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Method of establishing mouse genome mutant embryonic stem cell library

An embryonic stem cell and cell technology, which is applied in the field of establishing a mouse genome-wide mutant embryonic stem cell bank, can solve the problems of labor-intensive, time-consuming, and slow progress of mutant embryonic stem cell cloning.

Inactive Publication Date: 2004-11-24
MABPLEX INT LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the progress of mutant embryonic stem cell clones obtained in this research is slow and cannot really support the needs of functional genomics research. The main reason for this phenomenon is that the commonly used method of identifying mutant cell clones one by one is still a time-consuming and labor-intensive strategy

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Construction of gene trap vectors

[0053] The mother plasmid is pSIR (Clontech Company), and the screening gene is the Neo gene without polyA signal; PGK is used as the promoter of Neo. The 3' end of the vector is connected with an RNA splicing donor sequence (SD), and the 5' end of the vector is connected with an RNA splicing acceptor sequence (SA). The plasmid construction method was carried out as usual to obtain a gene trap vector with the following structure: LTR-SA-PGK-Neo-SD-LTR.

[0054] The maternal plasmid used is a self-inactivating retroviral plasmid, so when the pseudovirus produced by the plasmid is used to infect the cells and inserted into the carrier on the chromosome, the 5' end LTR is automatically inactivated, and the gene Neo is screened. Expression is thus not disturbed by LTR.

Embodiment 2

[0055] Example 2: Establishment of mutant embryonic stem cell bank

[0056] In this embodiment, the pseudovirus infection method is adopted, and the packaging cell line Phoenix of Orbigen Company is used.

[0057] The packaging cells were cultured in a 100mm culture dish filled with DME / 10% FBS medium until the cells grew to a density of 60-70% (2-4×10 6 ), transfect 10-15ug of the gene trap vector plasmid established in Example 1 with the calcium phosphate co-precipitation method, and after 48 hours, collect the cell supernatant, which can be stored at -80°C after centrifugation; it can also be used to contain G418 (0.5 mg / ml) medium for culturing and monoclonal screening of the above-mentioned transfected cells. One week later, select cell clones, measure the titer of the cell culture supernatant after expanding the culture, and select the cell line that can produce high titer, which can generally reach 10 per ml of culture supernatant. 5 -10 6 of virus particles.

[005...

Embodiment 3

[0060] Example 3: Preparation of mutant embryonic stem cell bank cDNA

[0061] For each mutant embryonic stem cell subbank established in Example 2, a corresponding cDNA was established, and a total of 500-1000 copies of cDNA were prepared correspondingly; the preparation method of relevant cell mRNA and cDNA was carried out according to the standard method, using polydT and random primers as reverse recorded primers.

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Abstract

The method of utilizing gene capture technology in establishing mouse genome mutant embryonic stem cell library includes the following steps: introducing gene capture carrier, which has the from-5'-to-3' structure of LTR-RNA splice acceptor sequence-promoter-screening gene-RNA splice donor sequence-LTR, with the screening gene containing no polyA area, into embryonic stem cell; screening survival embryonic stem cell with the screening gene; and merging the survival embryonic stem cell to obtain mutant embryonic stem cell library. The present invention also provides the identification method of mutant gene site. The present invention lays the foundation for obtaining gene mutant mouse in large scale and high flux.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically relates to a method for establishing a mouse whole-genome mutant embryonic stem cell bank. Background technique [0002] The complete sequence determination of the human genome and some other important animal and plant genomes has been or will be completed in the near future, and more species genome sequencing projects are underway. However, these works are only the first step in understanding the genome functions of organisms. Since most of the gene functions are not yet known, with the rapid accumulation of massive sequence data, people are faced with the great challenge of how to identify the biological functions represented by these sequence data , the development of bioinformatics has provided unprecedented convenience for the analysis of genome sequence to predict the function of genes. [0003] However, experimental research, especially in vivo gene function research is ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/09C12N15/10C12N15/11C12Q1/68
Inventor 顾华黄芳王铸钢
Owner MABPLEX INT LTD
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