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A primer set, kit and method for detecting the copy number of frt sites in flp-in host cell line

A host cell and flp-in technology, applied in the field of genetic engineering, can solve the problems of a large amount of genomic DNA, time-consuming, laborious, etc., achieve good specificity, improve detection efficiency, and save DNA consumption

Active Publication Date: 2021-11-02
BEIJING DCTY BIOTECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The traditional detection method of exogenous gene copy number is Southern blot, but it is time-consuming, laborious and requires a large amount of genomic DNA

Method used

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  • A primer set, kit and method for detecting the copy number of frt sites in flp-in host cell line
  • A primer set, kit and method for detecting the copy number of frt sites in flp-in host cell line
  • A primer set, kit and method for detecting the copy number of frt sites in flp-in host cell line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Identification of copy number of FRT site in Flp-In host cell line by fluorescent quantitative PCR

[0053] Genomic DNA preparation

[0054] The obtained host cell line Flp-In-K562 was constructed according to the operation steps of the Flp-InTMComplete System (Product No.: K601001) kit of Thermo Fisher Company. Genomic DNA of the host cell line Flp-In-K562 transformed with the pFRT / lacZeo plasmid was extracted. Genomic DNA was extracted with reference to the QIAGEN QIAamp DNA mini kit, and the DNA quality was detected by 1% agarose gel electrophoresis. 2 O was uniformly diluted to 50ng / μl. Genome electrophoresis results such as Figure 10 As shown, the extracted DNA bands are complete without tailing and smearing phenomenon, and the OD260 / 280 value of the measured sample is 1.9, which meets the experimental requirements in the range of 1.8-2.0.

[0055] The fluorescent quantitative PCR reaction system is shown in Table 2.

[0056] Table 2 Fluorescent quantitative ...

Embodiment 2

[0072] The repeatability detection of method in embodiment 1

[0073] Re-extract the sample DNA, measure the copy number of the sample again according to the method in Example 1, and observe whether the copy number calculation method twice is repeatable. The results are shown in Table 4.

[0074] Table 4 Secondary detection results of copy number of 7 Flp-In-K562 host cell line samples

[0075] Sample serial number LAC mean CT value UCR average CT value ΔCt copy number 5 22.968 21.056 1.912 1 6 22.702 20.903 1.799 1 23 23.522 21.635 1.887 1 30 22.936 21.322 1.614 1 58 23.153 21.184 1.969 1 63 22.131 21.594 0.537 1 76 20.014 21.195 -1.181 5

[0076] It can be drawn from Table 4 that the results of the seven samples are completely consistent. It shows that the method for measuring the copy number of the FRT site in the Flp-In-K562 host cell line provided by the present invention has stable results an...

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Abstract

The invention provides a primer set, a kit and a method for detecting the copy number of FRT sites in a Flp-In host cell line, and belongs to the technical field of genetic engineering. The primer set includes a primer pair for detecting LAC genes and an internal reference gene for detecting The primer pair; the internal reference gene is the intron UCR of the human gene SYNCRIP; the sequence number of the internal reference gene in the NCBI database is NG_031848.1; the kit and method provided by the invention can realize the FRT locus copy number Compared with other gene copy number detection methods, the detection not only has simple operation and accurate results, but also has the advantages of good repeatability, high sensitivity, good specificity, low cost, convenience and flexibility.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a primer set, a kit and a method for detecting the copy number of an FRT site in a Flp-In host cell line. Background technique [0002] Flp-In Complete System (Flp-In Complete System) can integrate and express the target gene at a specific site in the genome of mammalian cells. The Flp-In system involves introducing the Flp recombination target (FRT) site into the genome of a selected mammalian cell line, and then mediated DNA recombination at the FRT site by Flp recombinase to integrate the expression vector containing the gene of interest into the genome middle. [0003] The main components of the Flp-In system include three parts: the first part is the Flp-Ino target site vector pFRT / lacZeo, which is used to prepare a host cell line containing the FRT integration site, and the resulting Flp-In host cell line contains Complete FRT site, and express lac...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/686C12N15/11
CPCC12Q1/686C12Q2600/166C12Q2563/107C12Q2545/114
Inventor 焦顺昌张嵘卫静袁翰
Owner BEIJING DCTY BIOTECH CO LTD
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