDONG ACADEMY OF AGRI SCI
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