Porcine adipose tissue-specific chimeric promoters

A tissue-specific, chimeric promoter technology, applied in the field of animal genetic engineering, can solve the problem that adipose tissue is not very clear

Inactive Publication Date: 2012-07-25
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is not very clear whether this fragment can be used as an adipose tissue-specific high-efficiency expression promoter

Method used

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  • Porcine adipose tissue-specific chimeric promoters
  • Porcine adipose tissue-specific chimeric promoters
  • Porcine adipose tissue-specific chimeric promoters

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Acquisition of the sequence adi-pro containing a CRE binding site in the pre-promoter region of the first exon of the porcine adipose tissue-specific expression gene adiponectin.

[0052] 1. Main reagents:

[0053] Phenol (Sinopharm Chemical Reagent Co., Ltd.), chloroform (Sinopharm Chemical Reagent Co., Ltd.), isoamyl alcohol (Sinopharm Chemical Reagent Co., Ltd.), plasmid extraction kit (purchased from OMEGA Company), pMD-18T vector (Bao Bio Engineering Dalian Co., Ltd.), LA Taq polymerase (Purchased from Bao Biological Engineering Dalian Co., Ltd.), dNTP (purchased from Fermentas Company), DL-2000Marker (purchased from Fermentas Company), UNIQ-10 Column DNA Gel Recovery Kit (purchased from Beijing Biotech Biotechnology Co., Ltd.)

[0054] 2. Preparation of the main solution:

[0055] (1) Preparation of related solutions for blood sample collection

[0056] 1M Tris.cl stock solution: Weigh 121.4g Tris base and dissolve it in 800mL double distilled water,...

Embodiment 2

[0097] Embodiment 2: Construction of regulatory element adi-pro transfection vector

[0098] 1. Main reagents

[0099] DL10000Marker and T4 DNA ligase were purchased from Bao Bio-Engineering Dalian Co., Ltd.; restriction endonuclease KpnI and Mlu I were purchased from NEB Company; plasmid mini-quick extraction kit was purchased from Yuanpinghao Biological Company; Endo-free Plasmid Mini KitII plasmid small The extraction kit was purchased from OMEGA Company; the pGL3-Basic eukaryotic expression vector was purchased from Promega Company

[0100] 2. Double enzyme digestion of the dual luciferase reporter vector pGL3-Basic

[0101] Digest the pGL3-Basic vector with Kpn I and Mlu I, double digestion system 20 μl: 2 μl Buffer 2, 0.5 μl Kpn I, 0.5 μl Mlu I, 1 μlpGL3-Basic vector, 0.2 μl BSA, add sterilized ultrapure water to 20 μl. Enzyme digestion 4-6h. The digested products were recovered and purified with UNIQ-10 Column DNA Gel Recovery Kit (purchased from Beijing Biotech Bio...

Embodiment 3

[0106] Example 3: Activity Analysis of adi-pro Regulatory Elements

[0107] 1. Main reagents:

[0108] Fetal bovine serum (FBS, purchased from GIBCO, USA); horse serum (HBS, purchased from GIBCO, USA); DMEM high-glucose medium (purchased from GIBCO, USA); liposome Lipofectamine2000 (purchased from invitrogen); -Luciferase Reporter AssaySystem (purchased from Promega Company)

[0109] 2. Main consumables: cell culture 24-well plate (Corning, USA), cell culture flask, disposable plastic pipette, disposable filter with pore size of 0.22 μm, disposable needle syringe, sealing film (Parafilm), rubber gloves, ELISA plate (Corning, USA)

[0110] 3. Preparation of main reagents:

[0111] (1) 1×PBS preparation: weigh 8g of NaCl, 0.2g of KCl, NaCl 2 HPO 4 12H 2 O 1.54g, KH 2 PO 4 Dissolve 0.19g in 1000mL distilled water, adjust the pH to 7.0, and autoclave.

[0112] (2) Oil Red O dye solution: Weigh 0.5g Oil Red O (purchased from Amersco) and add 100mL of isopropanol to stand ...

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Abstract

The invention belongs to the technical field of animal genetic engineering, and specifically relates to cloning, functional verification and application of two porcine adipose tissue-specific chimeric promoters. According to the invention, the activity of an adi-pro regulating element is verified, and different chimeric promoters containing a pCMVIE promoter and an SV40 enhancer are constructed through a chimeric method, so that the activity of the adi-pro regulating element is greatly improved and new specific expression promoter resources are provided for genetic engineering and molecular breeding. The nucleotide sequences of the promoters are respectively as shown in the sequence tables SEQ ID NO: 4 (sequence identity number: 4) and SEQ ID NO: 5.

Description

technical field [0001] The invention belongs to the technical field of animal genetic engineering. It specifically involves the construction, functional verification and application of a porcine adipose tissue-specific promoter, and improves its transcriptional activity by constructing a chimeric promoter, constructing two chimeric promoters without destroying its adipose tissue specificity sex. Background technique [0002] A promoter is a component of a gene, and in genetics it refers to a deoxyribonucleic acid (deoxyribonuclein acid, DNA) sequence that enables gene transcription. The promoter can be recognized by ribonucleic acid (ribonuclein acid, RNA) polymerase, transcription regulator, etc. and combine with it to form a transcription initiation complex region, thereby initiating gene transcription. The promoter is usually located upstream of the gene, which controls the start site of gene transcription and the degree of expression, and is necessary for precise and e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113
Inventor 蒋思文李芳
Owner HUAZHONG AGRI UNIV
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