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Reporter gene carrier for detecting activity of AP2alpha transcription factor

A reporter gene and resistance gene technology, which is applied in the field of luciferase reporter gene retroviral vector and its construction, can solve the problems of increased combination and inability to reflect the inhibition truthfully, and achieve simple and convenient operation, low price and good stability Effect

Inactive Publication Date: 2013-09-18
CHILDRENS HOSPITAL OF CHONGQING MEDICAL UNIV
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

Two dominant-negative mutant adenoviruses with deletion of bHLH region and deletion of TAD region were respectively constructed by deletion mutation method, infected MSCs, and EMSA detection found that the binding of dnAP2α-△TAD to AP2α-specific cis-acting element DNA sequence was significantly increased , since dnAP2α-△TAD retains the DNA-binding domain, EMSA cannot faithfully reflect the inhibition of AP2α transcriptional activity by this mutant

Method used

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  • Reporter gene carrier for detecting activity of AP2alpha transcription factor
  • Reporter gene carrier for detecting activity of AP2alpha transcription factor
  • Reporter gene carrier for detecting activity of AP2alpha transcription factor

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Example 1: Access to AP2α-specific cis-acting elements

[0037] Design an AP2α-specific cis-acting element consisting of 4 tandem GCCCGCGG nucleotide sequences. The upstream and downstream sequences correspond to the nucleotide sequences of SEQ ID NO.1 & SEQ ID NO.2 respectively, using annealing buffer (Beiyuntian company) in vitro annealing into double strands (100ul system: 5ug each of upstream and downstream sequences, 95°C for 5 min, 72°C for 2min, 60°C for 2min, 50°C for 2min, 40°C for 2min, 20°C for 2min).

Embodiment 2

[0038] Example 2 : Construction and identification of pBGLuc-AP2α-RE reporter gene vector

[0039] Retroviral vector pBGLuc was double digested with BamH Ⅰ and Mlu Ⅰ (40 μl system: 2 μl of BamHI, 2 μl of Mlu Ⅰ, 4 μl of 10× buffer, 5 μg of DNA, added sterilized water to 40 μl, 6-8 hr at 37°C) and then purified. DNA double-strand directional ligation in Example 1 (ligation system 20 μl: DNA double-strand 5 μl, restriction plasmid 5 μl, DNA ligase 10 μl, 1 hr at 16°C) ( figure 1 ). The ligation product was transformed into competent Escherichia coli DH5α, using the sequence complementary to the BSD gene sequence (SEQ ID NO.3) and the sequence complementary to the sequence of the AP2α cis-acting element (SEQ ID NO.4) as primers, colony PCR screening (touch-down PCR system: 10ul of 2×PCR reaction solution, 1ul of 10ng / ul upstream and downstream primers each, colony as template, add water to 20ul; PCR parameters: 95°C for 5 min; 93°C for 20s, 65~55°C for 20s, 72°C for 20s, Lower...

Embodiment 3

[0040] Example 3 :: pBGLuc-AP2α-RE reporter gene vector for detection of AP2α transcription factor activity

[0041] The purpose of this example is to test the application of the pBGLuc-AP2α-RE reporter gene vector obtained in Example 2 as a molecular tool. Rat MSCs were seeded in 24-well plates, 1×10 5 / well, set 3 duplicate wells, culture overnight, when the density was 60%, transfect the cells with the pBGLuc-AP2α-RE plasmid with liposomes. After 24 hours, add Ad-AP2α, Ad-dnAP2α-△bHLH, and Ad-dnAP2α-△TAD adenovirus respectively. After 48 hours of infection, the positive rate of RFP was observed under an inverted fluorescent microscope to reach 60-70%. 1umol / L ATRA induction set up blank control group (control group) and ATRA induction control group at the same time. After 24 hours of ATRA induction, take 20ul of the culture supernatant from each well to detect the activity of secreted luciferase, add 10ul of Luciferin reaction solution (reaction buffer: substrate = 100:...

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Abstract

The invention provides a reporter gene carrier which comprises an AP2alpha specific cis acting element. The carrier is characterized in that the AP2alpha specific cis acting element consists of four GCCCGCGG nucleotide sequences connected in series, wherein the four GCCCGCGG nucleotide sequences are connected in series through a nucleotide sequence TT. The reporter gene carrier designed by the invention is easy to prepare, simple to operate and good in repeatability, and can effective transfect different mammalian cells. The carrier is more accurate and reliable than EMSA (Electrophoretic Mobility Shift Assay) when is used to detect activity change of a dominant negative mutant-mediated transcription factor. The carrier is suitable for in vitro study of biological activity of the AP2alpha transcription factor in various mammalian cells.

Description

[0001] technical field [0002] The invention belongs to the field of biotechnology, and in particular relates to a luciferase reporter gene retroviral vector carrying a transcription factor AP2α cis-acting element and a construction method thereof. technical background [0003] Transcription factor AP2 is a transcriptional activator with a molecular weight of 52-kDa. It is located in the nucleus and has a unique protein molecular structure. It usually binds to DNA-rich GC elements in the form of dimers, specifically regulates gene expression, and specifically regulates Gene expression is involved in the growth and development of vertebrates, cell proliferation and differentiation, and can bidirectionally regulate the occurrence of tumors, and itself is also affected by various signaling molecules and proteins. Studies have found that the AP2 transcription factor is particularly sensitive to retinoic acid and can be regulated by the retinoic acid signaling pathway. In retino...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/65C12Q1/68
Inventor 李廷玉毕杨陈洁龚敏魏小平侯娜丽
Owner CHILDRENS HOSPITAL OF CHONGQING MEDICAL UNIV
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