Yak intramuscular precursor fat immortalized cell line and preparation method thereof

Abstract

The invention discloses a yak intramuscular precursor fat immortalized cell line and a preparation method thereof. The method comprises the following steps: (1) obtaining yak primary intramuscular precursor fat cells; (2) performing SV40T lentivirus infection on the precursor fat cells in the yak muscle and screening cell strains: performing SV40T lentivirus infection by utilizing a 1 / 2 small-volume infection method to obtain immortalized cells after the precursor fat cells in the yak primary muscle are infected with SV40T, namely immortalized cells for short; puromycin is adopted to screen immortalized cells; and diluting the screened cells by using a limited dilution method, and carrying out enlarged culture to obtain the immortalized cell line. The yak intramuscular precursor fat immortalized cell line constructed by the invention has genetic homogeneity, the heterogeneity influence of cell populations is eliminated, and an in-vitro cell model is provided for subsequent research related to yak intramuscular fat cells.

Description

Technical Field

[0001] The invention relates to a method for preparing an immortalized cell line, in particular to a yak intramuscular pre-fat immortalized cell line and a preparation method thereof. Background Art

[0002] The information provided in this section is for the purpose of generally presenting the background to the technology related to the present invention. To the extent described in this section, the work of the presently named inventors and the aspects of the description that may not constitute prior art at the time of filing are neither explicitly nor implicitly admitted to be prior art to the present disclosure.

[0003] Yaks are mainly distributed in the Qinghai-Tibet Plateau at an altitude of 2000-5000m and its adjacent high mountains or subalpine areas. They have strong adaptability to high altitude, severe cold and strong ultraviolet rays, and are an important source of production and living materials for farmers and herdsmen in the Qinghai-Tibet Plateau. However, compared with ordinary cattle of the same age, yaks grow slowly, have slow muscle development and low fat deposition efficiency, resulting in poor tenderness and rough taste of yak meat, which is not conducive to improving the economic benefits of yak meat in the Qinghai-Tibet Plateau. Therefore, improving the muscle development and fat deposition efficiency of yak beef, and improving the tenderness and taste of yak beef are of great significance to promoting the sustainable development of yak breeding industry and improving economic benefits.

[0004] Intramuscular fat (IMF) is the fat in skeletal muscle, which is composed of fat droplets (low content) in muscle cells and adipose tissue between muscle fibers. Intramuscular fat content is one of the key factors in evaluating meat quality, and has a significant correlation with meat color, tenderness, water retention, flavor and juiciness. Changes in IMF content are mainly related to the number and size of adipocytes, involving a series of processes such as preadipocyte proliferation, preadipocyte differentiation, adipocyte maturation and decreased muscle growth.

[0005] Normal cells in animals go through a series of processes including proliferation, differentiation, aging and apoptosis. The number of these cells is in a dynamic equilibrium, which allows the animal to grow and metabolize normally. However, the proliferation capacity of animal cells in vitro is limited. They cannot proliferate and replicate indefinitely. After a certain stage, they will be static, which is called the Hayflick limit. Cell immortalization refers to the process of cell culture in vitro. Due to changes in their own genes or various external stimuli, cells overcome the Hayflick limit, thereby avoiding the aging and death process, and can be cultured for a long time and divide and proliferate indefinitely.

[0006] At present, most of the research on yak intramuscular adipocyte differentiation still relies on primary preadipocytes cultured in vitro. Although primary preadipocytes have the advantage of being very close to preadipocytes in vivo in terms of morphology, structure and functional activities, they can be used as a research model for adipocyte differentiation to a certain extent. However, the culture of primary intramuscular preadipocytes itself has insurmountable shortcomings and deficiencies: (1) Primary cells cannot be passaged indefinitely. During the research process, researchers need to repeatedly isolate cells from tissues to establish a culture system. This process is very cumbersome, time-consuming and costly; (2) Primary cells are heterogeneous, and it is very difficult to separate preadipocytes from various fibroblasts; (3) Primary cells come from different individuals, and there are differences in the genetic background between different batches of cells, which makes it difficult to obtain stable and reliable results. Due to these shortcomings and deficiencies of the primary preadipocyte culture system, the in-depth research on yak intramuscular adipocyte differentiation has been seriously hindered.

[0007] The ideal research model for adipocyte differentiation is the immortalized preadipocyte cell line. Compared with primary cells cultured in vitro, the immortalized preadipocyte cell line has both unlimited proliferation ability and the characteristics of normal preadipocytes. It can provide a large number of stable, uniform, and consistent cell sources, eliminating the effects of different developmental stages, different physiological conditions, and different cell populations, and ensuring the repeatability and comparability of the research.

[0008] Currently, there are four main methods for establishing immortalized cell lines: (1) immortalizing cells through physical and chemical methods. This method has low efficiency and is prone to cause genomic damage to cells, with a high possibility of cancer. (2) obtaining immortalized cells from cancer tissues through spontaneous mutations. The cells obtained by this method may lose many characteristics of the source cells. (3) transfecting cells with the telomerase catalytic subunit gene (hTERT). The activated telomerase uses itself as a template to continuously synthesize telomeric DNA to maintain the length of telomeres and the stability of chromosomes, allowing cells to cross the M2 phase and acquire the ability to divide and proliferate indefinitely, thereby achieving immortalization. (4) Virus method. Some viruses such as SV40 virus (Simian vacuolating virus 40 or Simian virus40), Epstein-Barr virus (EBv), human papillomavirus (HPVs), etc. can activate telomerase activity, making cells immortal. Viral DNA can be integrated into the cell genome and continuously expressed as the cell replicates. Summary of the invention

[0009] The purpose of the present invention is to provide a yak intramuscular prefat immortalized cell line and a preparation method thereof, which solves the problems of difficult separation of existing yak intramuscular prefat cells, limited number of passages, differences between different batches, etc. The constructed yak intramuscular prefat immortalized cell line has genetic uniformity, eliminates the influence of heterogeneity of cell populations, and the number of chromosomes of the yak intramuscular prefat immortalized cells passaged to the 30th generation is normal. When passaged to the 30th generation, the yak intramuscular prefat immortalized cells still have the ability to normally differentiate into mature adipocytes, and the differentiation ability is stronger than that of primary cells, thereby providing an in vitro cell model for subsequent research on yak intramuscular adipocytes.

[0010] In order to achieve the above object, the present invention provides a method for preparing a yak intramuscular pre-fat immortalized cell line, the method comprising:

[0011] (1) Obtaining primary intramuscular preadipocytes from yaks

[0012] The longissimus dorsi muscle tissue sample of the yak was rinsed with PBS buffer containing 3% streptomycin-penicillin double antibody, and the visible fascia and blood vessels were removed with sterile scissors in a PBS moist environment, and the remaining tissue was cut into pieces; digestive fluid was added to the cut tissue for digestion;

[0013] After digestion, filter through 100μm, 70μm and 40μm sieves in sequence, take the filtrate and centrifuge at 2000r / min, pour out the supernatant, add red blood cell lysis solution to lyse at room temperature, and centrifuge at 2000r / min; resuspend the precipitate in serum-free DMEM / F12 basal medium, and centrifuge at 1800r / min and 1500r / min in sequence, add growth medium to the precipitate and resuspend it, then transfer it to a culture dish, culture in a 37℃ constant temperature incubator with 5% CO2, replace with fresh growth medium after adherence, the growth medium is 1% double antibody + 10% fetal bovine serum + 89% DMEM / F12 basal medium, and then replace the medium every 2 days, culture until passage and cryopreservation, and obtain yak primary intramuscular preadipocytes, referred to as primary cells;

[0014] (2) Infection of yak intramuscular preadipocytes with SV40T lentivirus and screening of cell lines

[0015] The obtained primary cells with good growth status were cultured overnight in a 37°C, 5% CO2 incubator. When the cell confluence rate reached 50%, the control lentivirus infection was first performed using the 1 / 2 small volume infection method. The packaging system and infection ability of the control lentivirus were completely consistent with those of the SV40T lentivirus. The difference was that the vector plasmid of the control lentivirus carried the GFP gene, while the vector plasmid of the SV40T lentivirus carried the SV40T gene. After the optimal MOI value was obtained by infection with the control lentivirus, the SV40T lentivirus infection was performed: before infection, the original culture medium of the cells was aspirated, half of the normal amount of the culture medium was added, and the virus stock solution was added to the cells according to the optimal MOI value. After infection for 4 hours, the culture medium was supplemented to the normal amount, and the infection was continued. After infection for 24 hours, the culture medium containing the virus was aspirated, replaced with fresh complete culture medium, and continued to be cultured at 37°C to obtain immortalized cells after yak primary intramuscular preadipocytes were infected with SV40T, referred to as immortalized cells;

[0016] The cells infected with the SV40T lentivirus are cultured until the cell fusion rate is 55-65%, and the culture medium is replaced with a complete culture medium containing 3 μg / mL puromycin to kill the primary cells in the infected cells, and the immortalized cells can grow normally, so as to screen the immortalized cells;

[0017] The cells infected with SV40T virus and screened by the above-mentioned puromycin were digested with trypsin, the cell pellet after centrifugation was blown and mixed, and the digested cell suspension was filtered using a 40μm cell filter to remove cell clumps and obtain a more uniform single cell suspension; then, the limiting dilution method was used for dilution and plating to ensure that each well contained a single cell, and cell lines with good growth status were selected for cell amplification, and 3μg / mL puromycin was continued to be applied for maintenance screening and culture. When the cell confluence reached 90%, the cells were digested to a 24-well plate and 3μg / mL puromycin was continued to be applied for maintenance screening and culture. According to the screening and culture method, the cells were digested to a 12-well plate, a 6-well plate, a T25 culture flask, and a 90mm cell culture dish in turn for expansion culture to obtain monoclonal immortalized cells.

[0018] Preferably, the optimal MOI value is 30.

[0019] Preferably, the longissimus dorsi muscle tissue sample of the yak is selected from the longissimus dorsi muscle tissue between the 12th and 13th ribs of a fattening yak of about 4 years old.

[0020] Preferably, the volume ratio of the tissue to the digestive fluid is 1:2.

[0021] Preferably, the digestion solution is 0.1% type I collagenase and 0.1% type II collagenase in a volume ratio of 2:1.

[0022] Preferably, the digestion is carried out in a 37° C. water bath for 4 h, with shaking every 5 min.

[0023] Preferably, after the digestion is completed, the filtrate is filtered through 100 μm, 70 μm and 40 μm sieves in sequence, the filtrate is centrifuged at 2000 r / min for 5 min, the supernatant is discarded, and red blood cell lysis solution is added to lyse at room temperature for 5 min, and then centrifuged at 2000 r / min for 5 min; the precipitate is resuspended in serum-free DMEM / F12 basal culture medium, and gradient centrifuged at 1800 r / min and 1500 r / min for 5 min in sequence, the precipitate is resuspended in complete culture medium and transferred to a culture dish, cultured in a constant temperature incubator at 37°C with 5% CO2, and fresh growth culture medium is replaced after adhesion for 1.5 hours, and then the culture medium is replaced every 2 days until passage and cryopreservation to obtain yak primary intramuscular preadipocytes.

[0024] Preferably, SV40T lentivirus infection is performed using the 1 / 2 small volume infection method: before infection, the original culture medium of the cells is aspirated, 50 μL of culture medium is added, and the virus stock solution is added to the cells at an MOI value of 30. After 4 hours of infection, 50 μL of culture medium is added again. 24 hours after infection, the culture medium containing the virus is aspirated, replaced with fresh complete culture medium, and cultured at 37°C is continued.

[0025] Preferably, the cells infected with the SV40T lentivirus are cultured until the cell fusion rate reaches 55-65%, and then the culture medium is replaced with a complete culture medium containing 3 μg / mL puromycin for 48 hours.

[0026] Preferably, the screened cells are diluted by limiting dilution and plated into a 96-well plate to ensure that each well contains a single cell.

[0027] Another object of the present invention is to provide a yak intramuscular pre-fat immortalized cell line obtained by the preparation method.

[0028] The yak intramuscular preadipocyte immortalized cell line and preparation method thereof of the present invention solve the problems of difficult separation of existing yak intramuscular preadipocytes, limited passage times, and differences between different batches, and have the following advantages:

[0029] (1) In the process of SV40T lentivirus transfection, the method of the present invention found that the cell death rate was higher after adding Polybrene, and judging from the expression of GFP fluorescent protein, for yak intramuscular preadipocytes, the use of Polybrene or not had little effect on the transfection efficiency of SV40T lentivirus, so the present invention did not add Polybrene when transfecting SV40T lentivirus, thereby improving the survival rate of immortalized cells. Moreover, since yak primary cells are prone to aging, which will seriously affect the transfection efficiency of SV40T lentivirus, the present invention also screened the primary cells used to prepare immortalized cells in the early preliminary experiment process, and screened cells with better growth status for subsequent virus infection experiments, thereby improving the transfection efficiency of SV40T virus. In addition, the virus packaging system used in the present invention is mainly based on a three-plasmid system, consisting of psPAX2, pMD2.G, and pHBLV series plasmids, which greatly improves the expression efficiency and transfection efficiency of the SV40T gene; when transfecting the SV40T lentivirus (HBLV-SV40T-3xflag-Puro), a control lentivirus with a GFP label (HBLV-ZsGreen-Puro) is also transfected as a control group, so that the transfection efficiency of the SV40T virus can be observed and judged more intuitively;

[0030] (2) The present invention uses SV40T virus to infect primary yak intramuscular preadipocytes by a 1 / 2 small volume infection method, and uses puromycin to screen SV40T positive cells. When the SV40T virus is stably expressed, monoclonal positive cells are screened and cultured by a limiting dilution method, thereby constructing a yak intramuscular preadipocyte immortalized cell line, providing an in vitro cell model for subsequent research on yak intramuscular adipocytes;

[0031] (2) The present invention improves the infection efficiency of SV40T virus by using the 1 / 2 small volume infection method for virus infection. Compared with low volume virus infection, the 1 / 2 volume method uses a relatively large amount of virus. More virus particles increase the chance of contact with cells, allowing the virus to more effectively adsorb to cell surface receptors, thereby entering the cell and initiating the infection process. At the same time, the 1 / 2 volume method is also closer to the physiological infection state. During the natural infection process in vivo, the virus usually gradually enters the cell and spreads the infection. The 1 / 2 volume method simulates an infection mode in vitro that is closer to the physiological state by using a relatively large amount of virus;

[0032] (3) The present invention uses the limiting dilution method to screen monoclonal immortalized cell lines, and uses a cell filter to filter the digested cell suspension to remove cell clumps and obtain a more uniform single cell suspension, thereby improving the accuracy of monoclonal cell screening, ensuring the genetic uniformity of the immortalized cell line and eliminating the influence of heterogeneity of the cell population, thereby obtaining an immortalized yak intramuscular preadipocyte cell line with good growth status, stable expression of the SV40T gene, stable passage and high differentiation efficiency. The monoclonal cell line is derived from the amplification of a single cell, and the integration position and copy number of the target gene of all cells are basically the same, which makes them highly consistent in gene expression level and pattern, eliminating the influence of other irrelevant cells, thereby ensuring the accuracy and repeatability of subsequent experimental results;

[0033] (4) The present invention further verified the proliferation ability and adipogenic differentiation ability of immortalized yak intramuscular precursor cells by using experiments such as Oil Red O staining, Bodipy staining, EdU staining, CCK-8, and chromosome karyotype analysis, which fully proved that the immortalized yak intramuscular precursor adipocytes were successfully constructed in this study;

[0034] (5) The chromosome numbers of the immortalized yak intramuscular precursor cells obtained by the method of the present invention at the 30th generation are mostly normal, without any abnormalities. Moreover, the immortalized yak intramuscular precursor fat cells obtained by the present invention still have the ability to differentiate into mature adipocytes normally at the 30th generation, and the differentiation ability is stronger than that of the primary cells;

[0035] (6) The immortalized yak intramuscular pre-fat cell line constructed by the present invention can provide an in vitro cell model for the research on the quality of yak beef: the intramuscular fat content is closely related to the tenderness and juiciness of the meat. By studying intramuscular fat cells, it is helpful to have a deeper understanding of the molecular mechanism of yak intramuscular fat deposition, thereby providing theoretical support for improving the edible quality of yak beef; secondly, the study of yak intramuscular fat cell line can help determine which fatty acids and metabolites play a key role in the unique flavor of yak beef, thus providing a basis for the development of yak beef products with distinctive flavor; the intramuscular fat cell line can also be used in feed research and development, in the process of cell culture By adding various feed ingredients and additives, their effects on the growth, differentiation and metabolism of yak fat cells can be tested, thereby providing a scientific basis for the development of efficient and high-quality feed suitable for yaks, improving feed utilization and reducing breeding costs; finally, the yak intramuscular precursor adipocyte line can also be used to screen and cultivate excellent breeds, understand the formation and development mechanism of yak intramuscular fat cells, and the expression and regulation of related genes, which is of great significance for the genetic breeding of yaks. For example, by studying and screening genes related to intramuscular fat cells, genetic markers related to high intramuscular fat content can be found to assist breeding work. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 These are the results of screening the MOI value of the lentivirus in Experimental Example 2 of the present invention; A: the expression of the control lentivirus GFP protein after the cells were infected with the MOI value; B: the expression of the control lentivirus GFP protein at different time points after infection.

[0037] Figure 2 This is the identification result of the immortalized cells of generation P10 in Experimental Example 2 of the present invention.

[0038] Figure 3 This is the identification result of the immortalized cells of generation P15 in Experimental Example 2 of the present invention.

[0039] Figure 4 This is the identification result of the immortalized cells at generation P20 in Experimental Example 2 of the present invention.

[0040] Figure 5 This is the identification result of the immortalized cells at generation P30 in Experimental Example 2 of the present invention.

[0041] Figure 6 This is the chromosome karyotype analysis of the immortalized cells at generation P30 in Experimental Example 2 of the present invention.

[0042] Note: Figures 2 to 5 The scale bars are all 200 μm. A is the apparent morphology of immortalized cells (SV40T+) and primary cells (SV40T-); B is the expression of SV40T gene in immortalized cells and primary cells; C is the comparison of the number of EdU-positive cells in immortalized cells and primary cells; D is the absorbance value at 450 nm after CCK-8 incubation of immortalized cells and primary cells for 1 hour; E is Bodipy staining of immortalized cells before and after differentiation induction; F is Oil Red O staining of immortalized cells before and after differentiation induction; in E and F, Ⅰ is the immortalized cells after differentiation induction, Ⅱ is the immortalized cells before differentiation induction, and Ⅲ is the primary cells after differentiation induction; Figure 6 This is the chromosome karyotype of immortalized cells (100×). DETAILED DESCRIPTION

[0043] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] Note: The experimental methods in the following experimental examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following experimental examples, unless otherwise specified, can all be obtained from commercial channels.

[0045] In the present invention, all features defined in the form of numerical ranges or percentage ranges, such as values, quantities, contents and concentrations, are only for brevity and convenience. Accordingly, the description of numerical ranges or percentage ranges should be considered to have included and specifically disclosed all possible secondary ranges and individual values ​​within the range (including integers and fractions).

[0046] The features mentioned in the present invention can be combined arbitrarily, as long as there is no contradiction in the combination of these features, all possible combinations should be considered as the scope of this specification. Each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.

[0047] The culture medium used in the experimental examples of the present invention is as follows:

[0048] (1) Growth medium: 1% double antibody (penicillin-streptomycin solution) + 10% fetal bovine serum + 89% DMEM / F12 basal medium;

[0049] (2) Oleic acid induction medium: growth medium + 100 μM oleic acid.

[0050] Experimental Example 1 Isolation and culture of primary intramuscular preadipocytes from adult yaks

[0051] The longissimus dorsi muscle tissue between the 12th and 13th ribs of fattening yaks around 4 years old was repeatedly rinsed with PBS buffer containing 3% streptomycin-penicillin double antibody, and then immediately placed on ice and brought back to the laboratory; the collected tissue samples were washed with PBS buffer containing 3% streptomycin-penicillin double antibody, and the visible fascia and blood vessels were removed with sterile scissors in a PBS moist environment, and the remaining tissue was cut into 1mm 3 Minced meat of different sizes; add digestive solution into the chopped tissue at a volume ratio of 1:2, which is 0.1% collagenase type I and 0.1% collagenase type II at a volume ratio of 2:1. Digest in a 37°C water bath for 4 hours, shaking every 5 minutes.

[0052] After digestion, filter through 100 μm, 70 μm and 40 μm filter sieves in sequence, take the filtrate and centrifuge at 2000 r / min for 5 min, discard the supernatant, add 2 mL of red blood cell lysis buffer to lyse at room temperature for 5 min, and centrifuge at 2000 r / min for 5 min; resuspend the precipitate in serum-free DMEM / F12 basal medium, and centrifuge at 1800 r / min and 1500 r / min for 5 min in sequence, add growth medium to resuspend the precipitate and transfer to a culture dish, culture in a 37°C constant temperature incubator with 5% CO2, replace fresh growth medium after adhesion for 1.5 h, and then replace medium every 2 days until culture and cryopreservation, to obtain yak primary intramuscular preadipocytes, hereinafter referred to as primary cells.

[0053] Experimental Example 2: Infection of yak intramuscular preadipocytes with SV40T lentivirus and screening of cell lines

[0054] Since the general transfection efficiency of SV40T lentivirus is low, the existing SV40T lentivirus transfection process generally requires the aid of the transfection aid Polybrene. The inventors of the present invention have found in their research that the cell mortality rate is high after adding Polybrene to yak intramuscular preadipocytes infected with SV40T lentivirus, and judging from the expression of GFP fluorescent protein, for yak intramuscular preadipocytes, the use of Polybrene or not has little effect on the transfection efficiency of SV40T lentivirus. Therefore, in the present invention, Polybrene is not added when transfecting SV40T lentivirus, thereby improving the survival rate of immortalized cells.

[0055] 1. Primary cell screening

[0056] Due to the individual differences and heterogeneity between primary cells, there are great differences in the growth status of different batches of cells; at the same time, yak primary cells are prone to aging, which will seriously affect the transfection efficiency of SV40T lentivirus; in order to improve the infection efficiency of SV40T lentivirus, different batches of primary yak intramuscular preadipocytes were resuscitated in the pre-experimental stage and cultured in an incubator for 48 hours. During this process, the morphology and growth status of cells from different batches were compared and observed, and the primary cells with the best growth status were selected for subsequent SV40T lentivirus infection.

[0057] 2. Exploration of MOI value gradient of SV40T lentivirus

[0058] The SV40T lentivirus purchased from Shanghai Hanheng Biotech Co., Ltd. was used. The lentivirus three-plasmid system includes three key plasmids: psPAX2, pMD2.G and pHBLV. The target gene SV40T is located on the pHBLV plasmid. In addition, a control lentivirus with a GFP tag was used. The packaging system and infection ability of the control lentivirus with a GFP tag are exactly the same as those of the SV40T lentivirus. The only difference is that the vector plasmid of the control lentivirus carries the GFP gene, while the vector plasmid of the SV40T lentivirus carries the SV40T gene. The specific experimental process is as follows:

[0059] After the screening, the primary cells with good growth status were digested and counted, and then plated in 96-well plates (2×10 4 Cells were cultured in a 37°C, 5% CO2 incubator overnight. When the cell confluence reached 50%, the control lentivirus (lentivirus containing a GFP tag) and SV40T lentivirus (purchased from Shanghai Hanheng Biotechnology Co., Ltd.) were infected using the 1 / 2 small volume infection method. The MOI value was explored using the control lentivirus to more intuitively observe the infection efficiency of the lentivirus and to prevent the green fluorescence produced by the GFP tag from affecting subsequent fluorescent staining experiments. Before infection, the original culture medium of the cells was aspirated, and 50 μL of growth medium was added. The MOI values ​​were 5, 10, 20, and 30. The control lentivirus was added to the cells. After 4 hours of infection, 50 μL of growth medium was added. 24 hours after infection, the culture medium containing the virus was aspirated, and replaced with fresh growth medium. The culture was continued at 37°C. The MOI value with the least damage to the cells and the highest infection efficiency was selected, and then the SV40T lentivirus was infected with this MOI value through the above infection method to obtain immortalized cells after yak primary intramuscular preadipocytes were infected with SV40T, hereinafter referred to as immortalized cells.

[0060] like Figure 1 As shown in the figure, after 48 hours of infection of yak intramuscular preadipocytes with control lentivirus at different MOI values, the GFP expression efficiency can be preliminarily observed using a fluorescence microscope, thereby indirectly obtaining the infection efficiency of viruses at different MOI values, see Figure 1 A. As the MOI value increases, the fluorescence brightness of GFP gradually increases, so 30 MOI was selected for subsequent experiments; see Figure 1 B. At 12h, 24h, and 36h after infection with 30 MOI, the fluorescence brightness of GFP gradually increased, indicating that the lentivirus used in the present invention can be stably expressed in yak intramuscular preadipocytes.

[0061] 3. Determine the optimal concentration of puromycin (purchased from Thermo Fisher)

[0062] Yak primary intramuscular preadipocytes (primary cells) and yak intramuscular preadipocytes infected with SV40T were plated in 48-well plates (7 × 10 cells per well). 4 cells) and cultured in a 37°C, 5% CO2 incubator. When the cell fusion rate was about 60%, the culture medium was replaced with a growth medium containing different concentrations of puromycin (1.0, 2.0, 3.0, 4.0, 5.0, 6.0 μg / mL) for 48 h. The lowest concentration that could kill more than 90% of the primary cells while keeping the immortalized cells in good growth state was selected for subsequent experiments.

[0063] The results showed that after adding different concentrations of puromycin to primary cells and SV40T-infected cells, when the puromycin concentration was 1 and 2 μg / mL, the mortality rate of primary cells was 40-80%. After adding a final concentration of 3 μg / mL and above, almost all primary cells died, while immortalized cells could grow normally, indicating that the optimal concentration of puromycin is 3 μg / mL.

[0064] 4. Screening and expansion of monoclonal stable strains

[0065] The cells infected with SV40T virus and screened by the above-mentioned puromycin were digested with trypsin, the cell precipitate after centrifugation was blown and mixed, and then the digested cell suspension was filtered using a 40μm cell filter to remove cell clumps and obtain a more uniform single cell suspension, thereby improving the accuracy of monoclonal cell screening; then the limiting dilution method was used to dilute and plate to a 96-well plate to ensure that each well contained a single cell, 24 cell lines with good growth status were selected for cell amplification, and puromycin was continued to be applied for maintenance screening and culture, when the cell confluence reached 90%, the cells were digested to a 24-well plate and puromycin was continued to be applied for maintenance screening and culture, and according to this step, the cells were digested to a 12-well plate, a 6-well plate, a T25 culture flask, and a 90mm cell culture dish in turn for expansion culture, thereby obtaining a yak intramuscular preadipocyte immortalized cell line.

[0066] 5. Identification of immortalized cell line of yak intramuscular preadipocytes

[0067] When the monoclonal cells obtained by limiting dilution were stably subcultured to P10, P15, P20, and P30, their SV40T expression (RT-qPCR), proliferation ability (CCK-8, EdU), differentiation ability (Oil Red O staining, Bodipy staining) and chromosome karyotype were identified.

[0068] 1) RT-qPCR:

[0069] RNA from monoclonal cells and primary cells was extracted using the Trizol method, and RNA was reverse transcribed into cDNA using the Takara kit (Takara, RR047A) according to the instructions. Premix EX TaqTM II kit (Takara, RR820A) was used to detect the expression level of SV40T in monoclonal cells and primary cells.

[0070] 2) CCK-8:

[0071] Monoclonal cells and primary cells were inoculated into 96-well plates and cultured with fresh growth medium. CCK-8 assay was performed 24h, 48h, 72h and 96h after inoculation: 10μL CCK-8 reagent was added to each well, incubated in a 37℃ incubator for 1h, and the OD value was measured at a wavelength of 480nm.

[0072] 3) EdU:

[0073] Monoclonal cells and primary cells were inoculated into 12-well plates. When the cell density reached 80%, the cells were stained with EdU according to the instructions of the EdU kit (Biyuntian Biotechnology, C0075S), and photographed using a fluorescence microscope.

[0074] 4) Oil Red O staining:

[0075] Monoclonal cells were inoculated into 12-well plates. When the cell fusion rate reached 70%, the growth medium was replaced with oleic acid-induced differentiation medium. After inducing differentiation for 48 hours, the cells were fixed with 4% paraformaldehyde for 30 minutes, then stained with Oil Red O working solution for 30 minutes, washed with PBS, and photographed under a fluorescent inverted microscope.

[0076] 5) Bodipy staining:

[0077] Monoclonal cells were inoculated into 12-well plates. When the cell fusion rate reached 70%, the growth medium was replaced with oleic acid-induced differentiation medium. After inducing differentiation for 48 hours, the cells were fixed with 4% paraformaldehyde for 30 minutes, then stained with Bodipy working solution for 30 minutes, washed with PBS, and then stained with DAPI working solution for 10 minutes. After washing with PBS, the cells were placed under a fluorescent inverted microscope for photography.

[0078] 6) Chromosome karyotype analysis:

[0079] The monoclonal cells were inoculated into 6-well plates. When the cells reached the logarithmic growth phase, they were treated with 0.3 μg / mL colchicine for 4 h. The treated cells were digested with trypsin and then subjected to hypotonic treatment with KCl solution. The cells were then fixed with methanol-acetic acid solution (methanol: acetic acid = 3:1). After fixation, the cells were dropped onto slides and finally stained with Giemsa stain for 20 min. After being washed with running water, the cells were observed under a microscope and photographed.

[0080] The results are as follows Figures 2 to 5 As shown (the scale bars in the figures are all 200 μm), A is the apparent morphology of immortalized cells (SV40T+) and primary cells (SV40T-); B is the expression of SV40T gene in immortalized cells and primary cells; C is the comparison of the number of EdU-positive cells in immortalized cells and primary cells; D is the absorbance value at 450nm after CCK-8 incubation of immortalized cells and primary cells for 1 hour; E is Bodipy staining of immortalized cells before and after differentiation induction; F is Oil Red O staining of immortalized cells before and after differentiation induction; in E and F, Ⅰ is the immortalized cells after differentiation induction, Ⅱ is the immortalized cells before differentiation induction, and Ⅲ is the primary cells after differentiation induction; Figures 2 to 5 The identification results of immortalized cells at generations P10, P15, P20, and P30 are shown in order; Figure 6 This is the chromosome karyotype of immortalized cells (100×).

[0081] See also Figures 2 to 5 A in the figure shows a comparison of the morphology of immortalized cells and primary cells after infection with SV40T. It can be seen from the figure that the monoclonal immortalized cells are relatively consistent with the primary cells, both of which grow by adhering to the wall, and the cells are multipolar or bipolar fibroblast-like.

[0082] See also Figures 2 to 5 In B, RT-qPCR results showed that the SV40T gene was stably and highly expressed in immortalized cells but was hardly expressed in primary cells.

[0083] See also Figures 2 to 5 In C and D, the proliferation ability assay experiment shows that the proliferation ability of monoclonal immortalized cells is stronger than that of primary cells.

[0084] See also Figures 2 to 5 In Figures E and F, the differentiation ability test experiment shows that the monoclonal immortalized cells gradually change from fibrous to round after oleic acid-induced differentiation, and have obvious lipid droplet accumulation. The yak intramuscular precursor fat immortalized cells obtained by the present invention still have the ability to normally differentiate into mature adipocytes when they are passaged to the 30th generation, and the differentiation ability is stronger than that of the primary cells.

[0085] See also Figure 6The karyotype analysis of the 30th generation immortalized preadipocytes showed that the number of chromosomes in most cells was 60, and their karyotype was consistent with the yak chromosome map, indicating that the chromosomes of the cultured cells had not undergone obvious mutations.

[0086] The above results show that the present invention successfully constructed a monoclonal cell line of yak intramuscular preadipocytes using SV40T lentivirus, and the cell line has strong proliferation ability and the ability to differentiate into mature adipocytes.

[0087] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. A method for preparing a yak intramuscular pre-fat immortalized cell line, characterized in that: The method includes: (1) Obtaining primary intramuscular preadipocytes from yaks The longissimus dorsi muscle tissue sample of the yak was rinsed with PBS buffer containing 3% streptomycin-penicillin double antibody, and the visible fascia and blood vessels were removed with sterile scissors in a PBS moist environment, and the remaining tissue was cut into pieces; digestive fluid was added to the cut tissue for digestion; After digestion, filter through 100μm, 70μm and 40μm filter sieves in turn, take the filtrate and centrifuge at 2000r / min, pour out the supernatant, add red blood cell lysis solution to lyse at room temperature, and centrifuge at 2000r / min; resuspend the precipitate in serum-free DMEM / F12 basal medium, and centrifuge at 1800r / min and 1500r / min in turn, add complete medium to resuspend the precipitate and transfer it to a culture dish, culture in a 37℃ constant temperature incubator with 5% CO2, replace with fresh growth medium after adherence, the growth medium is 1% double antibody + 10% fetal bovine serum + 89% DMEM / F12 basal medium, and then replace the medium every 2 days, culture until passage and cryopreservation, and obtain yak primary intramuscular preadipocytes, referred to as primary cells; (2) Infection of yak intramuscular preadipocytes with SV40T lentivirus and screening of cell lines The obtained primary cells with good growth status were cultured overnight in a 37°C, 5% CO2 incubator. When the cell confluence rate reached 50%, the control lentivirus infection was first performed using the 1 / 2 small volume infection method. The packaging system and infection ability of the control lentivirus were completely consistent with those of the SV40T lentivirus. The difference was that the vector plasmid of the control lentivirus carried the GFP gene, while the vector plasmid of the SV40T lentivirus carried the SV40T gene. After the optimal MOI value was obtained by infection with the control lentivirus, the SV40T lentivirus infection was performed: before infection, the original culture medium of the cells was aspirated, half of the normal amount of the culture medium was added, and the virus stock solution was added to the cells according to the optimal MOI value. After 4 hours of infection, the culture medium was supplemented to the normal amount, and the infection was continued. After 24 hours of infection, the culture medium containing the virus was aspirated, replaced with fresh growth culture medium, and continued to be cultured at 37°C to obtain immortalized cells after yak primary intramuscular preadipocytes were infected with SV40T, referred to as immortalized cells; The cells infected with the SV40T lentivirus are cultured until the cell fusion rate is 55-65%, and the culture medium is replaced with a growth medium containing 3 μg / mL puromycin to kill the primary cells in the infected cells, and the immortalized cells can grow normally, so as to screen the immortalized cells; The cells infected with SV40T virus and screened by the above-mentioned puromycin were digested with trypsin, the cell sediment after centrifugation was blown and mixed, and then the digested cell suspension was filtered using a 40μm cell filter to remove cell clumps and obtain a more uniform single cell suspension; then, the cells were diluted by limiting dilution method and plated on a 96-well plate to ensure that each well contained a single cell, 24 cell lines with good growth status were selected for cell amplification, and puromycin was continued to be applied for maintenance screening and culture, when the cell confluence reached 90%, the cells were digested into a 24-well plate and puromycin was continued to be applied for maintenance screening and culture, and according to this step, the cells were digested into a 12-well plate, a 6-well plate, a T25 culture flask, and a 90mm cell culture dish for expansion culture, thereby obtaining a yak intramuscular preadipocyte immortalized cell line.

2. The preparation method according to claim 1, characterized in that: The longissimus dorsi muscle tissue sample of the yak is selected from the longissimus dorsi muscle tissue between the 12th and 13th ribs of a fattening yak about 4 years old.

3. The preparation method according to claim 1, characterized in that: The volume ratio of the tissue to the digestion fluid is 1:

2.

4. The preparation method according to claim 1, characterized in that: The digestion solution is 0.1% type I collagenase and 0.1% type II collagenase in a volume ratio of 2:

1.

5. The preparation method according to claim 1, characterized in that: The digestion was carried out in a 37°C water bath for 4 h, with shaking every 5 min.

6. The preparation method according to claim 1, characterized in that: After the digestion, the cells were filtered through 100 μm, 70 μm and 40 μm sieves in sequence, the filtrate was centrifuged at 2000 r / min for 5 min, the supernatant was discarded, red blood cell lysis solution was added to lyse at room temperature for 5 min, and the cells were centrifuged at 2000 r / min for 5 min; the precipitate was resuspended in serum-free DMEM / F12 basal culture medium, and gradient centrifuged at 1800 r / min and 1500 r / min for 5 min in sequence, the precipitate was added with growth medium to be resuspended and transferred to a culture dish, cultured in a 37°C constant temperature incubator with 5% CO2, and fresh growth medium was replaced after adhesion for 1.5 hours, and the culture medium was replaced every 2 days until passage and cryopreservation to obtain yak primary intramuscular preadipocytes.

7. The preparation method according to claim 1, characterized in that: Use the 1 / 2 small volume infection method to infect the cells with SV40T lentivirus: before infection, remove the original culture medium of the cells, add 50 μL of culture medium, add the virus stock solution to the cells at an MOI value of 30, add 50 μL of culture medium 4 hours after infection, and 24 hours after infection, remove the culture medium containing the virus, replace it with fresh complete culture medium, and continue to culture at 37°C.

8. The preparation method according to claim 1, characterized in that: The cells infected with the SV40T lentivirus were cultured until the cell fusion rate was 55-65%, and the culture medium was replaced with a growth medium containing 3 μg / mL puromycin for 48 hours.

9. The preparation method according to claim 1, characterized in that: The screened cells were diluted by limiting dilution method and plated into 96-well plates to ensure that each well contained a single cell.

10. The yak intramuscular preadipose immortalized cell line obtained by the preparation method according to any one of claims 1 to 9.

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