Gene search vector, random gene mutation control method and application thereof

A gene regulation and gene technology, applied in the field of random gene mutation, can solve the problem of not improving the long-term survival time of pancreatic cancer patients

Inactive Publication Date: 2012-10-24
THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the past two decades, important progress has been made in research on its occurrence, development, diagnosis, and treatment, and the ratio of the average annual mortality rate to the average annual incidence rate has declined, but it still cannot improve the long-term survival time of patients with pancreatic cancer

Method used

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  • Gene search vector, random gene mutation control method and application thereof
  • Gene search vector, random gene mutation control method and application thereof
  • Gene search vector, random gene mutation control method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1 main experimental material

[0048] Restriction endonuclease and T4 DNA ligase were purchased from NEB Company; primers, sequencing and Taq polymerase were purchased from Invitrogen Company; plasmid extraction kit and gel recovery kit were purchased from OMEGA Company; Aspc-1 cell line (purchased from Cell Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences), fetal bovine serum, DMEM medium, and PBS were purchased from Hyclone Company, FugenHD was purchased from Roche Company, various cell culture consumables were purchased from Corning Company, puromycin (puromycin), Luciferin The substrate was purchased from Invivo Company, neomycin (neomyicn, G418) was purchased from Merck Company, and thymus-deficient nude mice (BALB / c nu / nu) female mice were purchased from China National Institute for the Control of Pharmaceutical and Biological Products.

Embodiment 2

[0049] Example 2: Construction of gene search vector

[0050] The gene search vector in the present invention includes several parts such as tetracycline response element, neomycin resistance gene, plasmid replication origin and piggbac transposon, these core parts need to be spliced ​​together, the general process is shown in image 3 A, specifically includes the following parts:

[0051] 1. The acquisition of the TATA-14Tet fragment, which includes the eukaryotic gene transcription initiation point TATA box and 2 tetracycline response elements; use restriction endonucleases SmaI, XhoI and EcoRI to single-cut and double-cut pUHD13-3 plasmid respectively 38 ,Methods as below:

[0052] SmaI single-cut plasmid pUHD13-3, the reaction system is shown in Table 1 below:

[0053] Table 1:

[0054]

[0055] The reaction conditions are: incubate at 25°C for 2h.

[0056] XhoI and EcoRI double cut plasmid pUHD13-3, the reaction system is shown in Table 2 below:

[0057] Table 2: ...

Embodiment 3

[0078] Example 3: Construction of a genome-wide random mutation library

[0079] The construction of the random mutation library involved in the present invention plays a role by randomly inserting the core elements on the piggyBac transposon-based gene search vector of the cell genome. This work includes constructing a vector expressing the transcriptional activator tTA, establishing a Tet- Pancreatic cancer cell lines with off expression regulation system, randomly integrated gene search vectors into Tet-off cell lines, details are as follows:

[0080] 1. Construct a plasmid expressing the transcriptional activator tTA. In order to establish a Tet-off cell line that highly expresses tTA, first insert the strong promoter CAG in front of the tTA coding gene. The coding sequence of the transcriptional activator tTA was amplified from the pTet-off / hygro plasmid (purchased from Clontech) using primer tTA-P1 and primer tTA-P2. Expression vector pCAG-PBase 41 After transformation...

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Abstract

The present invention relates to a gene search vector, a random gene mutation control method and application thereof. Specifically, the present invention relates to a vector for gene search system, a random gene control method, a method for screening high transfer human tumor cell lines, a method for screening a gene for improving human tumor cell line transfer, a whole-genome random mutation library obtained by using the random gene control method, pharmaceutical application of the gene obtained by using the method for screening gene for improving human tumor cell line transfer, pharmaceutical application of an inhibitor of the gene, and pharmaceutical application of an inhibitor of an expression product of the gene. The gene obtained by the method of the invention, the inhibitor of the gene, the expression product of the gene and the inhibitor of the expression product of the gene can be applied to development of specific antitumor drug.

Description

technical field [0001] The invention belongs to the field of random gene mutation methods. Specifically, the present invention relates to a carrier used in a gene search system, a method for random gene regulation, a method for screening highly metastatic human tumor cell lines, a method for screening genes that increase the metastasis of human tumor cell lines, and a gene obtained by using the random gene regulation method. Whole-genome random mutation library and the pharmaceutical use of the gene obtained by screening the gene obtained by screening the method for improving the metastasis of human tumor cell lines, the pharmaceutical use of the inhibitor of the gene, and the pharmaceutical use of the inhibitor of the protein expression product of the gene use. Background technique [0002] With the successful completion of the Human Genome Project, the field of life science research has entered the post-genome era. [0003] The completion of genome sequence determination...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/63C12N5/10C12N15/10C07K16/18C40B40/08A61K48/00A61K39/395A61P35/00
Inventor 李利民常德
Owner THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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