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Establishing method of sheep testicle cell immortalized cell line and application of method

A technology of immortalized cell line and construction method, applied in the field of construction of immortalized cell line of sheep and sheep testis cells, can solve the problems of abnormal chromosome function, change, up-regulation of telomerase level, etc., achieve strong resistance and reduce the amount of use , the effect of immortalized cell stabilization

Pending Publication Date: 2019-04-19
ANHUI AGRICULTURAL UNIVERSITY
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Problems solved by technology

Hockemeyer et al. found that immortalization is divided into two processes. First, it is caused by the mutation of telomerase, and the promoter (TERT) upstream of the telomerase gene is mutated, but the mutation level is very low, which is not enough to make cancer cells immortal. , but it can delay cell aging, and the level of telomerase will gradually increase; in the second step, the chromosomes containing short telomeres may malfunction, prompting changes in the genome, so that the cells achieve immortality

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  • Establishing method of sheep testicle cell immortalized cell line and application of method
  • Establishing method of sheep testicle cell immortalized cell line and application of method
  • Establishing method of sheep testicle cell immortalized cell line and application of method

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Embodiment Construction

[0029] Below in conjunction with accompanying drawing, the present invention is described in further detail:

[0030] The specific implementation plan is as follows:

[0031] 1) Construction of recombinant plasmid pLOV-CMV-hTERT:

[0032] According to the whole gene sequence of telomerase (GenBank: KT923469.1), a pair of PCR-specific primers for amplifying the hTERT gene was designed, and the sequence is as follows:

[0033] hTERT-Nhe I-F: 5'-CTAGCTAGCTAGATGCCGCGCGCTCCCCGCT-3',

[0034] hTERT-Not I-R: 5'-TTGCGGCCGCTCAGTCCAGGATGGTCTTGAA-3'.

[0035] The PCR system is: 25 μL of 2×PFU Mixture, 1 μL of upstream and downstream primers (10 μmol / L), 0.5 μL (100 ng) of pBabe-neo-hTERT plasmid, and ddH2O to 50 μL. PCR amplification program: pre-denaturation at 94°C for 3 min, denaturation at 94°C for 30 s, annealing at 63°C for 45 s, extension at 72°C for 4 min, 31 cycles, and post-extension at 72°C for 5 min. The PCR product was gel-cut for gel recovery. The purified product and the...

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Abstract

The invention discloses an establishing method of a sheep testicle cell immortalized cell line. The method approximately includes the following steps of firstly, establishing recombinant plasmids; secondly, preparing slow virus sample particles; thirdly, establishing and screening an hTERT gene cell line; fourthly, expressing an hTERT gene and detecting the mRNA transcriptional level of the hTERTgene; fifthly, detecting the expression of hETRT protein. By means of the establishing method, the research status of the shortage of existing cell lines can be easily improved, and the resource use and popularization are facilitated. By establishing the sheep testicle cell immortalized cell line, the use amount of experimental animals is reduced, and a more stable experimental research object isprovided.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a method for constructing an immortalized cell line of sheep testicular cells and an application thereof. Background technique [0002] Immortalization refers to the ability of cells to escape the crisis of proliferation spontaneously or affected by external factors, so as to obtain the ability to proliferate indefinitely. Spontaneous cell immortalization probability is small, 10 for rodents -5 ~10 -6 , the probability of human cells is lower, less than 10 -12 . The proliferation and culture of normal animal cells in vitro is limited. This natural limit is called the Hayflick limit. The process of cell growth and metabolism requires a complex regulatory network. Once the network is wrong, cells will cross the natural limit and form Immortalization. The process of cell division and proliferation will go through the senescent M1 phase and the lethal M2 ph...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N5/10C12N15/54C12R1/91
CPCC12N5/0683C12N9/1276C12N15/86C12Y207/07049C12N2740/15043C12N2510/04C12N2800/107
Inventor 王桂军刘光清周晓雅周祺邢雪许泽军顾香雪黄荣
Owner ANHUI AGRICULTURAL UNIVERSITY
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