Preparation method of metaphase of porcine cell chromosome

Abstract

The invention discloses a preparation method of a metaphase of a porcine cell chromosome, and belongs to the technical field of genetics. The preparation method comprises the following steps: compounding colchicine and nocodazole which are microtubule polymerization inhibitors into a composition, culturing a pig cell line for 3 hours by using a culture medium containing 0.1 mu g / mL colchicine and 0.2 mu g / mL nocodazole, retarding cell division, and preparing the metaphase of the pig cell chromosome through the steps of digestion, hypotonic treatment, fixation, dropping, dyeing and the like. Experimental results show that the preparation method provided by the invention overcomes the problem of excessive condensation of chromosomes caused by single colchicine, the test efficiency is improved while the excellent morphology of the chromosomes is ensured, and the combination of colchicine and nocodazole produces an outstanding synergistic effect. The invention provides a universal, efficient and optimized method for preparing the metaphase of the porcine cell chromosome, and lays a foundation for detection and breeding of structural variation of the porcine chromosome.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetics, in particular to a method for preparing metaphase of pig cell chromosomes. BACKGROUND

[0002] In the pig breeding industry, the growth rate, meat quality, reproductive performance and other economic traits of live pigs directly affect the industry's efficiency. At the same time, pigs play an important role in biomedical research. Due to the high similarity of pigs' physiological structure, metabolic process and genome to humans, pigs have become an ideal animal model for studying human diseases, drug development and organ transplantation. Therefore, it is particularly crucial to explore the genetic characteristics of pigs in depth.

[0003] Chromosome karyotype analysis is an important technical means in the field of genetics. Each species has its unique chromosome karyotype, including the number, size and morphology of chromosomes. Through chromosome karyotype analysis, species can be accurately identified, genetic relationships can be distinguished, and various genetic diseases can be diagnosed and identified. By comparing the chromosome karyotypes of different species or different populations of the same species, the evolution of chromosomes can be revealed. By analyzing the number changes, morphological differences and banding characteristics of chromosomes, the evolution mechanism of chromosomes in the evolution process of species can be understood. Previous studies have found that changes in the number of chromosomes are likely to cause slow growth and development, reduced reproductive capacity and other problems in pigs. Structural variations in chromosomes, such as deletions, duplications and inversions, can affect gene expression and regulation, ultimately affecting the economic traits of pigs. Therefore, in-depth exploration of pig chromosome karyotypes can help reveal the genetic basis of pigs and provide a solid theoretical basis for pig breed improvement and genetic breeding.

[0004] Notably, chromosomal rearrangements, as a special case of chromosomal structural variation, occur in a variety of mammals, and translocations are particularly common in domestic pigs compared to other species. Chromosomal rearrangements are structural chromosomal abnormalities caused by one or more chromatin breaks and rearrangement or ectopic exchange of chromatin fragments, and obvious changes in the carrier karyotype can usually be seen under an optical microscope. Although there is a large-scale rearrangement of genetic material within the genome, usually on the scale of millions of base pairs, there is usually no associated observable symptoms when chromosomal rearrangements occur. However, it is known that chromosomal rearrangements can cause a decrease in the fertility of carriers, and the loss of piglets caused by chromosomal rearrangements varies among different carriers and is affected by a variety of factors. During meiosis, derivative chromosomes must meet the need for homologous chromosome pairing and form various complex structures between derivative chromosomes and their counterparts. During meiosis, the asymmetric segregation of these chromosomes causes genetic imbalance in a portion of gametes. Due to the presence of genetic imbalance, embryos carrying lethal mutations or embryos with genetic imbalance will die prematurely during development after fertilization. Chromosomal rearrangements, most notably reciprocal translocations, are one of the main causes of reproductive dysfunction in domestic pigs, and previous studies have shown that they occur spontaneously in 1 / 200 live-born pigs. It is estimated that if rearrangement carriers are allowed to breed, about 50% of offspring will inherit rearrangements, which will increase the incidence of chromosomal rearrangements in pig populations over time.

[0005] Cytogenetics plays a crucial role in this series of work, which can precisely identify chromosomal rearrangements and other chromosomal abnormalities in the pig genome, and through these identifications, better boars can be selected for breeding services, directly reducing the incidence of chromosomal structural variations in pig populations. Obtaining high-quality metaphase chromosomes is the primary and key technical bottleneck in cytogenetic experiments. The quality of this process is complexly affected by many factors such as cell cycle synchronization efficiency, spindle microtubule depolymerization degree, hypotonic and fixation effect. Among them, the use of chemical reagents to intervene in the cell cycle and efficiently and controllably block cells in the metaphase of mitosis is a decisive step in the entire process. Currently, the main metaphase induction fluid mainly relies on microtubulin inhibitors.

[0006] There is no mature method for preparing pig cell metaphase chromosomes in the field of pig genetics, and there are relatively few studies on the mechanism of chromosomal structural variation, accurate diagnostic methods, origins, and effects on meiosis and the genome itself, which are insufficient to provide solid support for the healthy breeding and genetics research of the entire pig population. Therefore, there is an urgent need to provide a universal, efficient and optimized method for inducing pig chromosome metaphase. SUMMARY

[0007] The application aims to provide a preparation method of pig cell chromosome metaphase, so as to solve the problems in the prior art. The application combines colchicine and nocodazole to form a composition. The combination of colchicine and nocodazole produces an outstanding synergistic effect. The application provides a universal, efficient and optimized method for preparing pig cell chromosome metaphase, and lays a foundation for the detection and breeding of pig chromosome structure variation.

[0008] To achieve the above object, the application provides the following solutions.

[0009] The application provides a composition for preparing pig cell chromosome metaphase, which comprises colchicine and nocodazole.

[0010] Further, the mass ratio of the colchicine to the nocodazole is 1:2.

[0011] The application also provides an application of the above composition in the preparation of pig cell chromosome metaphase.

[0012] The application also provides a preparation method of pig cell chromosome metaphase, which comprises the step of blocking the cell cycle by using the above composition.

[0013] Further, the method comprises the following steps.

[0014] The complete culture medium and the composition are mixed to prepare a blocking culture medium.

[0015] The pig cell line is cultured, and when the cells grow to 70%-80%, the cells are washed, the blocking culture medium is added, and the cells are cultured, digested, centrifuged and collected, subjected to hypotonic treatment, fixed after the hypotonic treatment, and dropped on a slide after the fixation, and then stained to obtain the pig cell chromosome metaphase.

[0016] Further, the final concentration of the colchicine in the blocking culture medium is 0.1 μg / mL, and the final concentration of the nocodazole is 0.2 μg / mL.

[0017] Further, the culture time is 3 h.

[0018] Further, the hypotonic solution for the hypotonic treatment is 0.075M KCl aqueous solution, the temperature for the hypotonic treatment is 37℃, and the time is 40 min.

[0019] Further, the volume of the cell suspension is 20 μL, and the height of the dropping on the slide is not less than 50 cm.

[0020] The application also provides an application of the above composition in the karyotype analysis of pig cells for non-disease diagnosis or treatment purposes.

[0021] The present invention discloses the following technical effects:

[0022] This invention combines the microtubule polymerization inhibitor colchicine and nocodazole into a composition. Pig cell lines were cultured for 3 hours in a medium containing 0.1 μg / mL colchicine and 0.2 μg / mL nocodazole to arrest cell division. Following digestion, hypotonicity, fixation, slide preparation, and staining, metaphase phases of pig chromosomes were obtained. Experimental results show that the preparation method provided by this invention overcomes the problem of excessive chromosome condensation caused by colchicine alone, while also improving experimental efficiency while maintaining excellent chromosome morphology. The combined use of colchicine and nocodazole produced a significant synergistic effect. This invention provides a universal, efficient, and optimized method for preparing metaphase phases of pig chromosomes, laying the foundation for the detection and breeding of pig chromosome structural variations. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The results show the effects of colchicine-induced metaphase chromosomes in porcine kidney epithelial cells; where A represents porcine kidney epithelial cells treated with 0.5 μg / mL colchicine for 6 h; B represents porcine kidney epithelial cells treated with 1 μg / mL colchicine for 6 h; and C represents porcine kidney epithelial cells treated with 5 μg / mL colchicine for 6 h.

[0025] Figure 2 The study aimed to induce metaphase of chromosomes in porcine primary small intestinal epithelial cells using colchicine. Specifically, A consisted of porcine primary small intestinal epithelial cells treated with 0.5 μg / mL colchicine for 6 h; B consisted of porcine primary small intestinal epithelial cells treated with 1 μg / mL colchicine for 6 h; and C consisted of porcine primary small intestinal epithelial cells treated with 5 μg / mL colchicine for 6 h.

[0026] Figure 3 The study aimed to induce metaphase of chromosomes in porcine primary small intestinal epithelial cells using nocodazole. Specifically, A consisted of treatment with 0.1 μg / mL nocodazole for 6 h; B consisted of treatment with 0.2 μg / mL nocodazole for 6 h; and C consisted of treatment with 0.5 μg / mL nocodazole for 6 h.

[0027] Figure 4 The combined use of colchicine and nocodaazole to induce metaphase of chromosomes;

[0028] Figure 5 Metaphase of chromosomes in porcine kidney epithelial cells after 24 h of colchicine treatment. Detailed Implementation

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0031] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0032] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0033] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0034] Colchicine, a classic spindle inhibitor, specifically binds to tubulin, preventing its polymerization into microtubules and thus disrupting spindle formation, causing cell arrest at metaphase. However, its effect is irreversible and it easily leads to excessive chromosome condensation and shortening, which limits its application in scenarios requiring high-resolution analysis. Nocodazole, on the other hand, is another highly effective microtubule polymerization inhibitor, with its greatest advantage being its reversible action. By precisely controlling its duration of action, longer and better-dispersed chromosomes can usually be obtained, facilitating more detailed genetic observations. Theoretically, the combination of colchicine and nocodazole could potentially produce a synergistic effect through their different action kinetics, thereby achieving better regulation of chromosome morphology while ensuring high arrest efficiency.

[0035] Previous studies lacked a mature method for preparing metaphase chromosomes in pigs. This invention aims to overcome the shortcomings of existing technologies and provide a universal solution for pig cells, contributing to a deeper understanding of the mechanisms underlying pig chromosome structural variations. Analysis of a large number of metaphase samples can explore the relationship between chromosome structural variations and gene expression and regulation.

[0036] In practical applications, this invention can be applied to pig breeding. During the selection process, chromosomal structural variations are detected in candidate pigs, and individuals with normal chromosomes are selected for reproduction. This avoids the transmission of chromosomes carrying structural variations in the pig herd, thereby effectively reducing the incidence of chromosomal structural variations in pigs, improving the genetic quality and overall health of the pig herd, and promoting the sustainable development of the pig industry.

[0037] Example 1

[0038] I. Experimental Methods

[0039] 1. Culture of porcine cell lines

[0040] Porcine kidney epithelial cells and primary porcine small intestinal epithelial cells were extracted from liquid nitrogen and rapidly transferred to a 37°C water bath to dissolve into a cell suspension. The cell suspension was then transferred to 15 mL centrifuge tubes, and 1 mL of fresh complete culture medium was added in equal proportions. The complete culture medium was a system supplemented with 10% fetal bovine serum (Gibco, 10099-141) and 1% penicillin-streptomycin antibiotics (Gibco, 15140-122). The cells were centrifuged in a horizontal centrifuge (1000 rpm, 5 min), the supernatant was discarded, and the cells were resuspended in 5 mL of fresh complete culture medium. The suspension was then transferred to a culture flask and placed in a 37°C incubator containing 5% CO2.

[0041] 2. Blocking cell cycle arrest at metaphase

[0042] 2.1 Colchicine blockade

[0043] When the cells reached 70%-80% confluence, they were washed three times with PBS for 5 min each time. Then, they were added with prepared complete culture medium containing colchicine (purchased from Beijing Solarbio Science & Technology Co., Ltd., IC7670) at final concentrations of 0.5, 1, and 5 μg / mL, respectively. After culturing for 6 h, the cells were digested with 0.25% trypsin. After adding 1 mL of complete culture medium to stop the digestion, the cell suspension was collected and centrifuged (1000 rpm, 5 min), and the supernatant was discarded.

[0044] 2.2 Nocodazole blockade

[0045] When the cells reached 70%-80% confluence, they were washed three times with PBS for 5 min each time, and then filled with complete culture medium containing nocodaazole (purchased from MedChemExpress, HY-13520) at final concentrations of 0.1, 0.2 and 0.5 μg / mL, respectively. After culturing for 6 h, the cells were digested with 0.25% trypsin, and after adding 1 mL of complete culture medium to stop the digestion, the cell suspension was collected and centrifuged (1000 rpm, 5 min), and the supernatant was discarded.

[0046] 2.3 Combined use of the two

[0047] When the cells reach 70%-80% confluence, wash them three times with PBS for 5 min each time. Add the prepared complete culture medium with a final concentration of 0.1 μg / mL colchicine and 0.2 μg / mL nocodazole. Culture for 3 h and then digest with 0.25% trypsin. Add 1 mL of complete culture medium to stop the digestion. Collect the cell suspension and centrifuge (1000 rpm, 5 min). Discard the supernatant.

[0048] 3. Cell chromosome slide preparation

[0049] 3.1 Hypoosmolarity

[0050] Prepare 0.075 M KCl hypotonic solution (prepared with sterile water) and preheat it in a water bath at 37°C for 30 min. Add 1 mL of hypotonic solution to the cell pellet and gently pipette to form a uniform cell suspension. Add the remaining preheated hypotonic solution to centrifuge tubes (7-10 mL of hypotonic solution is needed per T25 flask depending on the cell density) and place them in a 37°C water bath for hypotonic treatment for 40 min. Gently shake the centrifuge tubes periodically during this time.

[0051] 3.2 Fixed

[0052] Prepare 10 mL of fixative (methanol:glacial acetic acid volume ratio 3:1) and pre-chill at 4°C. First, add 1 mL of pre-chilled fixative to a centrifuge tube after the hypotonic phase is complete, gently pipette from top to bottom 10 times, centrifuge at 1000 r / min for 5 min, and carefully remove the supernatant, leaving a small amount of supernatant to avoid excessive cell loss. Add 5 mL of fresh pre-chilled fixative, gently pipette to form a single-cell suspension, and fix at 4°C for 30 min, centrifuge at 1000 r / min for 5 min. Repeat this fixation operation twice, centrifuging at 1000 r / min for 5 min, and remove the supernatant. A total of 3 fixation cycles are required.

[0053] 3.3 drops

[0054] Resuspend the cells in the cell pellet with 300-600 μL of fixative. Take a clean, adhesive slide (pre-cooled at -20°C), and drop 20 μL of cell suspension onto the slide at a height of 50 cm or more above the slide to break up the chromosomes. Place the slide in a 75°C oven to dry for 1-3 hours, and store at -20°C. It can be used in the short term.

[0055] 4. Chromosome staining

[0056] Stain porcine kidney cells and primary small intestinal epithelial cells with DAPI for 10 min in the dark, wash three times with PBS for 5 min each time, add 20 μL of anti-fluorescence quenching agent, mount the slides, and store at 4°C.

[0057] II. Experimental Results

[0058] 1. Treatment of porcine kidney epithelial cells with different colchicine concentrations to induce metaphase of chromosomes

[0059] The results are as follows Figure 1 As shown in Figures A, B, and C, treating porcine kidney epithelial cells with 0.5, 1, and 5 μg / mL colchicine for 6 h resulted in relatively clear metaphase chromosome morphology and major constriction locations under a fluorescence microscope after DAPI staining, with good dispersion. However, during cell line stabilization, chromosome breakage and fusion may be induced, forming multiple small chromosome fragments. Fusion may connect previously independent chromosomes, leading to a discrepancy between the actual chromosome number and the actual number.

[0060] 2. Induction of metaphase of chromosomes in porcine primary small intestinal epithelial cell lines

[0061] 2.1 Colchicine-induced metaphase of chromosomes

[0062] Domestic pigs have 38 chromosomes, including 18 pairs of autosomes and one pair of sex chromosomes. This invention stains porcine small intestinal epithelial cells treated with 0.5, 1, and 5 μg / mL colchicine for 6 h. The results are as follows: Figure 2 As shown in Figures A, B, and C, the chromosome number was accurate after induction with the three concentrations of reagents, and the chromosome morphology was intact, with no obvious deletions or breaks observed. The 1 μg / mL chromosomes exhibited a clear rod-like structure, with a relatively prominent centromere position and a suitable ratio of long to short arms. Figure 2 (Medium B). Although the high concentration group showed a uniform distribution of fluorescence signals on the chromosomes, the chromosomes were short and stout, which is closely related to the irreversibility of colchicine, leading to excessive chromosome condensation and shortening. Figure 2 (C). Therefore, a colchicine concentration of 1 μg / mL was chosen for subsequent experiments.

[0063] 2.2 Nocodazole induces metaphase of chromosomes

[0064] like Figure 3 As shown in Figures A, B, and C, the experimental results indicate that all nocodazole treatment groups (0.1, 0.2, and 0.5 μg / mL) accurately yielded metaphase cells with a chromosome number of 2n=38, demonstrating its reliable arrest efficiency. Nocodazole exhibits a unique concentration-dependent effect on chromosome morphology: among which, such as Figure 3 As shown in Figure B, the medium concentration treatment of 0.2 μg / mL yielded the best results, producing chromosomes significantly longer than those treated with colchicine. This morphological advantage allowed for clear and distinct differentiation between the largest mesocentric chromosome (chromosome 1) and the largest telocentric chromosome (chromosome 13) in the porcine karyotype, a feat difficult to achieve in colchicine specimens where chromosomes are generally short and thick. It is worth noting that, as... Figure 3 As shown in Figure C, when the concentration of nocodazole increased to 0.5 μg / mL, the chromosomes showed a tendency to excessively condense and become shorter, which was worse than the medium concentration group. This reveals the key role of precise control of drug concentration in obtaining the ideal morphology.

[0065] In summary, the nocodaazole concentration scheme provided by this invention successfully overcomes the problem of excessive chromosome condensation caused by the traditional colchicine method.

[0066] 2.3 Colchicine and nocodazole in combination to induce metaphase of chromosomes

[0067] Based on the optimization results of colchicine and nocodaazole used alone, this invention further combines their optimal concentrations in a synergistic manner. For example... Figure 4As shown, the results indicate that this combined treatment regimen successfully obtained high-quality metaphase chromosomes while significantly shortening the induction time (3 h). The morphological improvement of the obtained chromosomes was comparable to that of the nocodazole treatment group. This result confirms that the combination of colchicine and nocodazole produced a good synergistic effect, not only overcoming the problem of excessive chromosome condensation caused by colchicine alone, but also improving experimental efficiency while ensuring excellent chromosome morphology.

[0068] III. Comparative Analysis

[0069] Cell lines play a crucial role in biological research such as mammalian gene function, disease models, and drug screening. This invention primarily investigates a method for preparing metaphase chromosome specimens in porcine-related cells. Compared to conventional methods, this invention significantly improves upon the concentrations and durations of colchicine and nocodazole, the duration of hypotonic treatment, and the height of the droplet, and constructs a method that combines colchicine and nocodazole.

[0070] The conventional method involves treating mammalian cells with 15 μg / mL colchicine for 24 h before collecting the samples. In preliminary experiments, this invention found that high colchicine concentrations and long treatment times do indeed increase the number of metaphase chromosomes, but... Figure 5 As shown, when this method is applied to cells, it causes excessive condensation of chromosomes, resulting in a shortened morphology that makes it difficult to observe the main constriction and other fine structures.

[0071] In hypotonic solutions, the solute concentration is lower than that inside the cells, causing water to enter the cells along the concentration gradient and swell. Our research has shown that if the hypotonic time is too short, cell swelling is insufficient, making it difficult for chromosomes to rupture during slide preparation and resulting in poor dispersion.

[0072] Choosing the appropriate droplet height ensures that the force of the droplet impacting the slide is moderate. This allows the cells to fully rupture, enabling the chromosomes to spread well on the slide while maintaining their integrity and ensuring even dispersion. This facilitates clear observation of the chromosomes' morphology, structure, and number under the microscope. If the height is too high, chromosomes may be lost, making counting and identification difficult. If the height is too low, cells may not rupture sufficiently, and chromosomes may not spread and disperse effectively, resulting in chromosome aggregation and overlap.

[0073] Therefore, this invention addresses the aforementioned problems by improving upon existing methods to produce dispersed and clearly defined metaphase chromosomes. Experimental results show that controlling the concentration of colchicine at 0.5-1 μg / mL and shortening the induction time to 6 h is more conducive to obtaining well-morphologically sound metaphase chromosomes; controlling the concentration of nocodazole at 0.1-0.2 μg / mL and the induction time to 6 h is conducive to obtaining more refined metaphase chromosomes; the combined group also controlled within a low concentration range, and high-quality metaphase chromosomes could be obtained after 3 h of induction. In addition, cell swelling occurs when the cell hypotonicity time is around 40-60 min, which facilitates chromosome rupture after slide preparation; a slide height of 40-50 cm can obtain relatively dispersed and spread metaphase chromosomes, avoiding chromosome overlap.

[0074] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A composition for preparing metaphase of porcine chromosomes, characterized in that, Including colchicine and nocodazole.

2. The composition according to claim 1, characterized in that, The mass ratio of colchicine to nocodazole is 1:

2.

3. The use of the composition according to claim 1 or 2 in the preparation of metaphase of porcine cell chromosomes.

4. A method for preparing metaphase of porcine chromosomes, characterized in that, The step includes using the composition of claim 1 or 2 to block the cell cycle.

5. The preparation method according to claim 4, characterized in that, Includes the following steps: Mix the complete culture medium and the composition to prepare a retardation culture medium; Culture the pig cell line until the cells grow to 70%-80%, wash them, add the aforementioned inhibition medium, culture, digest, centrifuge to collect the precipitate, perform hypotonic treatment, fix them after hypotonic treatment, take cell suspension droplets after fixation, stain them, and obtain the metaphase of the pig cell chromosomes.

6. The preparation method according to claim 5, characterized in that, In the retardation culture medium, the final concentration of colchicine is 0.1 μg / mL; and the final concentration of nocodazole is 0.2 μg / mL.

7. The preparation method according to claim 5, characterized in that, The incubation period is 3 hours.

8. The preparation method according to claim 5, characterized in that, The hypotonic solution used for the hypotonic treatment was a 0.075M KCl aqueous solution; the hypotonic treatment temperature was 37℃ and the time was 40 min.

9. The preparation method according to claim 5, characterized in that, The volume of the cell suspension is 20 μL; the height of the droplet is not less than 50 cm.

10. The use of the composition of claim 1 or 2 in porcine cell chromosome karyotype analysis for non-disease diagnosis or treatment purposes.

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