Construction and application of Stard6 gene knockout mouse animal model

Abstract

The invention relates to the technical field of animal models, in particular to construction and application of a Stard6 gene knockout mouse animal model, and the Stard6 gene knockout mouse animal model is obtained by mating a Stard6-flox mouse with a Star8-Cre mouse. A Stard6 gene knockout mouse model is constructed, and it is found that gene level expression and protein level expression of a specific Stard6 gene knockout mouse in male germ cells are remarkably reduced, so that testis development and spermatogenesis of a male mouse of the Stard6 gene knockout mouse are abnormal. Therefore, the problems that a common animal model cannot target a target gene, interference of other factors exists, an animal model with whole body knockout is prone to embryo death and the like are solved.

Description

Technical Field

[0001] This application relates to the field of animal model technology, and in particular to the construction and application of Stard6 gene knockout mouse animal models. Background Technology

[0002] Stard6 is a protein with the START (associated lipid transfer) domain of the acute regulatory protein of steroid synthesis (StAR), a member of the START family, and plays a crucial role in intracellular cholesterol transport. Stard6 is expressed in various tissues and cell types. Some studies indicate that Stard6 is specifically expressed in testicular tissue, primarily in mature sperm cells, but its specific regulatory mechanisms and physiological functions require further investigation. Clinical sample analysis shows that Stard6 expression levels in the testicular tissue of some patients with asthenospermia and azoospermia are significantly lower than in healthy individuals, suggesting a possible association with abnormal sperm quality. Currently, it is unclear whether abnormal Stard6 expression is a direct cause of decreased sperm quality or a secondary consequence of reproductive system diseases. Furthermore, systematic research data on whether Stard6 affects other reproductive-related physiological processes by regulating cholesterol transport is lacking. The establishment of a Stard6 gene knockout mouse model provides a crucial tool for studying the mechanism of Stard6 in male germ cells.

[0003] Existing animal models have the following problems: traditional modeling methods are often accompanied by a variety of complex factors such as damage to other organs and tissues, making it impossible to accurately target the physiological function of male reproductive organs, and the experimental data lacks credibility; systemic gene knockout of Stard6 may result in embryonic lethality or complex phenotypes in multiple tissues and organs due to gene deletion, making it difficult to accurately target the function of the Stard6 gene in male reproductive organs and its developmental stages. Summary of the Invention

[0004] This application provides the construction and application of the Stard6 gene knockout mouse model. By using CRISPR-Cas9 technology and the Cre-LoxP gene recombination system to specifically knock out the Stard6 gene in spermatogonia, it solves the problems of ordinary animal models being unable to target the target gene, the existence of other factors interfering, and the easy embryonic lethality of systemic knockout animal models.

[0005] This application provides a method for constructing a Stard6 gene knockout mouse model. The method involves mating Stard6-flox mice with Stra8-cre mice to obtain male germ cell-specific Stard6 gene knockout mice, namely Stard6-CKO, which is the Stard6 gene knockout mouse model.

[0006] Optionally, the Stard6 gene knockout mice are made using CRISPR-Cas9 technology and the Cre-LoxP gene recombination system.

[0007] Optionally, the Stard6-flox mouse is obtained by generating a (flox / +) heterozygous mouse from a mouse embryonic stem cell through homologous recombination to produce the Stard6-flox allele, and then mating the (flox / +) heterozygous mice together.

[0008] Optionally, the construction method includes the following steps: (1) First, the Stard6-flox mice were mated with Stra8-cre mice to obtain Stard6(flox / +)- mice and (flox / +) Stra8-cre mice; (2) Then, female Stard6(flox / +) Stra8-cre mice are mated with male Stard6(flox / flox) mice to obtain male Stard6 Stra8-cre mice, which are Stard6 gene knockout mice.

[0009] Optionally, in step (2), the changes in the genome structure of Stard6 gene knockout mice are confirmed by routine PCR using the toe or tail genes.

[0010] Optionally, in step (2), the expression level of the Stard6 gene in the testes of male mice was detected by real-time quantitative PCR.

[0011] Optionally, in step (2), the expression level of Stard6 protein in the testes of male mice was detected by Western Blot in Stard6 gene knockout mice.

[0012] Optionally, the toe gene identification of the Stard6 gene knockout mouse model is performed and the toes are labeled at around two weeks of age, and the tail gene identification can be used for mice whose toes have been clipped and labeled.

[0013] This application also proposes the application of a method for constructing a Stard6 gene knockout mouse model, the application of which includes the study of the regulatory mechanisms of male germ cell development and spermatogenesis.

[0014] Optionally, the applications also include clinical treatment and drug development for male infertility.

[0015] Therefore, the beneficial effects of the embodiments of this application are as follows: (1) In the embodiments of this application, a male spermatogonial-specific Stard6 gene knockout model was constructed, providing an animal model related to the Stard6 gene and male reproductive function. The regulatory role of the Stard6 gene in male germ cells is still unclear. This model targets the Stard6 gene, filling a gap in research methods and providing a key tool for in-depth exploration of its function.

[0016] (2) The efficient gene editing capability of CRISPR-Cas9 technology in this application embodiment rapidly constructs Stard6-flox mice, improving the efficiency of gene editing in embryonic stem cells. The use of the Cre-LoxP system to achieve conditional gene knockout avoids problems such as embryonic lethality or unclear phenotype that may be caused by systemic knockout, and achieves precise regulation of Stard6 gene function during spermatogenesis.

[0017] (3) The embodiments of this application use Cre tool mice regulated by the Stra8 gene, which can specifically express Cre recombinase in male germ cells from birth to before meiosis without affecting the function of the endogenous Stra8 gene. After mating with Stard6-flox mice, this tool mouse can mediate the splicing and recombination of the Stard6 gene fragment in the offspring spermatogonia, ensuring the cell specificity of gene knockout.

[0018] (4) The gene identification method in this application involves sampling the toes of mice at two weeks of age, which can achieve permanent numbering, facilitate genotype screening and cage separation, and also take into account animal welfare. Mice at this stage have stable survival, better tolerance than newborn mice, and better pain response and recovery than individuals older than three weeks.

[0019] (5) Based on the tissue gene PCR identification, the embodiments of this application carried out subsequent verification by real-time fluorescence quantitative PCR and Western Blot, respectively confirming the Stard6 gene knockout efficiency at the DNA, RNA and protein levels. The combination of the three verification methods greatly ensured the reliability and accuracy of the experimental results.

[0020] This solves the problems of ordinary animal models being unable to target the target gene, the existence of other interfering factors, and the risk of embryonic death in whole-body knockout animal models.

[0021] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0022] Figure 1 A flowchart for constructing a Stard6 gene knockout mouse model provided in this application embodiment; Figure 2This is a diagram showing the genotype identification results of the Stard6 gene knockout mouse provided in Example 2 of this application; Figure 3 This is a real-time fluorescence quantification result image of Stard6 gene knockout mice provided in Example 3 of this application; Figure 4 This is a Western Blot result of the Stard6 gene knockout mouse provided in Example 4 of this application. Detailed Implementation

[0023] The technical solutions of this application will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0024] The reagents and equipment used in the embodiments of this disclosure are all conventional and commercially available.

[0025] Example 1 Construct a Stard6 gene knockout mouse model.

[0026] To further clarify the role of the Stard6 gene in testicular development, Suzhou Cyagen Biotech Co., Ltd. was commissioned to generate the fluxed Stard6 allele in mouse embryonic stem cells through homologous recombination. LoxP sites were inserted at both ends of the exons to obtain Stard6 (flox / +) heterozygous mice. After mating with Stra8-Cre tool mice, the exon between the two loxP sites was targeted and excised using Cre recombinase, thereby constructing spermatogonial-specific Stard6 gene knockout mice.

[0027] Stard6(flox / +) heterozygous mice were used to create Stard6(flox / flox) mice. Stard6(flox / flox) mice were mated with Stra8-cre mice to obtain Stard6(flox / +)- mice and Stard6(flox / +) Stra8-cre mice. Then, female Stard6(flox / +) Stra8-cre mice were mated with male Stard6(flox / flox) mice to obtain male Stard6-flox Stra8-cre mice, which are the Stard6 gene knockout mice.

[0028] Example 2 Mouse genotyping (1) Extracting DNA DNA was extracted from toe tissues of newborn mice at approximately 2 weeks of age using the toe-labeling method and amplified by PCR. The amplified samples were then identified as Stra8-cre and Stard6-flox by agarose gel electrophoresis. 500 μL of pre-prepared tissue lysis buffer was added to centrifuge tubes containing toe tissue, followed by 20 μL of 20 mg / mL proteinase K solution. The tissue was gently pipetted to ensure complete immersion in the lysis buffer, preventing adhesion to the centrifuge tube walls. The centrifuge tubes were placed in a 55°C water bath with shaking at 50-60 rpm overnight. During digestion, the tissue was observed every 6 hours to ensure complete lysis. If significant tissue residue remained, the digestion time could be extended by 1-2 hours until the solution was uniformly turbid and free of visible solid particles.

[0029] (2) PCR amplification First, prepare a mixture of 10 μL 2x Taq Master Mix, 7 μL ddH2O, 1 μL upstream primer, and 1 μL downstream primer, and add it to the PCR tubes. Then, add 1 μL of DNA template to each tube. The PCR amplification program is set as follows: 95℃ pre-denaturation for 5 min; (95℃ denaturation for 30 s, 60℃ annealing for 30 s; 72℃ extension for 30 s) × 35; 72℃ final extension for 5 min.

[0030] (3) Agarose gel electrophoresis Accurately weigh 2.0 g of agarose powder into a 100 mL clean Erlenmeyer flask, add 100 mL of 1×TAE electrophoresis buffer, and gently shake to fully disperse the agarose powder and avoid local clumping. Place the Erlenmeyer flask in a microwave oven on medium-high heat, shaking it every 30 seconds to ensure the agarose particles are heated evenly. When the solution is clear and transparent with no visible particles, stop heating and allow the Erlenmeyer flask to cool to room temperature until it feels slightly warm to the touch. Then, slowly pour the cooled agarose solution into a pre-assembled gel tray, immediately insert a suitable gel comb, and let it stand at room temperature for 20-30 minutes until the agarose gel is completely solidified. Carefully remove the gel comb to complete gel preparation. After PCR amplification, use a pipette to add 5 μL of the amplification product to the wells of the agarose gel. Set the voltage to 110 V and the electrophoresis time to 10 minutes. Observe the results using a gel imaging system. The wild-type PCR result shows a single 165 bp band after amplification with FR primers. The PCR product from homozygous mice with the LoxP marker showed a single 220 bp band after amplification with FR primers. The PCR product from heterozygous mice showed bands at both 165 bp and 220 bp with FR primers. The PCR product with Stra8-cre primers showed a single band at 500 bp.

[0031] Example 3 Real-time quantitative PCR for detecting gene expression levels Step 1: RNA extraction (1) Grinding: First, place a 1.5 mL RNase-free EP tube on ice, remove the tissue and place it in a pre-cooled EP tube, then add 300 μL of Trizol lysis buffer and grind thoroughly on ice. Then add another 700 μL of Trizol lysis buffer and incubate on ice for 10 min for lysis.

[0032] (2) After lysis, add 200 μL of chloroform to the EP tube, shake to mix, and let stand on ice for 10 min. Then centrifuge at 13000 rpm and 4℃ for 15 min.

[0033] (3) Remove the EP tube, avoiding shaking. Take 400 μL of the upper aqueous phase liquid, put it into a new EP tube and add 400 μL of isopropanol, gently invert to mix. Let stand at 4℃ for 30 min, then centrifuge at 13000 rpm at 4℃ for 10 min.

[0034] (4) Remove the supernatant and retain the white precipitate at the bottom of the EP tube, which is the solid RNA. Dilute 1 mL of 75% ethanol with DEPC water and gently wash the precipitate with a pipette, being careful not to disturb the precipitate during the process. Then centrifuge at 12600g, 4℃ for 5 min.

[0035] (5) Remove the supernatant, invert the EP tube, and blot the liquid off the EP tube wall with absorbent paper. Then add an appropriate amount of DEPC water to the EP tube to dissolve the RNA. Finally, use a UV spectrophotometer to measure the RNA concentration. An A260 / 280 ratio between 1.8 and 2.0 indicates that the extracted RNA has high purity. Store at -80℃.

[0036] Step 2: Reverse transcription Before starting the reverse transcription experiment, all reagents (5×RT buffer, specific primer, reverse transcription enzyme, and nuclease-free water) must be removed from the -20°C freezer and slowly thawed on ice to avoid repeated freeze-thaw cycles that could reduce reagent activity. Once completely thawed, gently mix and briefly centrifuge to allow the reagents to settle at the bottom of the tube. Simultaneously, pre-chill the enzyme-free 500μL LEP tubes, pipette tips, and other consumables in an ice box. Strictly adhere to RNase-free operating procedures throughout the process to prevent RNA degradation from affecting reverse transcription efficiency. First, prepare a 3μL Mix of 5×RT buffer, primer, and enzyme according to the amount of reverse transcription sample. Calculate the required RNA template volume V based on the RNA sample concentration; the volume of nuclease-free water is 7-V. Then, add nuclease-free water, mix, and RNA template sequentially to the 500μL LEP tube, bringing the total reaction volume to 10μL. After thoroughly vortexing the reaction mixture, reverse transcription was initiated. The reaction program was set as follows: 37℃-15min; 98℃-5min; store at 4℃. The cDNA generated from reverse transcription was used for real-time quantitative PCR or stored at -20℃ for later use.

[0037] Step 3: Real-time quantitative PCR First, prepare a mix by mixing 10 μL SYBR Green, 7 μL Nuclease-Free Water, 1 μL upstream primer, and 1 μL downstream primer. Then, add 19 μL of the mix and 1 μL of cDNA to each reaction tube, for a total of 20 μL. Place the reaction tubes in a real-time quantitative PCR instrument and perform the reaction according to the real-time quantitative PCR reaction program. The real-time quantitative PCR reaction program is as follows: 95℃-10 min; (95℃-15s, 65℃-30s, 72℃-30s) × 40. Real-time quantitative PCR data are analyzed using 2... -∆∆Ct The relative content of the target gene was calculated. The results showed that the expression level of the Stard6 gene in Stard6 knockout mice was significantly lower than that in wild-type mice.

[0038] Example 4 Western blot analysis of protein expression Testicular tissue was homogenized with RIPA lysis buffer and centrifuged at 10000-12000g for 5 min at 4°C. The supernatant was collected. A small amount of the supernatant was used to determine the protein concentration using the BCA method. The protein samples were then adjusted to the same concentration. The protein samples were mixed with 5× loading buffer at a ratio of 4:1 and heated at 99°C for 10 min to denature the protein. After cooling, the samples were frozen at -80°C for storage.

[0039] (1) Electrophoresis Prepare the electrophoresis gel by removing it from the gel preparation rack, installing and fixing it in the electrophoresis tank, and slowly pouring freshly prepared electrophoresis buffer into the tank until the inner tank buffer completely covers the sample wells and is slightly higher than the outer tank buffer. Remove the comb from the electrophoresis gel vertically upwards, and add the samples to the sample wells in the order designed for the experiment, adding protein markers to both sides of the sample wells. Turn on the power and run the gel at 90V for approximately 30 minutes. After the protein markers separate in the gel, adjust the voltage to 120V. Electrophoresis is complete when the bromophenol blue indicator migrates to the bottom of the gel.

[0040] (2) Electro-rotation Before transfer, prepare 1× transfer buffer and pre-cool it in a -20℃ freezer. Before transfer, soak the PVDF membrane in methanol for 2 minutes. Open the transfer clamp in a tray containing 1× transfer buffer and prepare the "transfer sandwich" in the following order: red plate - sponge - filter paper - PVDF membrane - gel - filter paper - sponge - black plate. Be careful not to break the gel when carefully prying open the glass plate with a gel cutter. Remove this part of the gel and place it on thin filter paper, being careful to avoid air bubbles during this process. Place the transfer clamp in the transfer tank, put an ice box in the tank, and fill the transfer tank with the pre-cooled 1× transfer buffer. Submerge the transfer tank in crushed ice, turn on the power, and set the transfer conditions to a constant current of 600mA for 90 minutes.

[0041] (3) Closed After electroporation, the PVDF membrane was removed and placed back-to-back in a pre-prepared 5% milk blocking solution. It was then sealed on a shaker at room temperature for 3 hours to eliminate non-specific binding sites on the PVDF membrane.

[0042] (4) Antibody incubation After blocking, the bands were washed with TBST buffer, sealed with Stard6 primary antibody solution, and incubated overnight at 4°C. After primary antibody recovery, the bands were washed three times with TBST buffer for 10 min each time. Then, the bands were added to a secondary antibody solution of the corresponding species as the primary antibody, sealed, and incubated at 4°C for 4 h. After secondary antibody recovery, the bands were washed three times with TBST buffer for 10 min each time.

[0043] (5) Development Mix ECL luminescent solution A and solution B in equal volumes until homogeneous. Incubate the strip in the mixed luminescent solution for 1 minute. After incubation, place the strip in an exposure machine for development.

[0044] (6) Gray-scale analysis Image J software was used to perform grayscale quantitative analysis of protein bands. The results showed that the expression level of Stard6 protein in Stard6 knockout mice was significantly lower than that in wild-type mice.

[0045] Example 5 Mouse phenotyping (1) Mouse tissue weight Twenty-four wild-type male mice and twenty-four Stard6 knockout male mice of the C57 / BL6J strain were selected at ages 3, 5, 7, and 10 weeks, with six mice at each age. Body weight, liver weight, kidney weight, and testis weight were measured for both wild-type and Stard6 knockout mice at 3, 5, 7, and 10 weeks of age. The weight and tissue weight percentages at each age are shown in the table below. Stard6 knockout mice showed significantly reduced reproductive capacity in terms of body weight and testis weight compared to wild-type mice.

[0046] (2) Mouse reproductive capacity test Two-month-old wild-type male mice and Stard6 knockout male mice were caged with sexually mature wild-type female mice at a male-to-female ratio of 1:3, with 6 wild-type males and 6 Stard6 knockout males in each cage. The birth dates and numbers of newborn mice in each cage were observed and recorded. The results are shown in the table below. The average litter size and average number of pups in Stard6 knockout mice were significantly lower than those in wild-type mice, indicating a significant decrease in reproductive capacity.

[0047] In summary, although embodiments of this application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of this application. All equivalent changes and improvements made within the scope of this application shall still fall within the patent coverage of this application.

Claims

1. A method for constructing a Stard6 gene knockout mouse animal model, characterized in that, The method is to mate the Stard6-flox and Stra8-cre mice to obtain the male germ cell-specific Stard6 gene knockout mouse Stard6-CKO, which is the Stard6 gene knockout mouse animal model.

2. The method of claim 1, wherein the method of constructing a Stard6 knockout mouse animal model is characterized by, The Stard6 gene knockout mouse is prepared by CRISPR-Cas9 technology and a Cre-LoxP gene recombination system.

3. The method of claim 1, wherein the method of constructing a Stard6 knockout mouse animal model is characterized by, The Stard6-flox mouse is obtained by homologous recombination in mouse embryonic stem cells to generate the Stard6-flox allele to obtain the (flox / +) heterozygous mouse, and the (flox / +) heterozygous mouse is mated to obtain the Stard6-flox mouse.

4. The method of claim 1, wherein the method of constructing a Stard6 knockout mouse animal model is characterized by, The construction method comprises the following steps: (1) First, mate the Stard6-flox mouse of claim 3 with the Stra8-cre mouse to obtain the Stard6 (flox / +) mouse and the (flox / +) Stra8-cre mouse; (2) Then, mate the female Stard6 (flox / +) Stra8-cre mouse with the male Stard6 (flox / flox) mouse to obtain the male Stard6 Stra8-cre mouse, which is the Stard6 gene knockout mouse.

5. The method for constructing the Stard6 gene knockout mouse model according to claim 4, characterized in that, The step (2) Stard6 gene knockout mouse is identified by toe or tail gene conventional PCR to confirm the change in the genomic structure.

6. The method of claim 4, wherein the mouse animal model is constructed by knocking out the Stard6 gene. The step (2) Stard6 gene knockout mouse is detected by real-time fluorescent quantitative PCR to detect the expression content of the Stard6 gene in the testis of the male mouse.

7. The method for constructing the Stard6 gene knockout mouse model according to claim 4, characterized in that, The step (2) Stard6 gene knockout mouse is detected by Western Blot to detect the expression content of the Stard6 protein in the testis of the male mouse.

8. The method of claim 5, wherein the mouse animal model is constructed by knocking out the Stard6 gene. The toe gene identification is performed on the mouse about two weeks after birth, and the mouse is labeled.

9. Use of the method for constructing a Stard6 knockout mouse model according to any one of claims 1 to 8, characterized in that, The application includes the research on the regulation mechanism of the development state of the male germ cell and the process of spermatogenesis.

10. The use of the Stard6 knockout mouse animal model according to claim 9, characterized in that, The application also includes the clinical treatment and drug research of male infertility.

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