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 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.
Bravo
Susana B
SB
Department of Physiology, School of Medicine and Clinical Hospital, University of Santiago de Compostela (USC), IDIS, Santiago de Compostela, Spain. susanabelen.bravo@usc.es
Garcia-Rendueles
Maria E R
ME
Perez-Romero
Sihara
S
Cameselle-Teijeiro
Jose
J
Rodrigues
Joana S
JS
Barreiro
Francisco
F
Alvarez
Clara V
CV
D002478:Cells, Cultured; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D013961:Thyroid Gland / Q000166:cytology*; D014162:Transfection
D011506:Proteins; D012313:RNA