Expression of exogenous proteins and short hairpin RNAs in human primary thyrocytes

Recently, it has been shown that commercial human thyroid lines were in fact derived from colon, mammary carcinoma, or melanoma. Others have demonstrated the absence of a common pattern of gene expression between available thyroid cancer cell lines and tumors from patients. Thus, it is important to...

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Bibliographic Details
Published in:Analytical biochemistry 2010-05, Vol.400 (2), p.219-228
Main Authors: Bravo, Susana B., Garcia-Rendueles, Maria E.R., Perez-Romero, Sihara, Cameselle-Teijeiro, Jose, Rodrigues, Joana S., Barreiro, Francisco, Alvarez, Clara V.
Format: Article
Language:English
Subjects:
Cells, Cultured
Gene Expression
Gene Transfer Techniques
Genetic Vectors
Human primary culture
Humans
Lentivirus
Nucleofection
Proteins - genetics
Proteins - metabolism
RNA - genetics
RNA - metabolism
shRNA
Thyroid
Thyroid cancer
Thyroid Gland - cytology
Transfection
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Summary:Recently, it has been shown that commercial human thyroid lines were in fact derived from colon, mammary carcinoma, or melanoma. Others have demonstrated the absence of a common pattern of gene expression between available thyroid cancer cell lines and tumors from patients. Thus, it is important to use several primary cells with a common pathological origin to achieve reproducible results, and it is necessary to find common methods for manipulation of protein expression in such various cultures. We have standardized a transfection method for efficient expression of exogenous proteins in human primary thyroid cultures. We compared lipid-based techniques with three electroporation systems (Electroporator PulseAgile [PA]-4000, Microporator MP-100, and Nucleofector II). Nucleofection was unquestionably the most efficient even for promoter regulation studies, and it was effective in cultures from different origins as normal thyroid, papillary carcinoma, or lymphoid node metastasis. We also standardized, through lentiviral infection, the short hairpin RNA downregulation of protein expression generating human thyrocytes with low levels of p27KIP1 as a model system.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2010.01.034