Bovine testis sertoli cell carcinoma cell, and application thereof in separation and culture of poxviruses
A technology of testicular Sertoli cells and Sertoli cells, applied in the direction of tumor/cancer cells, viruses, animal cells, etc., can solve the problems of high production costs, carrying other pathogens, low production efficiency, etc., to simplify the production process, reduce production costs, The effect of reducing test costs
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Embodiment 1
[0038] [Example 1] Discovery and isolation of newborn bovine testicular Sertoli cell carcinoma cells (BTSCC)
[0039] 1.1 Transfection of NBSC cells with pCI-neo-hTERT plasmid
[0040] The limited passage cell line (NBSC) of bovine testis Sertoli cells (NBSC), which can be limited to 20 passages, was isolated and purified from the cultured bovine testis primary cells, and the eukaryotic expression plasmids encoding hTERT and neo genes were transferred by lipofection method pCI-neo-hTERT was introduced into the established limited-passage cell line NBSC, and the NBSC limited-passage cell line was immortalized. The pCI-neo-hTERT expression vector map and enzyme digestion identification results are as follows figure 1 shown. Gradient concentration of G418 was used to screen, and anti-G418 positive clones were obtained: hTERT-NBSC ( figure 2 ). RNA was extracted from the screened hTERT-NBSC cells, and the expression of exogenous hTERT in transfected cells was detected by RT-PC...
Embodiment 2
[0058] [Example 2] Newborn bovine testicular Sertoli cell carcinoma cell BTSCC serum dependence test
[0059] The serum dependence of bovine testicular Sertoli cell carcinoma BTSCC cells was detected by MTT method, as follows:
[0060] (1) The 30th generation, 40th generation, 50th generation, 60th generation BTSCC and the 12th generation BTSCC cells or NBSC cells were divided into 1×10 4 cells / well were seeded in a 96-well culture plate.
[0061] (2) 24 hours after inoculation, the cell solution was replaced with DMEM / F12 culture medium of 50mL / L FBS, 100mL / L FBS, and 200mL / L FBS respectively. Make 3 replicate wells for each gradient, and set 3 blank wells at the same time.
[0062] (3) Incubate for 14 hours in an incubator at 37° C. with a condition ratio of 5% CO2 and saturated humidity.
[0063] (4) Add 10 μL of MTT (10 mg / mL) reaction solution, and continue to incubate at 37° C. for 2 to 4 hours until obvious needle-like purple crystals can be seen under the microscope...
Embodiment 3
[0067] [Example 3] Passage of BTSCC cell and its proliferation test to ORFV and GTPV
[0068] The BTSCC cells were continuously subcultured, and the time for each generation of cells to grow into a monolayer was recorded. For each passage of 5 generations, the ORFV cell virulence (lgTCID) of the same generation was inoculated. 50 ≥6.0) and ORFV cytotoxicity (lgTCID 50 ≥4.0), observe the CPE of ORFV and GTPV on the BTSCC cell line, and measure its TCID 50 .
[0069] Experiments have confirmed that when BTSCC cells are subcultured to 65 generations, the cell lines still maintain vigorous vitality. And it maintains a high proliferative ability for ORFV and GTPV viruses, the cells produce CPE more regularly, and the titer of ORFV virus (lgTCID 50 ) are above 6.20; GTPV virus titer (lgTCID 50 ) are above 4.20, the results are shown in Table 3.
[0070] Table 3 Passaging of BTSCC and its proliferation to ORFV
[0071]
[0072] In summary, the present invention successfully ...
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