Establishing method of immortal AM cell line

A technique for establishing a method and a cell line, applied in biochemical equipment and methods, botanical equipment and methods, cells modified by introducing foreign genetic material, etc., can solve the problem of inability to ensure the stable expression of the target gene and the inability to guarantee the malignancy of AM-1 Transformation and other issues, to achieve stable and reliable results of cell lines

Inactive Publication Date: 2010-08-11
陶谦
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Problems solved by technology

[0004] Harada [25] and this research group [26] The liposome method was used to transfect HPV16E6 / E7 and SV40 large T antigen (SV40) respectively to try to establish immortalized AM cell lines. Each of the two experiments has certain shortcomings. For example, the introduction of the HPV16 gene fragment by the scholar Harada is relatively large, which cannot guarantee whether AM-1 has undergone malignant transformation, and the biological behavior of inoculating tumors in vivo has not been reported; The plastid transfection method cannot guarantee the stable expression of the target gene; in addition, both HPV6 and SV40 are DNA viruses, which do not participate in the occurrence and development of AM, but the above research provides experimental basis for the study of AM cell immortalization. possibility and experience

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  • Establishing method of immortal AM cell line
  • Establishing method of immortal AM cell line
  • Establishing method of immortal AM cell line

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Experimental program
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Embodiment Construction

[0024] 1. Construction of pLXSNneo-hTERT retroviral vector

[0025] 1. Main materials and reagents

[0026] 1.1 Plasmid pCIneo-hTERT was donated by Professor Weinberg RA of Massachusetts Institute of Technology. It contains the ampicillin (AMP) resistance marker gene, and the length of the hTERT gene fragment is about 3kb.

[0027] 1.2pLXSNneo retroviral vector is preserved by our research group. The total length of the vector is 5.9kb, and the screening marker is neomycin derivative G418.

[0028] 1.3 Escherichia coli DH5a-α, PA317 cell lines and NIH3T3 cell lines were stored in -80°C refrigerator by our research group.

[0029] 1.4 Main reagents:

[0030]

[0031] 1.5 Experimental consumables: 6-well cell culture plate, 25cm 2 Culture bottle, LB culture plate, 15ml centrifuge tube, cell cryopreservation tube, American Corning Company; 0.45um microporous membrane, hemocytometer, 1.0ml / 1.5ml Eppendorf (EP) tube, 0.5ml flat cap PCR tube, 0.5 ul-10ul, 200ul, 1000ul steril...

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Abstract

The invention provides an establishing method of an immortal AM cell line, which establishes the immortal AM cell line by constructing a retrovirus carrier to infect cell activation telomerase. The establishing method comprises the following processes: constructing the pLXSNneo-hTERT retrovirus carrier, and carrying out AM cell primary culture and AM cell immune cell chemical identification to establish a stable and reliable immortal AM cell line. The cell line meets the features of epithelial cells, can ensure the stable expression of a target gene and provides a valuable in-vitro model for researching AM development and invasion features.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically relates to a method for establishing an immortalized AM cell strain by constructing a retrovirus vector and infecting cells to activate telomerase. Background technique [0002] Ameloblastoma (AM) is the most common benign odontogenic tumor of the jaw, accounting for about 60% of odontogenic tumors and 10% of all jaw tumors [1-5] . Clinically, AM mostly occurs in young adults, foreign scholars Reichart et al. [6] According to 3677 cases of AM reported in the research literature, the average age of onset of AM is 35 years old, and the male to female incidence ratio is about 1.4:1. 80% of AM occurs in the mandible, and the mandibular angle and its adjacent parts are the most common. Although AM is a benign tumor, it has locally invasive biological behavior, and the postoperative recurrence rate is high, and distant metastasis is occasionally seen [7-8] , so it is also called "b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N5/10
Inventor 陶谦
Owner 陶谦
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