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Transposable element carrier expressing pig telomerase reverse transcriptase, building method thereof and application in building pig immortalization cell line

An immortalized cell line and transposon technology, applied in the field of biotechnology research, can solve the problems of biological safety of virus sequences, inability to effectively integrate foreign genes, and differences in immortalization efficiency.

Active Publication Date: 2014-07-16
ZHONGCHONG XINNUO BIOTECH TAIZHOU CO LTD
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Problems solved by technology

[0005] At present, the human TERT gene is commonly used for animal cell immortalization, which is a foreign gene for animal cells, and its immortalization efficiency may also be different from that of animal TERT
In addition, gene introduction methods mainly include expression plasmid transfection and recombinant virus transduction. The former contains resistance genes and requires long-term screening, while the latter cannot effectively integrate foreign genes. Although retroviruses and lentiviruses can mediate Foreign gene integration, but gene integration is not only random, but also the inserted virus sequence has biological safety hazards

Method used

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  • Transposable element carrier expressing pig telomerase reverse transcriptase, building method thereof and application in building pig immortalization cell line
  • Transposable element carrier expressing pig telomerase reverse transcriptase, building method thereof and application in building pig immortalization cell line
  • Transposable element carrier expressing pig telomerase reverse transcriptase, building method thereof and application in building pig immortalization cell line

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Embodiment Construction

[0025] Source of biological material:

[0026] 1. pcDNA3 vector: imported from Invitrogen Company of the United States and preserved in our laboratory.

[0027] 2. PK-15 cells: imported from the US ATCC (ATCC CCL-33), preserved in our laboratory.

[0028] 3. pMD18-T vector: introduced from KaTaRa Company.

[0029] 4. pT2 / HB transposon vector and pSB16 transposase vector: imported from Addgene Corporation of the United States. Literature: Cui Z, Geurts AM, Liu G, Kaufman CD, and Hackett PB. Structure-function analysis of the inverted terminal repeats of the sleeping beauty transposon. J Mol Biol. 2002, 318:1221–1235.

[0030] 5. pEGFP-N1 vector: introduced from BD Biosciences Clontech, USA. Literature: Prasher DC, Eckenrode VK, Ward WW, et al. Primary structure of the aequorea victoria green fluorescent protein. Gene, 1992, 111(2): 229-233.

[0031] 6. Escherichia coli DH5a: imported from BD Biosciences Clontech, USA, and preserved in our laboratory.

[0032] 7. Six-week-o...

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Abstract

The invention provides a building method of a transposable element carrier expressing pig telomerase reverse transcriptase, and application in building a pig immortalization cell line, and belongs to the field of biotechnology research. The building method includes the steps of cloning of a promoter of a pig protein translation elongation factor 1a and detection of transcriptional activity, building of an SB transposable element expression vector and exogenous gene integration, pig telomerase reverse transcriptase cDNA cloning, activity detection and building of the pig fibroblast immortalization cell line. Compared with other methods, the pig immortalization cell line built through the transposable element carrier comprises non-transformed normal cells, does not contain resistance genes, virogenes or oncogenes, and is suitable for cell physiological function researching, pig virus separation cultivation and vaccine production.

Description

technical field [0001] The invention relates to the field of biotechnology research, in particular to the construction of a transposon vector expressing porcine telomerase reverse transcriptase and its application in establishing porcine immortalized cell lines. Background technique [0002] Primary cells derived from normal tissues can grow and divide in vitro. However, not only are the sources limited and difficult to prepare, but they will also senescence and die after repeated passages, which limits the application of cell culture technology. Cell lines can overcome the shortcomings of primary cells, and are also an indispensable tool for the study of cell biology, virus isolation and culture, and vaccine production. However, the types of pig cell lines at home and abroad are extremely limited. [0003] Cell immortalization refers to the process in which cells cultured in vitro escape from the crisis of aging under the influence of spontaneous mutation or environmental f...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/66C12N5/10
Inventor 孙怀昌何姗张鑫宇夏晓莉
Owner ZHONGCHONG XINNUO BIOTECH TAIZHOU CO LTD
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