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A plasmid containing beef cattle dkk3 gene 3'utr sequence and dual luciferase reporter gene and its application

A dual-luciferase and sequence technology, applied in the field of molecular biology, can solve problems that have not been reported, and achieve good application prospects, high sensitivity, and low cost

Active Publication Date: 2022-07-15
INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research on DKK3 mainly focuses on the proliferation and invasion of cardiovascular and tumor cells, and the research on the meat quality and fat deposition of livestock has not been reported yet.

Method used

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  • A plasmid containing beef cattle dkk3 gene 3'utr sequence and dual luciferase reporter gene and its application
  • A plasmid containing beef cattle dkk3 gene 3'utr sequence and dual luciferase reporter gene and its application
  • A plasmid containing beef cattle dkk3 gene 3'utr sequence and dual luciferase reporter gene and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Construction of pmirGLO-DKK3-3'UTR dual-luciferase reporter plasmid

[0044] 1. Combined with the bioinformatics database TargetScan ( http: / / www.targetscan.org / vert_71 / ) and the NCBI database (https: / / www.ncbi.nlm.nih.gov / ) to obtain the target of bta-miR-25 binding to bovine DKK3 (SEQ ID NO. 2), which is located in the 3'UTR region of the DKK3 gene .

[0045] 2. The 3'UTR region amplification primers of DKK3 were designed with primer 5 software, and the restriction sites Pme I and XhoI were added; the MDBK cDNA was used as a template to amplify a fragment with a size of 321 bp (as shown in SEQ ID NO.1). ); the primer sequences are as follows:

[0046] DKK3-3'UTR-F:5'-gg gtttaaac ggagcctgtcagtggag-3' (SEQ ID NO. 5);

[0047] DKK3-3'UTR-R:5'-ccg ctcgag gtttagcagccctgttct-3' (SEQ ID NO. 6);

[0048] The underline is the restriction site, the base before the restriction site is the protection base, the restriction site of the forward primer (SEQ ID NO....

Embodiment 2

[0066] Example 2 Construction of DKK3 Gene Mutant and Deleted Vectors

[0067] Using the wild-type vector plasmid in Example 1 as a template, the predicted bta-miR-25 binding site was mutated or deleted by fusion PCR, and the primer sequences used were as follows:

[0068] DKK3-3'UTR-F:5'-GG GTTTAAAC GGAGCCTGTCAGTGGAG-3' (SEQ ID NO. 5)

[0069] DKK3-3'UTR-R:5'-CCG CTCGAG GTTTAGCAGCCCTGTTCT-3' (SEQ ID NO. 6)

[0070] DKK3-mut-F: 5'-TTGTGTGGGTAGATagatccTAGAAGTAGCTAAT-3' (SEQ ID NO. 7)

[0071] DKK3-mut-R: 5'-ATTAGCTACTTCTAggatctATCTACCCACACAA-3' (SEQ ID NO. 8)

[0072] DKK3-del-F: 5'-TTGTGTGGGTAGATTAGAAGTAGCTAAT-3' (SEQ ID NO. 9)

[0073] DKK3-del-R: 5'-ATTAGCTACTTCTAATCTACCCACACAA-3' (SEQ ID NO. 10)

[0074] The underline is the restriction site, the base before the restriction site is the protection base, the restriction site of the forward primer (SEQ ID NO.5) is Pme I, and the reverse primer (SEQ ID NO.6) The enzyme cleavage site is Xho I; lowercase letters are muta...

Embodiment 3

[0078] Example 3 The targeting effect of bta-miR-25 on the 3'UTR of DKK3 gene

[0079] 1. Cell transfection

[0080]MDBK cells were seeded into 48-well cell plates and transfected when the cells had grown to 80%. Add 0.1μg plasmid and 1μL miR-25-mimics to each well to dilute in 25μL opti-MEM, and 1μL Lipofectamine 2000 to dilute in 25μL opti-MEM. After 5min, the two parts were mixed and left at room temperature for 10-15min. in cell culture plates. In the control group, miR-25-NC (negative control) was replaced by miR-25-mimics in the same way. 24h after transfection, the cells were lysed with 1×PLB (Passive lysis buffer) and the cell lysate was collected to measure the dual luciferase activity.

[0081] 2. Dual-luciferase activity assay

[0082] Use the Dual-Reporter Assay System to detect the relative fluorescence activity of the fluorescent carrier in a multi-function microplate reader: Pipet 10 μL of cell lysate and add it to the microplate plate, first add 50 μL of LA...

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Abstract

The present invention provides a plasmid containing a beef cattle DKK3 gene 3'UTR sequence and a dual luciferase reporter gene and application thereof, the plasmid contains a target for binding bta-miR-25 and bovine DKK3, and its construction includes designing a DKK3 gene The 3'UTR region amplification primers were added with enzyme cleavage sites Pme I and Xho I, and the bovine kidney cell (MDBK) cDNA was used as a template for amplification, and the PCR product and pmirGLO Vector were respectively used with restriction enzymes Pme I and Xho 1 carry out double digestion, link the carrier pmirGLO Vector and DKK3 gene 3'UTR fragment purified after the digestion, transform, cultivate, PCR detection, sequencing, the bacterial liquid that the sequencing is correct is re-expanded and cultivated, and steps such as plasmid extraction, the present invention It provides a simple and easy tool for screening the miRNAs that regulate the expression of DKK3 and evaluating the regulation effect of miRNAs on the expression of DKK3, and has the advantages of high sensitivity, fast speed and low cost; the present invention also brings deeper insights into the regulation of DKK3 expression. Cognition can be applied to the study of the molecular regulation mechanism of the genetic improvement of livestock meat quality, and has a good application prospect.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a plasmid containing the 3'UTR sequence of beef cattle DKK3 gene and a dual luciferase reporter gene and its application. Background technique [0002] DKK3 (Dickkopf-3) is a secreted protein that acts by regulating the Wnt signaling pathway and is an inhibitor of the Wnt / β-catenin signaling pathway. At present, the research on DKK3 mainly focuses on the proliferation and invasion of cardiovascular and tumor cells, and the research on meat quality and fat deposition in livestock has not been reported yet. There are three main branches of Wnt signaling pathway: Wnt / β-catenin pathway, Wnt / Ca pathway 2+ pathway and Wnt / polarity pathway, among which Wnt / β-catenin pathway is a classic Wnt signaling pathway, which plays a key negative regulatory role in adipocyte differentiation. Accordingly, we speculate that DKK3 is likely to be involved in the regulation of adipogenesis ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/65C12N15/66C12N15/12C12Q1/66
CPCC12N15/85C12N15/65C12N15/66C07K14/4703C12Q1/66
Inventor 张凤陈明新熊琪刘洋张年陶虎李晓锋索效军杨前平张鹤山田宏熊军波陆姣云
Owner INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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