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Construction method, identification method and application of porcine FLJ small interference RNA expression vector

An expression vector, small interference technology, applied in recombinant DNA technology, microbial assay/inspection, introduction of foreign genetic material using a vector, etc. It can solve problems such as undetermined functions, and achieve the effect of a simple vector construction method.

Inactive Publication Date: 2012-06-27
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Human FLJ is a new gene screened out by sequencing in 2003, and its function has not yet been determined

Method used

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  • Construction method, identification method and application of porcine FLJ small interference RNA expression vector
  • Construction method, identification method and application of porcine FLJ small interference RNA expression vector
  • Construction method, identification method and application of porcine FLJ small interference RNA expression vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The construction and identification of the siRNA expression vector of embodiment 1, porcine FLJ, carry out the following steps successively:

[0038] 1. Target sequence selection: According to the full-length porcine FLJ gene sequence, follow the Tuschl rules and Cenix rules to design and screen the siRNA target sequence of FLJ (the position in the nucleotide sequence of the FLJ gene is indicated in brackets):

[0039] FS1 target sequence: 5'-AACTGTCGCTGGCCGACAGCA-3' (137-157bp)

[0040] FS2 target sequence: 5'-AAGCTGTTCATGCCCCGCAGC-3' (181-201bp)

[0041] FS3 target sequence: 5'-AAGGACGTCTACGGCTACTCC-3' (No. 286-306bp)

[0042] Select the 9-nucleotide loop "TTCAAGAGA", design and synthesize siRNA primers according to the information of the vector and the target sequence. The structural order of the synthetic primers is:

[0043]

[0044] The resulting 3 pairs of shRNA primers are:

[0045] fs1:

[0046] SENSE: 5′-GATCC CTGTCGCTGGCCGACAGCA TTCAAGAGA TGCTGTCGGCCA...

Embodiment 2

[0062] Embodiment 2, effective siRNA screening

[0063] 1. Using Lipofectamine TM 2000-mediated transfection of porcine intramuscular adipocytes with the recombinant interference vector of porcine FLJ constructed and screened in Example 1, irrelevant sequences (negative control) and blank control, respectively. The day before transfection, trypsinize the cells and count them, and plate the cells so that the density on the day of transfection is 80% to 90%. The DMEM cell culture medium contains 10% fetal bovine serum and does not contain antibiotics; for each well of cells, Dilute 4 μg DNA with 50 μl serum-free medium (OPTI-MEM I medium); for each well of cells, dilute 10 μl Lipofectamine with 50 μl OPTI-MEM I medium TM 2000 reagents. Lipofectamine TM After 2000 dilution, mix with the diluted DNA within 30min (excessive incubation time will reduce the activity); mix the diluted DNA and the diluted Lipofectamine TM 2000, incubate at room temperature for 20 min (the comple...

Embodiment 3

[0068] Example 3, Effect of FLJ-siRNA (fs1) on the expression of key functional genes of fat metabolism

[0069] The siRNA expression vector of fs1 with the best inhibitory effect was obtained by screening in Example 2, and its inhibitory effect on the expression of the target gene was detected according to the reaction system in Example 2. After the pig intramuscular adipocytes are subcultured for 48 hours, the density reaches 80-90% (volume density) on the day of transfection for treatment. The experiment was divided into three groups: control group (wt), transfected with empty vector, FLJ-siRNA group (neg), transfected with effective recombinant interference vector fs1 (fs1), and each treatment had 6 replicates. Real-time quantitative PCR was used to detect the mRNA expression of FAS, ACC, ATGL and HSL, the key functional genes of fat metabolism.

[0070] The results are as follows: After transfecting fs1 to interfere with the expression of FLJ gene, the expression of key ...

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Abstract

The invention discloses a construction method of a porcine FLJ small interference RNA expression vector. The construction method comprises the following steps of: 1) designing 3 pairs of siRNA primers of loop rings containing 9 nucleotides, wherein the 9 nucleotides are TTCAAGAGA; and 2) constructing an RNAi expression vector, namely after annealing the siRNA primers, connecting the siRNA primers with double-enzyme-cut RNAi vector and converting to obtain recombinant plasmid of the RNAi expression vector. The invention also provides a method for identifying the constructed porcine FLJ effective small interference RNA expression vector. The invention further provides application of the porcine FLJ effective small interference RNA expression vector constructed by the method. The porcine FLJ effective small interference RNA expression vector is used for researching the influence of the FLJ gene on fat deposition in porcine muscle.

Description

technical field [0001] The invention relates to the construction, identification and application of a porcine FLJ gene small interfering RNA (siRNA) effective expression carrier, which belongs to the application technology in the field of biology and modern agricultural technology. Background technique [0002] The FLJ gene is a new gene closely related to pig intramuscular fat deposition obtained through gene chip technology screening by our research group. Human FLJ is a new gene screened out by sequencing in 2003, and its function has not yet been determined. Based on the known EST sequence of the porcine FLJ gene, our research group cloned the full-length cDNA sequence of FLJ through RACE technology. The full-length cDNA sequence of the FLJ gene has 2793bp (GenBank accession number EU030283), including the 283bp 5' non-coding region (5 '-untranslated region, 5'-UTRs), 714bp coding region (coding region, CDs) and 1796bp 3' non-coding region (3'-UTR), the coding region tr...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/66C12Q1/68
Inventor 汪以真伍婷袁章琴
Owner ZHEJIANG UNIV
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