CN110904241A - Rapid identification method for transgenic mouse genotype - Google Patents
Rapid identification method for transgenic mouse genotype Download PDFInfo
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- 238000011830 transgenic mouse model Methods 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title claims abstract description 29
- 238000001502 gel electrophoresis Methods 0.000 claims abstract description 13
- 239000006166 lysate Substances 0.000 claims abstract description 13
- 238000012408 PCR amplification Methods 0.000 claims abstract description 11
- 239000002184 metal Substances 0.000 claims abstract description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 6
- 239000012634 fragment Substances 0.000 claims abstract 3
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- 241000581650 Ivesia Species 0.000 claims description 6
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- 239000011543 agarose gel Substances 0.000 claims description 5
- 238000006243 chemical reaction Methods 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- 210000003371 toe Anatomy 0.000 claims description 5
- 238000002474 experimental method Methods 0.000 claims description 3
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims 3
- 238000000137 annealing Methods 0.000 claims 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims 1
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- 230000003321 amplification Effects 0.000 abstract 1
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- 241000699660 Mus musculus Species 0.000 description 10
- 241000699666 Mus <mouse, genus> Species 0.000 description 7
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
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Abstract
The invention relates to a rapid identification method of transgenic mouse genotype, which comprises two independently developed lysates SG1Buffer and SG2Buffer, and comprises the processes of acquisition of transgenic mouse tissue DNA, amplification of target DNA fragments and gel electrophoresis of PCR products. According to the invention, the rat tail tissue is digested for 5-10min in a water bath or a metal bath at 95 ℃ by using a self-developed tissue lysate, the genome DNA in the mouse tissue is rapidly released, and the lysate is directly used as a template for PCR amplification without extraction and purification, so that the total time for preparing the transgenic mouse identification template is shortened to 10-15 min; the target DNA fragment is amplified by using PCR premixed solution 2 XPCR Master Mix (With Dye) according to the experimental system and conditions optimized by the method of the invention, and then the PCR product is subjected to gel electrophoresis to quickly obtain the identification result.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a rapid identification method of a transgenic mouse genotype.
Technical Field
The transgenic animal technology is an experimental technology which integrates an exogenous gene into an animal genome in a manual operation mode so that the transgenic animal can stably transmit the gene to offspring. Since the 21 st century, biotechnology is rapidly developed, genetic modification technology lines of transgenic mice become mature day by day, and due to the advantages of close relationship with human, low feeding cost and short reproduction period, the transgenic mice become the most common animal model for constructing human disease models at present, are widely applied to the fields of biology, immunology, pharmacy and the like, and become important powerful tools for mechanism discussion, drug screening, gene therapy and the like. Because the transgenic mouse is an experimental model which can be stably inherited and is obtained by a gene modification method, before the transgenic mouse is applied to carry out scientific research, the genotype of the mouse is generally required to be identified to distinguish the transgenic mouse from a wild mouse. The genotype identification of transgenic mouse relates to the processes of mouse DNA acquisition, PCR amplification of target DNA segment, DNA gel electrophoresis and the like.
Current traditional DNA extraction methods include phenol/chloroform extraction and high salt extraction. Although the methods can obtain tissue DNA with higher purity and higher yield, the methods have the defects of complicated operation steps (needing multiple times of centrifugation), overlong time consumption (needing overnight treatment for some operation steps), and the like, and relate to various reagents (all adopting chloroform, ethanol, protease K and other reagents); in addition, some of the commercially available tissue DNA extraction kits (e.g., Takara, Promega, and Vazyme kits) also consume a large number of collection columns, various reagents (e.g., lysis solution, neutralization solution, proteinase K, etc.), and multiple centrifugations, and the DNA extraction process (i.e., template preparation) also takes at least 45min to several hours. The problems increase the experiment cost (including reagent cost, material cost and labor cost) to a certain extent, thereby restricting the work efficiency of the genotype identification of the transgenic mice.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a rapid, efficient and simple method for identifying the genotype of the transgenic mouse. The autonomously developed tissue lysate digests rat tail tissues in a water bath or a metal bath at 95 ℃ for 5-10min, can quickly release genome DNA in the tissues, does not need extraction and purification, and can directly use a lysate for PCR amplification, thereby greatly reducing the steps of extracting the DNA and the time for obtaining the DNA, and shortening the total time for preparing the transgenic mouse identification template to 10-15 min. Then, the genotype identification can be rapidly and efficiently carried out on the transgenic mice by using a PCR premixed solution 2 XPCR Master Mix (Withdye) and an experimental system and conditions optimized by the method, and the experimental efficiency is greatly improved.
The method has the characteristics and advantages that the time for preparing the PCR identification template only needs 10-15min and is simple to operate, the time for preparing other reagent Kit templates in the market is longer, for example, One step mouse genotyping Kit of Vazyme company, the template preparation process comprises adding tissue lysate, incubating in 55 ℃ water bath for 20-30min after vortex oscillation, heating the sample in 95 ℃ or boiling water bath for 5min after the incubation is finished, and centrifuging at 12000rpm for 5min after the vortex products of the lysis are sufficiently and uniformly oscillated, and the time for preparing the template is at least more than 45 min; for example, TaKaRa MiniBEST Universal Genomic DNA Extraction kit Ver.5.0 from Takara corporation, the tissue lysis and DNA purification process involves adding multiple reagents, standing and centrifuging for multiple times, and the whole template preparation time is more than 2 hours. In addition, the invention has very good identification effect on different types (complete knockout, conditional knockout, fixed-point knock-in, random knock-in and the like) of transgenic mice, and the specific identification examples can be illustrated later.
The technical scheme adopted by the invention is as follows:
a method for extracting DNA of a transgenic mouse tissue and identifying a genotype comprises the following steps:
1) transgenic mouse tissues were cut and placed in a clean EP tube with the following sampling volumes:
mouse ear: punching a round hole with the diameter of 3mm or cutting an ear with the similar size
Rat toe: toes approximately 2mm long (length without fingernails)
And (3) rat tail: about 2mm of tail tip
2) To each EP tube containing the sample, 100. mu.L of SG1Buffer was added, and digested in a water or metal bath at 95 ℃ for 5-10 min. When the tissue is digested, it is necessary to completely immerse the tissue in the digestive juice. After digestion is completed, the tissue is still intact in appearance, but enough genomic DNA is released, so that the subsequent PCR experiment is not influenced;
3) after digestion, cooling to room temperature, then adding 8.6 μ L SG2Buffer into each EP tube, flicking the EP tube for 3-4 times, mixing uniformly, centrifuging at 8000g for 3min, and taking the supernatant as a PCR template.
4) The PCR reaction was carried out according to the following system and conditions:
PCR system
| Components | Volume of |
| ddH2O | 8μL |
| Primer 1 (10. mu.M) | 0.5μL |
| Primer 2 (10. mu. tM) | 0.5μL |
| Lysate template | 1μL |
| 2x PCR Master Mix | 10μL |
| Total volume | 20μL |
PCR amplification conditions
5) The PCR products were subjected to gel electrophoresis on a 1.5% agarose gel.
The following examples are given to illustrate the use of the invention
Example 1B6.129S-Sftpctm1(cre/ERT2)BlhIdentification of the transgenic mice
B6.129S-Sftpctm1(cre/ERT2)BlhThe identification of the/J transgenic mice is implemented by the following specific method:
1) cut about 2mm B6.129S-Sftpctm1(ere/ERT2)BlhThe mouse tail of the transgenic mouse is placed in a clean EP tube;
2) adding 100 μ L SG1Buffer into EP tube, and digesting in 95 deg.C metal bath for 5 min;
3) cooling to room temperature after digestion, then adding 8.6 mu L SG2Buffer into each EP tube, flicking the EP tube for 3-4 times, mixing uniformly, centrifuging at 8000g for 3min, and taking the supernatant as a PCR template;
4) PCR identification Using the following identification primers
5) PCR amplification was performed according to the following system and conditions
PCR system
| Components | Volume of |
| ddH2O | 7μL |
| 13007 primer (10. mu.M) | 0.5μL |
| 24999 primer (10. mu.M) | 1μL |
| 25000 primer (10. mu.M) | 0.5μL |
| Lysate template | 1μL |
| 2x PCR Master Mix | 10μL |
| Total volume | 20μL |
PCR amplification conditions
6) The PCR product obtained above was subjected to gel electrophoresis on a 1.5% agarose gel.
Example 2 identification of Rbpj Gene conditional knockout mice
The specific implementation method of the identification of the Rbpj gene conditional knockout mouse is as follows:
1) clipping the mouse tail of an Rbpj gene conditional knockout mouse with the diameter of about 2mm and placing the mouse tail in a clean EP tube;
2) adding 100 μ L SG1Buffer into EP tube, and digesting in 95 deg.C metal bath for 5 min;
3) cooling to room temperature after digestion, then adding 8.6 mu L SG2Buffer into each EP tube, flicking the EP tube for 3-4 times, mixing uniformly, centrifuging at 8000g for 3min, and taking the supernatant as a PCR template;
4) PCR identification Using the following identification primers
5) PCR amplification was performed according to the following system and conditions
PCR system
PCR amplification conditions
6) The PCR product obtained above was subjected to gel electrophoresis on a 1.5% agarose gel.
Example 3XM200862Ctsk-EGFP-P2A-iCre transgenic mouse identification
The specific implementation method of the identification of XM200862Ctsk-EGFP-P2A-iCre transgenic mice is as follows:
1) clipping mouse tails of XM200862Ctsk-EGFP-P2A-iCre transgenic mice with the diameter of about 2mm and placing the mouse tails in a clean EP tube;
2) adding 100 μ L SG1Buffer into EP tube, and digesting in 95 deg.C metal bath for 5 min;
3) cooling to room temperature after digestion, then adding 8.6 mu L SG2Buffer into each EP tube, flicking the EP tube for 3-4 times, mixing uniformly, centrifuging at 8000g for 3min, and taking the supernatant as a PCR template;
4) PCR identification Using the following identification primers
5) PCR amplification was performed according to the following system and conditions
PCR system
| Components | Volume of |
| ddH2O | 8μL |
| CTSK-TG-3tF1 primer (10. mu.M) | 0.5μL |
| CTSK-TG-3tR1 primer (10. mu.M) | 0.5μL |
| Lysate template | 1μL |
| 2x PCR Master Mix | 10μL |
| Total volume | 20μL |
PCR amplification conditions
6) The PCR product obtained above was subjected to gel electrophoresis on a 1.5% agarose gel.
Drawings
FIG. 1 shows B6.129S-Sftpctm1(cre/ERT2)BlhGel electrophoresis chart for identification of/J transgenic mice
1) Marker strip size from top to bottom: 2000bp \1000bp \750bp \500bp \250bp \100bp
2) 210bp and 327bp bands are amplified from 8 samples, and the identification result is heterozygote
FIG. 2 is a gel electrophoresis chart of the identification of Rbpj gene conditional knockout mice
1) Marker strip size from top to bottom: 1500bp \1000bp \900bp \800bp \700bp \600bp \500bp \400bp \300bp \200bp \100bp
2) And (3) identification result:
| genotype(s) | Mouse numbering |
| Negative mouse (WT) | 4，5，6，7 |
| Heterozygote (flox/+) | 1，3，8，11，13，15 |
| Homozygote (flox/flox) | 2，9，10，12，14，16 |
FIG. 3 is gel electrophoresis diagram of XM200862Ctsk-EGFP-P2A-iCre transgenic mouse identification
1) Marker strip size from top to bottom: 2000bp \1000bp \750bp \500bp \250bp \100bp
2) And (3) identification result:
XM200862 Ctsk-EGFP-P2A-icar transgenic mouse positive mouse number: 22, 23, 24.
Claims (8)
1. A method for rapidly identifying the genotype of a transgenic mouse is characterized by comprising the following steps: the invention uses independently developed tissue lysate SG1Buffer and SG2Buffer to quickly release genome DNA in mouse tissue, the lysate is directly used as a template for PCR amplification, a PCR premixed solution 2 xPCR Master Mix (With Dye) is used to amplify target DNA fragments according to an experimental system and conditions optimized by the method of the invention, and then gel electrophoresis is carried out, so that the genotype of the transgenic mouse can be quickly identified.
2. The method for rapidly identifying the genotype of the transgenic mouse according to claim 1, wherein the formula of the independently developed tissue lysate is as follows: SG1Buffer is 2.5-3.5mM EDTA (pH 8.0), 20-30mM NaOH, SG2Buffer is 40-50mM Tris-HCl (pH between 7.5-7.8).
3. The method for rapidly identifying the genotype of the transgenic mouse according to claim 1, which comprises the following experimental steps:
s1: shearing transgenic mouse tissues (mouse ears/mouse toes/mouse tails) and placing the tissues in a clean EP tube;
s2: adding 100 μ L SG1Buffer, and digesting in 95 deg.C water bath or metal bath for 5-10 min;
s3: after the S2 is cooled to room temperature, 8.6 mu L of SG2Buffer is added;
s4: taking 1-2 mu L of the S3 supernatant as a template for PCR reaction;
s5: carrying out PCR reaction;
s6: the product was subjected to gel electrophoresis, and visualized in a gel imager for band development.
4. The method for rapidly identifying the genotype of the transgenic mouse as claimed in claim 3, wherein the sampling site and the sampling amount of the transgenic mouse S1 are as follows:
mouse ear: punching a round hole with the diameter of 3mm or cutting off an ear with the similar size;
rat toe: toes about 2mm long (length without fingernails);
and (3) rat tail: a tail tip of about 2 mm.
5. The method of claim 3, wherein the digestion is performed according to the procedure of S2, and after the digestion is completed, the tissue remains intact in appearance, but sufficient genomic DNA is released to avoid affecting the subsequent PCR experiment.
6. The method for rapidly identifying the genotype of the transgenic mouse as claimed in claim 3, wherein the PCR reaction solution of S5 comprises: 2 × PCR Master Mix 10 μ L; 1 mu L of lysate template; primer 1 (10. mu.M) 0.5. mu.L; primer 2 (10. mu.M) 0.5. mu.L; ddH2O8 mu L; the total volume was 20. mu.L.
7. The method for rapidly identifying the genotype of the transgenic mouse as claimed in claim 3, wherein the PCR reaction conditions of S5 are as follows: pre-denaturation at 95 deg.C for 5 min; denaturation at 95 deg.C for 30 s, annealing at 55-65 deg.C (specifically based on the annealing temperature of specific primer) for 30 s, and extension at 72 deg.C for 30 s for 35 cycles; finally the temperature was extended at 72 ℃ for 7 min.
8. The method of claim 3, wherein the gel electrophoresis of S6 is performed using 1.5% agarose gel.
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Cited By (2)
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| CN112553301A (en) * | 2020-11-10 | 2021-03-26 | 东南大学 | Mouse DNA identification method |
| CN112725333A (en) * | 2021-02-07 | 2021-04-30 | 苏州大学 | Method for rapidly extracting animal genome DNA |
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| CN112725333A (en) * | 2021-02-07 | 2021-04-30 | 苏州大学 | Method for rapidly extracting animal genome DNA |
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