Panx3 gene overexpression red fluorescence lentiviral vector and building method and application thereof

A lentiviral vector and gene overexpression technology, applied in the field of genetic engineering, can solve the problem of mutual interference between overexpression vectors and functional research, and achieve the effect of high-efficiency expression

Inactive Publication Date: 2014-09-24
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to solve the problem of mutual interference between the overexpression vector and the function research in the research of the Panx3 gene, and provide a red fluorescent lentiviral vector for the overexpression of the Panx3 gene

Method used

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  • Panx3 gene overexpression red fluorescence lentiviral vector and building method and application thereof
  • Panx3 gene overexpression red fluorescence lentiviral vector and building method and application thereof
  • Panx3 gene overexpression red fluorescence lentiviral vector and building method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Construction of pCDNA3.1(+)-Panx3

[0040] Total RNA was extracted from human dental pulp cells, using the reverse-transcribed cDNA as a template, primers were designed according to the sequence of the Panx3 gene cDNA, and PCR amplification was performed with primers 1 and 2 as primers.

[0041] Primer 1: 5'-CCC AAGCTT ATGTCACTTGCACACACAG-3',

[0042] Primer 2: 5'-CGC AAGCTT TTTGACTCTTCGGCTCCA-3' (underlined bases are HindIII recognition sites).

[0043] The PCR reaction conditions were: 94°C for 3min, followed by 35 cycles of 94°C for 30s, 58°C for 40s, 72°C for 1min, and finally 72°C for 10min.

[0044]A linear DNA amplification product Panx3 (1396bp) can be obtained by PCR, with HindIII restriction sites at both ends. The Panx3 fragment and the pCDNA3.1(+) plasmid are simultaneously digested with HindIII, and recovered by the gel recovery kit (Kangwei Century Company), T4 DNA ligase for two-fragment ligation, and transform E.coli DH5α competent cells ...

Embodiment 2

[0045] Example 2 Construction of pCDNA3.1(+)-Panx3-RFP

[0046] The Thy-Brainbrow-1.0H (Addgene China agency company, Plasmid 18724) vector was used as a template, and the RFP red fluorescent protein fragment was amplified by PCR with primers 3 and 4 as primers.

[0047] Primer 3: 5'-TCC CCCGGG AGACCCAAGCTGGCTAGCG-3', recognized as dot by SmaI,

[0048] Primer 4: 5'-CCC TTCGAA CTGGCAACTAGAAGGCACAGTCG-3', BstBI recognition site.

[0049] The PCR reaction conditions were: 94°C for 3min, followed by 33 cycles of 94°C for 30s, 58°C for 30s, 72°C for 1min, and finally 72°C for 10min.

[0050] A linear DNA amplification product RFP (805bp) was obtained by PCR. One end contained a SmaI restriction site, and the other end contained a BstBI restriction site. SmaI and BstBI endonucleases were used to simultaneously digest the RFP fragment and pCDNA3.1(+)- The Panx3 recombinant plasmid was recovered by gel recovery kit (Kangwei Century Company), and the two fragments were connected...

Embodiment 3

[0051] Example 3 Construction of pCDNA3.1(+)-Panx3-CMV-RFP

[0052] Using pCDNA3.1(+) as a template, primers 5 and 6 were used to amplify the CMV promoter fragment by PCR,

[0053] Primer 5: 5'-CGC GGATCC GCTTGACCGACAATTGCAT-3', BamHI recognition site,

[0054] Primer 6: 5'-CGC GGATCC ATTTCGATAAGCCAGTAAGCAG-3', BamHI recognition site.

[0055] The PCR reaction conditions were: 94°C for 3min, followed by 33 cycles of 94°C for 30s, 58°C for 30s, 72°C for 1min, and finally 72°C for 10min.

[0056] A linear DNA amplification product CMV fragment (732bp) was obtained by PCR, with BamHI restriction sites at both ends, recovered from the gel extraction kit (Kangwei Century Company), and the fragment and pCDNA3.1( +)-Panx3-RFP recombinant plasmid, use T4 DNA ligase to connect the two fragments, and transform E.coli DH5α competent cells. The kit extracts the pCDNA3.1(+)-Panx3-CMV-RFP recombinant plasmid, digests it with restriction endonuclease NcoI-HF, and the digested product ...

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Abstract

The invention discloses a Panx3 gene overexpression red fluorescence lentiviral vector and a building method and application of the Panx3 gene overexpression red fluorescence lentiviral vector, and belongs to the field of gene engineering. According to the vector, a lentiviral vector pLL3.7 is used as a skeleton vector, a Panx3 gene, CMV promoter and RFP gene segment is inserted behind a CMV promoter of pLL3.7 in the direction from the 5' end to the 3' end, and the sequence of the segment is shown in SEQIDNO.1. The built Panx3 gene overexpression red fluorescence lentiviral vector has the advantages that the mutual interference problem of the overexpression vector and function research in Panx3 gene research is solved, a CMV serves as the promoter of the vector, eukaryon gene efficient expression can be guaranteed, RFP serves as a marker, the role of the Panx3 gene in intracellular calcium and ATP change after a half-channel is formed can be directly observed, the cell function is eventually affected, and a good tool is provided for research on the functions of the Panx3 gene.

Description

[0001] technical field [0002] The invention belongs to the field of genetic engineering and relates to the construction of a Panx3 gene overexpression lentiviral vector, in particular to a red fluorescent lentiviral vector for Panx3 gene overexpression and its construction method and application. Background technique [0003] Panx3 is a kind of gap junction protein discovered in vertebrates by Panchin et al. in 2000, and it is one of the most important gap junction protein families in vertebrates. It can form a gap protein channel, allowing ions and small molecules to pass through, and then connect adjacent cells and cells to the extracellular matrix, which is the basis for the synchronization of intercellular activities. At present, the function of the Panx3 gene is mainly manifested in the formation of hemichannels, which transmit signaling molecules such as calcium ions, cAMP, and ATP, and then affect a series of downstream cell proliferation, differentiation, and other...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/66
Inventor 黄翠付东杰王亚珂宋芳芳
Owner WUHAN UNIV
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