Primers and method for identifying the genotype of a GLD model twitcher mouse

Abstract

The twitcher mouse model is a main animal model for researching the pathogenesis and treatment method of GLD disease at present, and the model is caused by a spontaneous mutation of a C57BL / 6 mouse model GALC At present, a commonly used genotype identification method is time-consuming and expensive. In order to overcome the defect, the special identification primer is designed, the PCR method is adopted, and the restriction endonuclease Eco RV is used for single enzyme digestion verification, so that a rapid, efficient and economic genotype identification method is established, and a reference for the genotype identification of the twitcher mouse is provided.

Description

Technical Field

[0001] This invention belongs to the technical field, specifically relating to primers and methods for identifying the genotype of the GLD model Twitcher mouse. Background Technology

[0002] Globoid cell leukodystrophy (GLD), first reported by Knud Krabbe in 1916, has an incidence of approximately 1 in 100,000 live births. This disease is primarily caused by a mutation in the β-galactocerebrosidase gene (GALC), resulting in a deficiency of the GALC enzyme in lysosomes. This leads to the accumulation of the toxic substrates of GALC, galactosylceramide and psychosine, in the central and peripheral nervous systems. It is a rare lysosomal storage disorder with an autosomal recessive inheritance pattern. 85-90% of GLD patients are infantile, exhibiting normal behavior in the first few months after birth. Symptoms typically appear around six months of age, and death usually occurs before the age of two. Typical symptoms include restlessness, muscle weakness, feeding difficulties, non-infectious fever, rigidity, and developmental delays. As the disease progresses, persistent muscle weakness develops, affecting movement, chewing, swallowing, and breathing. Patients may also experience vision loss and seizures. 10-15% of GLD patients are late-onset, developing the disease in childhood, adolescence, or adulthood. Late-onset krabbe symptoms generally include muscle weakness, vision problems, and intellectual impairment, with greater variability in symptoms among patients.

[0003] The primary model for studying human GLD is the Twitcher mouse model, a naturally occurring mouse model with a mutation at position 1065 of the GALC gene, producing a premature stop codon. This leads to abnormal GALC gene expression and loss of GALC enzyme activity. Twitcher mouse symptoms appear on day 21 after birth; affected mice experience central white matter tract and peripheral nerve demyelination, weight loss, hind limb ataxia, kyphosis, and severe tremor, dying approximately 40 days after birth. Their biochemical and histological characteristics are similar to those of human GLD patients. Since its discovery, this model has been widely used in research on histological morphology, biochemical enzymology, and GLD treatment.

[0004] Currently, the only clinical treatment for GLD is hematopoietic stem cell transplantation. However, this method can only partially slow the progression of GLD and cannot cure the disease. Patients still experience progressive neurodegeneration and a reduced quality of life after hematopoietic stem cell transplantation, highlighting the urgent need for new treatments for GLD. In recent years, researchers have developed various treatment strategies in Twitcher mice, such as virus-mediated gene therapy, GALC enzyme replacement therapy, substrate reduction therapy, and anti-inflammatory therapy. Developing GLD treatments based on Twitcher mice requires early identification of the Twitcher mouse genotype. Especially with the development of gene therapy in recent years, accurate identification of the Twitcher mouse genotype immediately after birth is crucial for the effectiveness of gene therapy. Currently, the JAX laboratory recommends Sanger sequencing for Twitcher mouse genotyping. This method is time-consuming and expensive, often requiring samples to be sent to sequencing companies, which is inconvenient for researchers. Therefore, establishing a rapid, efficient, and economical method for Twitcher mouse genotyping is of significant practical importance and application value for GLD treatment and mechanistic research. Based on in-depth analysis of the GALC gene mutation sites in Twitcher mice, this invention designs special identification primers and introduces EcoR V restriction enzyme sites to identify the genotype of Twitcher mice through PCR and restriction endonuclease methods, thereby establishing a rapid, efficient and economical method for Twitcher mouse genotype identification. Summary of the Invention

[0005] To overcome the shortcomings of the above-mentioned technologies, this invention provides primers and a method for identifying the genotype of the GLD model Twitcher mouse. The primers incorporate the EcoR V restriction enzyme site, which not only amplifies the GALC gene but also allows for single-enzyme digestion to verify the amplification results. Furthermore, the identification method employed can rapidly and accurately identify the genotype of the Twitcher mouse.

[0006] The technical solution adopted in this invention is as follows:

[0007] This invention provides primers for identifying the genotype of the GLD model Twitcher mouse. The sequences of the primers are SEQ ID NO:1 and SEQ ID NO:2.

[0008] This invention also provides the application of primers for identifying the genotype of the GLD model Twitcher mouse in the identification of the Twitcher mouse genotype.

[0009] This invention also provides a method for identifying the genotype of the GLD model Twitcher mouse, comprising the following steps:

[0010] (1) Sampling: Twitcher mouse samples were cut and genomic DNA was extracted;

[0011] (2) PCR amplification: Using the genomic DNA obtained in step (1) as a template, the GALC gene is amplified by PCR using the identification primers described in claim 1 to obtain the amplification product;

[0012] (3) Single enzyme digestion verification: Take the amplification product obtained in step (2) and perform single enzyme digestion verification using restriction endonuclease;

[0013] (4) Result interpretation: mice with only one 306bp fragment after enzyme digestion are wild-type mice, mice with one 306bp fragment and one 249bp fragment are heterozygous mice, and mice with only one 249bp fragment are homozygous mice.

[0014] Preferably, the genome sample in step (1) is a mouse toe or tail, with a size of 0.2-0.3 cm.

[0015] Preferably, the PCR system in step (2) is 20 μL, containing 10 μL of 2×Master Mix, 2.0 μL of 10×Buffer, 3-3.5 μL of DNA template, 0.5-1 μL each of the front and rear primers, and 2.5-4 μL of ddH2O.

[0016] Preferably, the amount of DNA template used is 50-200 ng / μL.

[0017] Preferably, the PCR reaction program in step (2) is 93-95℃ for 8-10 min, 1 cycle; 93-95℃ for 20-30 s, 55-58℃ for 35-45 s, 65℃ for 30-60 s, 30-40 cycles; 65℃ for 5-10 min, 1 cycle; stored at 4℃.

[0018] Preferably, the PCR reaction program in step (2) is 94℃ for 10 min, 1 cycle; 94℃ for 30 s, 58℃ for 45 s, 65℃ for 1 min, 33 cycles; 65℃ for 5 min, 1 cycle; stored at 4℃.

[0019] Preferably, the restriction endonuclease in step (3) is EcoR V, and the single enzyme digestion verification conditions are digestion at 37℃ for 30-60 min.

[0020] Beneficial effects

[0021] This invention designs a method to specifically amplify the GALC gene, which exhibits a high rate of spontaneous mutations in Twitcher mice. Primers are designed to mutate the last base T of the original GATATT gene to C, resulting in homozygous mice producing the EcoR V restriction site GATATC in the PCR product. This restriction site can be introduced into the GALC gene PCR product via PCR amplification, and then the Twitcher mouse genotype can be rapidly and accurately verified using single-enzyme digestion. The identification primers and methods used in this invention are simple to operate, highly accurate, and low-cost, enabling genotype identification in Twitcher mice during early growth stages. This has significant practical value and application for studying treatment methods and mechanisms of GLD using Twitcher mice as a model. Attached Figure Description

[0022] Figure 1 This is an agarose gel electrophoresis image of the amplified GALC gene in Twitcher mice. In the image, M is the DNA Marker, and numbers 1-8 represent different mouse samples.

[0023] Figure 2 Agarose gel electrophoresis image for identifying Twitcher mouse genotypes using restriction endonucleases, where M is the DNA Marker and numbers 1-8 represent different mouse samples;

[0024] Figure 3 Sanger sequencing data for the mutation site in Twitcher mice;

[0025] Figure 4 Image of the Twitcher mouse;

[0026] Figure 5 This is a gait analysis diagram of the Twitcher mouse. Detailed Implementation

[0027] Twitcher mice (JAX Lab, NO.000845, p.W355*) were purchased from JAX Labs in the United States. They are a C57BL / 6 mouse model caused by a spontaneous mutation of the GALC gene. They are currently housed in the Specific Pathogen Free (SPF) animal facility of the Experimental Animal Center of Three Gorges University. They are fed fresh food and water daily, and the light and dark are alternated at 12h / 12h, strictly following the principles for the use of laboratory animals.

[0028] Main reagent kits and reagents:

[0029] Mouse gene identification kit ( The Mouse Genotyping Kit was purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0030] 2×Hieff Plus PCR Master Mix (With Dye) was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.

[0031] Restriction endonuclease EcoR V: purchased from Thermo Fisher Scientific

[0032] 50×TAE solution: Weigh 242g Tris and 37.2g Na2EDTA·2H2O into a beaker, then add 600mL of deionized water and stir thoroughly. Then add 57.1mL of glacial acetic acid, stir thoroughly, and then add deionized water to make up to 1L. Store at room temperature. Before use, dilute with deionized water to obtain a 1×TAE buffer solution.

[0033] Agarose powder was purchased from BioFroxx.

[0034] Example 1 Primer Design

[0035] (1) Analysis of the GALC gene sequence in Twitcher mice revealed that when the nonsense mutation at base 1065 (G) is changed to A, the mice transform from healthy to diseased mice, i.e., from GGTATT to GATATT. In homozygous mice, this sequence differs from the EcoRV restriction enzyme sequence by only one base; that is, the last base T of GATATT is mutated to C. The GALC gene has a Gene ID of 14420 on NCBI.

[0036] (2) Based on the analysis results of step (1), design the front and back primers, with primer sequences SEQ ID NO:1 and SEQ ID NO:2, respectively.

[0037] Example 2: Twitcher mouse sampling and genome extraction

[0038] (1) Select materials from 8 Twitcher mice born on the first day of life and fix them. Then, use surgical scissors disinfected with 75% alcohol to cut off the mouse toes or tails of about 0.2-0.3 cm to obtain samples.

[0039] (2) Place the sample obtained in step (1) into a PCR tube and add the lysis buffer from the mouse gene identification kit, where AD1 is 160 μL and AD2 is 40 μL, with a volume ratio of 4:1, to ensure that the mouse tissue is completely submerged.

[0040] (3) Place the PCR tubes from step (2) on a vortex mixer and mix well;

[0041] (4) Place the mixed sample solution in a PCR instrument for lysis. The reaction conditions are 55℃ for 115 min and 95℃ for 3 min. After the reaction is complete, tissue lysis products are obtained.

[0042] (5) The concentration of the tissue lysis product obtained in step (4) was determined and the concentration was adjusted to 50-200 ng / μL with ddH2O to prepare mouse genomic template DNA, which was stored at -20℃ for later use.

[0043] Example 3 PCR Amplification

[0044] (1) Using the tissue lysis product obtained in Example 2 as a template, and the primers designed in Example 2, the 2×Hieff primers were used to... PCR amplification was performed using the Plus PCR Master Mix (With Dye) kit. The reaction system is shown in Table 1, and the reaction procedure is shown in Table 2.

[0045] (2) Preparation of electrophoresis gel: Weigh 0.4 g of agarose powder into an Erlenmeyer flask, add 40 μL of 1×TAE electrophoresis buffer, and heat in a microwave oven to melt the agarose. After the solution cools to about 60°C, add 1 μL of ethidium bromide solution (final concentration 0.5 μg / mL), mix thoroughly, pour into a mold, and insert a comb. After the gel has completely solidified, remove the comb and place it in an electrophoresis tank containing TAE electrophoresis buffer for later use.

[0046] (3) Mix the amplified PCR product with 6×DNA Loading Buffer at a volume ratio of 5:1 and then spot the mixture onto the electrophoresis gel obtained in step (2). Electrophore at 100V for 40 minutes.

[0047] (4) After electrophoresis, the gel is transferred to a gel imaging analyzer for photographic analysis.

[0048] Table 1 PCR amplification system

[0049] Components Dosage DNA template (52.3 ng / μL) 3.5μL Forward Primer (10nM) 0.5μL Reverse Primer (10nm) 0.5μL 2×Master Mix 10μL <![CDATA[ddH2O]]> Make up to 20 μL

[0050] Table 2 PCR reaction procedure

[0051]

[0052]

[0053] The results are as follows Figure 1As shown, a DNA fragment appears between 250bp and 500bp. The target DNA fragment is 306bp in size, between 250bp and 500bp, which is consistent with the result. This indicates that the primer pair designed in Example 1 can successfully amplify the fragment containing the GALC mutation site in the genomes of 8 mice.

[0054] Example 4 Single enzyme digestion verification

[0055] The gene fragments amplified from the eight mice in Example 3 were verified by enzyme digestion. The specific steps are as follows:

[0056] (1) Take the PCR reaction product obtained in Example 3, add EcoR V to it, and then place it in a constant temperature water bath at 37℃ for 30 min to digest and perform single enzyme digestion verification. The verification system is shown in Table 3.

[0057] (2) After digestion, the sample is taken out and spotted into a 3% agarose gel and electrophoresed at 80V for 80min.

[0058] (3) After electrophoresis, the samples were transferred to a gel electrophoresis imaging system for photographing and analysis.

[0059] Table 3 Single enzyme digestion verification system

[0060] Components Dosage PCR products 2μL EcoR V 1μL 10×Buffer 2μL <![CDATA[ddH2O]]> Make up to 20 μL

[0061] The results are as follows Figure 2 As shown: mice 1, 3, 5, and 8 have only one 306bp fragment, indicating wild-type mice (base G at 1065 is not mutated); mice 2 and 7 show one 306bp and one 249bp fragment. Because the 57bp DNA fragment is too small to be easily observed after electrophoresis, the electrophoresis results show two bands, indicating heterozygous mice (base G at 1065 is partially mutated to A, and the other part is not mutated); mice 4 and 6 show one 249bp fragment. Similarly, the 57bp DNA fragment is too small to be easily observed after electrophoresis. Mice with only the 249bp fragment are homozygous mice (base G at 1065 is completely mutated to A).

[0062] Example 5: Sanger Sequencing

[0063] To further evaluate the accuracy of the method for identifying the Twitcher mouse genotype of this patented invention, based on the single enzyme digestion verification results in Example 4, PCR amplification products from mice 1, 4, and 7 were sent to Wuhan Bioengineering Co., Ltd. for Sanger sequencing. The sequencing results are shown below. Figure 3 The PCR amplification fragment sequence of wild-type mice is SEQ ID NO:3, and the PCR amplification fragment sequence of homozygous mice is SEQ ID NO:4.

[0064] like Figure 3 As shown, the 206th base in the PCR product of mouse No. 1 is G, indicating that the GALC gene has not undergone spontaneous mutation and is a wild-type mouse. The 206th base in the PCR product of mouse No. 4 is A, indicating that the GALC gene has undergone spontaneous mutation and is a homozygous mouse. The 206th base in the PCR product of mouse No. 7 contains both G and A, indicating that the GALC gene has partially undergone spontaneous mutation and is a heterozygous mouse. The Sanger sequencing results are consistent with the single enzyme digestion identification results shown in Example 4, demonstrating that the primer sequence designed in this invention and the identification method used can accurately and quickly complete the identification of the Twitcher mouse genotype.

[0065] Example 6: Phenotypic and Gait Analysis of Mice

[0066] Twitcher mice were observed every 3 days after birth. They developed tremors around 21 days old, followed by stable weight loss, muscle weakness, and hind leg paralysis. Around 30 days old, they developed kyphosis and typically died around 40 days old. The phenotype of Twitcher mice after 33 days of age is shown below. Figure 4 As shown.

[0067] Gait analysis was also performed on the Twitcher mice, and the specific steps are as follows:

[0068] (1) Twitcher mice on day 33 were fixed in place, and their forepaws were dipped in non-toxic red food coloring, and their hind paws were dipped in non-toxic blue food coloring:

[0069] (2) The mice treated in step (1) were placed in a self-made cardboard tactile paving with a length of 34cm, a width of 4cm and a height of 3cm. There were no obstacles in front of the tactile paving and the light was bright. A piece of white paper was placed at the bottom so that the mice could walk along the tunnel toward the light source. The paper with the colored paw prints was compared and analyzed.

[0070] The results are as follows Figure 5 As shown, compared with wild-type mice, homozygous mice have shorter and uneven gaits, and exhibit a dragging gait in their hind limbs. This indicates that homozygous mice gradually develop symptoms such as weight loss, muscle weakness and tremors, kyphosis, and hind limb paralysis after birth, while wild-type mice develop normally. This is consistent with the single enzyme digestion verification results in Example 4, demonstrating that the primers designed in Example 1 and the identification methods used in Examples 3 and 4 can accurately identify the genotype of Twitcher mice.

[0071] The above embodiments provide a further detailed description of the present invention. The advantages and features of the present invention will become clearer with the description. However, these examples are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions to the details and form of the technical solutions of the present invention can be made without departing from the spirit and scope of the invention, but such modifications and substitutions all fall within the protection scope of the present invention.

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Claims

1. A method for identifying the genotype of the GLD model Twitcher mouse, characterized in that: Includes the following steps: (1) Sampling: Twitcher mouse samples were cut and genomic DNA was extracted; (2) PCR amplification: Using the genomic DNA obtained in step (1) as a template, the GALC gene is amplified by PCR using the identification primers to obtain the amplification product; (3) Single enzyme digestion verification: Take the amplification product obtained in step (2) and perform single enzyme digestion verification using restriction endonuclease; (4) Result interpretation: mice with only one 306 bp fragment after enzyme digestion are wild-type mice, mice with one 306 bp fragment and one 249 bp fragment are heterozygous mice, and mice with only one 249 bp fragment are homozygous mice. The identification primers mentioned in step (2) are SEQ ID NO:1 and SEQ ID NO:

2.

2. The method for identifying the GLD model Twitcher mouse according to claim 1, characterized in that: The genome sample in step (1) is a mouse toe or tail, 0.2-0.3 cm in size.

3. The method for identifying the GLD model Twitcher mouse according to claim 1, characterized in that: The PCR system in step (2) is 20 μL, containing 10 μL of 2×Master Mix, 2.0 μL of 10×Buffer, 3-3.5 μL of DNA template, 0.5-1 μL each of the front and rear primers, and 2.5-4 μL of ddH2O.

4. The method for identifying the GLD model Twitcher mouse according to claim 1, characterized in that: The amount of DNA template used is 50-200 ng / μL.

5. The method for identifying the GLD model Twitcher mouse according to claim 1, characterized in that: The PCR reaction program for step (2) is as follows: 93-95℃ for 8-10 min, 1 cycle; 93-95℃ for 20-30 s, 55-58℃ for 35-45 s, 65℃ for 30-60 s, 30-40 cycles; 65℃ for 5-10 min, 1 cycle; store at 4℃.

6. The method for identifying the GLD model Twitcher mouse according to claim 5, characterized in that: The PCR reaction program for step (2) is as follows: 94℃ for 10 min, 1 cycle; 94℃ for 30 s, 58℃ for 45 s, 65℃ for 1 min, 33 cycles; 65℃ for 5 min, 1 cycle; stored at 4℃.

7. The method for identifying the GLD model Twitcher mouse according to claim 1, characterized in that: The restriction endonuclease in step (3) is EcoR V, and the single enzyme digestion verification conditions are digestion at 37℃ for 30-60 min.

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