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Manufacturing method of adjustable liver damage animal model and special DNA fragment thereof

A technology of DNA molecules and fragments, which is applied in the production of adjustable liver injury animal models and the field of special DNA fragments, which can solve the problems of slow speed, incomplete induction, and difficult operation

Active Publication Date: 2010-06-23
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

3) The operation is inconvenient, and the mice of 7-14 days are not easy to operate
3) Efficient and rapid induction: Dox can increase the expression of the target gene by 1000 times within 30 minutes, while other induction systems are slow and cannot be fully induced

Method used

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  • Manufacturing method of adjustable liver damage animal model and special DNA fragment thereof
  • Manufacturing method of adjustable liver damage animal model and special DNA fragment thereof
  • Manufacturing method of adjustable liver damage animal model and special DNA fragment thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Embodiment 1, the construction of pAlb-rtTA-polyA plasmid

[0069] 1. Construction of Psk-rtTA-polyA plasmid

[0070] 1. Digest the pTet-On-Advanced vector (Clontech Company, Catalog No.: 630930) with restriction endonucleases EcorI and HindIII at 37°C overnight. After the digestion, the digested product is subjected to 1% agarose gel electrophoresis. The rtTA-polyA fragment of about 1.2kb was recovered with Dingguo glue recovery kit. The recovered DNA fragments were dissolved in 10ul ultrapure water. The recovered DNA fragments were sequenced, see sequence 1 in the sequence listing, and the amino acid sequence encoded by the rtTA gene was shown in sequence 2 in the sequence listing.

[0071] 2. Use restriction enzymes EcorI and HindIII to digest the pBluscript vector (Fermentas company) overnight at 37°C. After the digestion, the digested product is subjected to 1% agarose gel electrophoresis, and about 3kb is recovered with the Dingguo gel recovery kit pSK vector b...

Embodiment 2

[0085] Embodiment 2, making TRE-PminCMV-uPA adenovirus

[0086] 1. Amplification of the prourokinase activator (uPA) gene

[0087] A pair of primers were designed according to the mouse uPA mRNA sequence (NM_008873) in Gene bank as follows:

[0088] Upstream primer (29bp): 5'-GGC GCTA ATGAAAGTCTGGCTGGCGAG-3';

[0089] Downstream primer (29bp): 5'-TCT GGTACC GAGAGGACGGTCAGCATGGG-3'.

[0090] Among the above primers, the underlined ones are restriction sites, and the italic boldfaced ones are Kozak sequences for enhanced expression. The upstream primer is located at the initiation ATG of uPA mRNA and contains an NheI restriction site. The downstream primer is located 76bp downstream of the stop codon and contains a Kpn I restriction site.

[0091]1. Extract kidney RNA of CD-1 mouse (purchased from Weitong Lihua) and reverse transcribe into cDNA. Using cDNA as a template, using the above primers, use TaKaRa's Pyrobest DNA Polymerase to perform PCR reaction.

[0092] 2....

Embodiment 3

[0142] Embodiment 3, preparation of liver injury mice

[0143] 1. Preparation of Alb-rtTA mice

[0144] (1) Digestion, recovery and purification of Alb-rtTA fragments

[0145] 1. Digest pAlb-rtTA-polyA plasmid overnight at 37°C with restriction enzymes SacI and KpnI.

[0146] 2. The digested products were subjected to 1% agarose gel electrophoresis. Electrophoresis results see Figure 5 . The 3.5kb band was cut out, and the DNA was recovered with a DNA gel recovery kit (Dingguo).

[0147] 3. Use Promega Wizard DNA clear-up kit (Cat: A 7280) to further purify the DNA fragment.

[0148] 4. TE (7mM Tris-HCl, 0.15mM EDTA, pH 7.5) dissolves the purified DNA.

[0149] (2) Preparation of DNA samples for pronuclear injection

[0150] To measure the OD value of the DNA sample, dilute the DNA to 2-5ng / ul with TE; centrifuge at 12000g×10min (4°C); carefully suck out the upper 1 / 3 part of the solution after centrifugation, divide it into several centrifuge tubes, freeze Store in -...

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Abstract

The invention discloses a manufacturing method of an adjustable liver damage animal model and a special DNA fragment thereof; a DNA fragment I is formed by sequentially connecting liver super promoters and antisense tetracycline activator coding genes of a Tet-on gene expression system from upstream to downstream; an DNI fragment II is formed by sequentially connecting tetracycline reaction factors, promoters and prourokinase activator coding genes of the Tet-off / Tet-on gene expression system from upstream to downstream; a recombination expression vector carrying the DNI fragment II is used for converting mammalian cells, the cells are cultivated to obtain recombinant adenovirus. The DNA fragment I is led in the animal to obtain a transgenosis first-construction animal; the transgenosis first-construction animal and scid / bg animal are back-crossed to obtain the animal carrying the DNA fragment I with the scid / bg background; the recombinant adenovirus is used for injecting the animal carrying the DNA fragment I with the scid / bg background, so as to obtain the adjustable liver damage animal model; in the model, the prourokinase activator expression is activated by doxycycline, the expression intensity is changed along with the change of dosage, the specificity is very high.

Description

technical field [0001] The invention relates to a method for making an animal model of liver injury that can be regulated and the special DNA fragment thereof. Background technique [0002] Hepatitis B virus (HBV) and hepatitis C virus (HCV) seriously threaten human health. There are about 360 million hepatitis B patients and 170 million hepatitis C patients in the world, and about 1.5 million patients die every year. In the past decade, great progress has been made in the treatment of these diseases, but there is still no effective cure, and the existing treatments are often accompanied by side effects, and the development of better antiviral drugs is still a top priority. [0003] At present, new antiviral drugs are mainly tested and evaluated at the cellular level, and their activity and toxicity must be tested in animal models before being used in clinical practice. Anti-HBV and HCV drugs can only be studied in chimpanzees. Because of economic and ethical reasons, chimp...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/12C12N15/63C12N15/861C12N7/01C07K14/47A01K67/027
Inventor 邓宏魁宋希军多曙光郭玉珊
Owner PEKING UNIV
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