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Quick identification method of genetically modified mouse genotypes

The technology of a transgenic mouse and an identification method is applied in the field of rapid identification of the genotype of the transgenic mouse, which can solve the problems of cumbersome operation steps, time-consuming, restricting the work efficiency of the identification of the genotype of the transgenic mouse, etc., and achieves simple operation and good identification. effect of effect

Pending Publication Date: 2020-03-24
广州铂晋生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these methods can obtain tissue DNA with higher purity and higher yield, there are cumbersome operation steps (requires multiple centrifugation), time-consuming (some operation steps need to be processed overnight), and involve multiple reagents (all using Chloroform, ethanol, proteinase K and other reagents) and other deficiencies; in addition, some existing tissue DNA extraction kits on the market (such as Takara, Promega and Vazyme and other companies’ kits) also need to consume a large number of collection columns, multiple reagents ( Such as lysate, neutralization solution, proteinase K, etc.) and multiple centrifugations, and the DNA extraction process (ie, template preparation) takes at least 45 minutes to several hours
The above problems all increase the experimental cost (including reagent cost, consumable cost and labor cost) to a certain extent, thus restricting the work efficiency of genotype identification of transgenic mice

Method used

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  • Quick identification method of genetically modified mouse genotypes
  • Quick identification method of genetically modified mouse genotypes
  • Quick identification method of genetically modified mouse genotypes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment example 1B6129

[0022] Implementation Case 1B6.129S-Sftpct m1(cre / ERT2)Blh Identification of / J transgenic mice

[0023] B6.129S-Sftpct m1(cre / ERT2)Blh The identification of / J transgenic mice, the specific implementation method is as follows:

[0024] 1) Cut about 2mm of B6.129S-Sftpct m1(ere / ERT2)Blh / J transgenic mouse tails were placed in a clean EP tube;

[0025] 2) Add 100 μL of SG1Buffer to the EP tube and place it in a metal bath at 95°C for 5 minutes for digestion;

[0026] 3) Cool to room temperature after digestion, then add 8.6 μL SG2Buffer to each EP tube, flick the EP tube 3-4 times to mix, centrifuge at 8000g for 3 min, and take the supernatant as a template for PCR;

[0027] 4) Use the following identification primers for PCR identification

[0028]

[0029] 5) Carry out PCR amplification according to the following system and conditions

[0030] PCR system

[0031] components volume wxya 2 o

7μL 13007 Primer (10 μM) 0.5μL 24999 Prime...

Embodiment example 2R

[0035] Implementation Case 2 Identification of Rbpj Gene Conditional Knockout Mice

[0036] The identification of Rbpj gene conditional knockout mice, the specific implementation method is as follows:

[0037] 1) Cut about 2mm of the Rbpj gene conditional knockout mouse tail and place it in a clean EP tube;

[0038] 2) Add 100 μL of SG1Buffer to the EP tube and place it in a metal bath at 95°C for 5 minutes for digestion;

[0039] 3) Cool to room temperature after digestion, then add 8.6 μL SG2Buffer to each EP tube, flick the EP tube 3-4 times to mix, centrifuge at 8000g for 3 min, and take the supernatant as a template for PCR;

[0040] 4) Use the following identification primers for PCR identification

[0041]

[0042] 5) Carry out PCR amplification according to the following system and conditions

[0043] PCR system

[0044]

[0045]

[0046] PCR amplification conditions

[0047]

[0048] 6) The PCR product obtained above was subjected to gel electrophores...

Embodiment example 3X

[0049] Implementation case 3 Identification of XM200862Ctsk-EGFP-P2A-iCre transgenic mice

[0050] The identification of XM200862Ctsk-EGFP-P2A-iCre transgenic mice, the specific implementation method is as follows:

[0051] 1) Cut the XM200862Ctsk-EGFP-P2A-iCre transgenic mouse tail about 2mm and place it in a clean EP tube;

[0052] 2) Add 100 μL of SG1Buffer to the EP tube and place it in a metal bath at 95°C for 5 minutes for digestion;

[0053] 3) Cool to room temperature after digestion, then add 8.6 μL SG2Buffer to each EP tube, flick the EP tube 3-4 times to mix, centrifuge at 8000g for 3 min, and take the supernatant as a template for PCR;

[0054] 4) Use the following identification primers for PCR identification

[0055]

[0056] 5) Carry out PCR amplification according to the following system and conditions

[0057] PCR system

[0058] components volume wxya 2 o

8μL CTSK-TG-3tF1 primer (10 μM) 0.5μL CTSK-TG-3tR1 primer (10 μM...

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Abstract

The invention relates to a quick identification method of genetically modified mouse genotypes. The quick identification method comprises two kinds of autonomous research and development lysate SG1 Buffer and SG2 Buffer, and comprises the steps of obtaining the DNA of genetically modified mouse tissue, amplifying target DNA fragments and performing gel electrophoresis on PCR products. The autonomous research and development tissue lysate can realize digestion of mouse tail tissue in water bath or metal bath of 95 DEG C for 5-10min, the genome DNA in the mouse tissue is rapidly released, wherein pyrolysis products are free from extraction and purification, and are directly used as a PCR amplification formwork, so that the total time for preparing the genetically modified mouse identification formwork is shortened to 10-15min. A PCR premixing solution 2*PCR Master Mix ( With Dye ) is used, experiment systems and conditions are optimized by the method, the target DNA fragments are amplified, then the PCR products are subjected to gel electrophoresis, and the identification results are quickly obtained.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for rapidly identifying genotypes of transgenic mice. [0002] technical background [0003] Transgenic animal technology refers to an experimental technique that integrates a foreign gene into the genome of an animal through manual manipulation, so that the transgenic animal can stably pass on the gene to its offspring. Since the beginning of the 21st century, with the rapid development of biotechnology, the genetic modification technology of transgenic mice has become increasingly mature, and because of their close relationship with humans, low feeding costs, and short reproductive cycles, transgenic mice have become the most commonly used at present. Animal models for the construction of human disease models are widely used in the fields of biology, immunology, and pharmaceuticals, and have become an important and powerful tool for mechanism exploration, dr...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6806
CPCC12Q1/6888C12Q1/6806
Inventor 张秋霞
Owner 广州铂晋生物科技有限公司
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