SiRNA for bovine trim5alpha gene silencing and application of siRNA

A technology of α gene and RNA interference, which is applied in the field of cell biology, can solve the problems of low transfection efficiency of plasmid vectors, low infection rate, low efficiency of direct liposome transfection of plasmid vectors, etc.

Inactive Publication Date: 2015-02-18
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The third is to construct shRNA viral expression vectors. Commonly used viral vectors include adenoviral vectors, adeno-associated viral vectors, and lentiviral vectors. The defect of low transfection efficiency
The infection rate of MDBK cells to mouse N-type retrovirus and human immunodeficiency virus type 1 (Human immunodeficiency virus type) virus is extremely low. When the infection index MOI is 1, the infection percentage is less than 1% (Si, Z. et al. .Proc.Natl.Aca

Method used

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  • SiRNA for bovine trim5alpha gene silencing and application of siRNA
  • SiRNA for bovine trim5alpha gene silencing and application of siRNA
  • SiRNA for bovine trim5alpha gene silencing and application of siRNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1. Construction of a human lentiviral vector carrying DNA encoding siRNA for specific silencing of the trim5α gene

[0052] 1. Screen the DNA sequence of the siRNA encoding the trim5α gene

[0053] Using bioinformatics to search NCBI GeneBank, the whole mRNA sequence of bovine trim5α gene (LOC505265) was obtained as the target sequence for siRNA design. Preliminary design and screening were carried out on the corresponding siRNA design website (http: / / rnaidesigner.lifetechnologies.com / rnaiexpress / ), to find possible target sequences with siRNA functions and corresponding DNA sequences encoding siRNA. Then compare the selected sense strand sequence and antisense strand sequence with the known gene sequence of the same species in GeneBank (http: / / www.ncbi.nlm.nih.gov / blast / ), and select the one with siRNA function The possible target sequences, respectively named SiRNA-1 to SiRNA-4. Include:

[0054] (1) trim5α-siRNA-1:

[0055] 5'-CTGGCAGAAGTCAAGACAA-3'

[00...

Embodiment 2

[0109] Example 2. MDBK cell trim5α gene silencing stable cell line screening

[0110] 1. Concentration test of puromycin in MDBK cells

[0111] (1) MDBK cells were treated with 5×10 4 / well into a 24-well plate. After 24 hours, add puromycin (purchased from Sigma, USA) to the following concentrations:

[0112] (2) 0.1 μg / ml; 2.5 μg / ml; 5 μg / ml; 7.5 μg / ml; 10 μg / ml.

[0113] (3) The medium was changed every 2 days, and puromycin was added again.

[0114] (4) Select the concentration at which all cells die within 3-4 days as the screening drug concentration.

[0115] 2. Obtaining of replication defective human lentivirus

[0116]Take any one of the five recombinant human lentiviral vectors prepared in Example 1, the three plasmids of the pH1 vector and the pH2 vector, and the Polyfect-V transfection reagent (purchased from Beijing Yingmaoshengye Biotechnology Co., Ltd.) with liposome transfection Human embryonic kidney cells HEK293T (purchased from Beijing Yingmao Shengye ...

Embodiment 3

[0161] Example 3. The effect of trim5α-337 siRNA on the transfer efficiency and expression level of foreign genes after infection of N14 stable cell line with human lentivirus

[0162] N14 cells were inoculated into 24-well cell culture plates at 30% confluence, and the number of cells per well was about 5×10 4 , cultured at 37°C for 18h. Add Polybrene to a final concentration of 5 μg / ml, add CMV-GFP-L.V lentivirus (purchased from Heyuan Biotechnology (Shanghai) Co., Ltd.) at MOI 100, discard the medium after 18 hours of infection, and add 500 μl fresh high Sugar DMEM medium. Fluorescent microscope pictures were taken 72 hours after infection. The gene transfer efficiency and expression efficiency of human CMV-GFP-L.V lentivirus in N14 cells and NC were detected by fluorescence microscopy. The specific steps include: placing the two kinds of cells on the stage of a fluorescent inverted microscope (ECLIPSE Ti-S, NIKON), and detecting them at an excitation wavelength of 488 n...

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Abstract

The invention provides an interference method for bovine trim5alpha gene silencing, a siRNA sequence, a siRNA human lentivirus expression vector, a replication-defective human lentivirus and a human lentivirus infected MDBK (madin-darby bovine kidney) cell. RNAi and the siRNA human lentivirus expression vector effectively interfere with the expression of a bovine trim5alpha gene, and the gene expression of the human lentivirus vector in the MDBK cell is significantly improved. The siRNA provides the new technical support for research on bovine virus disease related gene functions and transgenic cattle breeding.

Description

technical field [0001] The invention belongs to the field of cell biology, in particular, the invention relates to a siRNA for down-regulating expression of bovine trim5α and application thereof. Background of the invention [0002] Before the emergence of RNA interference technology, gene knockout (gene knockout) was the main research method of reverse genetics (reverse genetics), but its technical difficulty is high, the operation is complicated, and the cycle is long. Because RNA interference can use siRNA or siRNA expression vectors to quickly, economically and easily have high sequence specificity, it can specifically silence specific genes, obtain function loss or reduce mutant sequence-specific ways to knock out the expression of target genes, and now has It has become an important research tool for exploring gene function. In functional genomics research, loss of function or reduced mutation of a specific gene is required to determine its function, so RNA interferen...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/86C12N5/071
Inventor 刘艳红尚佑军孙德惠刘湘涛殷宏龚真莉祁淑芸李勇
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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