Expression of exogenous proteins and short hairpin RNAs in human primary thyrocytes Susana B. Bravo a, * ,1 , Maria E.R. Garcia-Rendueles a,1 , Sihara Perez-Romero a , Jose Cameselle-Teijeiro b , Joana S. Rodrigues a , Francisco Barreiro c , Clara V. Alvarez a, * a Department of Physiology, School of Medicine and Clinical Hospital, University of Santiago de Compostela (USC), IDIS, Santiago de Compostela, Spain b Department of Pathology, School of Medicine and Clinical Hospital, University of Santiago de Compostela (USC), IDIS, Santiago de Compostela, Spain c Department of Surgery, School of Medicine and Clinical Hospital, University of Santiago de Compostela (USC), IDIS, Santiago de Compostela, Spain article info Article history: Received 16 November 2009 Received in revised form 29 December 2009 Accepted 26 January 2010 Available online 1 February 2010 Keywords: Thyroid Human primary culture Transfection Nucleofection shRNA Lentivirus Thyroid cancer abstract Recently, it has been shown that commercial human thyroid lines were in fact derived from colon, mam- mary 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 pri- mary thyroid cultures. We compared lipid-based techniques with three electroporation systems (Electro- porator 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 stan- dardized, through lentiviral infection, the short hairpin RNA downregulation of protein expression gen- erating human thyrocytes with low levels of p27KIP1 as a model system. Ó 2010 Elsevier Inc. All rights reserved. The prevalence of proliferative disease of the thyroid is high in the human population. Many patients present with a papillary thy- roid carcinoma (PTC), 2 the most frequently presented of the two types of differentiated thyroid carcinoma (DTC), with the other type of DTC being follicular thyroid carcinoma (FTC). Although surgery alone or in combination with radioiodine is well established as the best first line of therapy, the large number of cases involved invari- ably leads to a range of requirements in terms of follow-up treat- ment due to either noncomplete remission or recurrence or metastasis. Moreover, it has been demonstrated that, contrary to most types of carcinoma, the incidence of PTC has been increasing during recent years, reaching 14.2 new cases per 100,000 in the gen- eral US female population in 2006 [1,2]. Because PTC is a well-man- aged, slow-growing cancer with a low mortality rate but a high rate of recurrence, its prevalence is approximately three to five times higher than its calculated incidence [3,4]. To design new therapies, it is essential to have a reliable cell culture method in which primary cells from patients can be stud- ied in terms of both their inherent mechanisms and their responses at the molecular level to potential new therapeutic agents at a range of concentrations. Recently, it has been shown that some commercially available human thyroid lines that are widely used in research are in fact derived from either colon, mammary carci- noma, or melanoma cells [5]. Other studies have demonstrated the absence of a common pattern of gene expression between available thyroid cancer cell lines and tumor cells from patients, especially if the cell lines have been either overly passaged or care- lessly cultured [6]. This problem is not exclusive to thyroid cell cul- ture, and a call for action has been proposed [7,8]. We have established a primary human cell culture bank from thyroid tissue obtained from surgery pieces, the Bank of Thyroid Tumors in Culture (BANTTIC) [9–11]. However, the utility of pri- 0003-2697/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2010.01.034 * Corresponding authors. Fax: +34 981 574145. E-mail addresses: susanabelen.bravo@usc.es (S.B. Bravo), clara.alvarez@usc.es (C.V. Alvarez). 1 These authors contributed equally to the study. 2 Abbreviations used: PTC, papillary thyroid carcinoma; DTC, differentiated thyroid carcinoma; FTC, follicular thyroid carcinoma; siRNA, small interfering RNA; NCS, newborn calf serum; TSH, thyroid-stimulating hormone; PBS, phosphate-buffered saline; RT–PCR, reverse transcription–polymerase chain reaction; M-MLV, Moloney murine leukemia virus; dNTP, deoxynucleoside triphosphate; cDNA, complementary DNA; TG, thyroglobulin; NIS, Na + /I – symporter; TSHR, TSH receptor; DAPI, 4’,6- diamidino-2-phenyindole; GFP, green fluorescent protein; NF-jB, nuclear factor– kappa B; tk, minimal thymidine kinase; TGFb, transforming growth factor beta; mw24, 24-multiwell; GFP+, transfected with GFP; EGTA, ethyleneglycoltetraacetic acid; DTT, dithiothreitol; ONPG, ortho-nitrophenolgalactopyranoside; MOI, multiplic- ity of infection; shRNA, short hairpin RNA; TBS, Tris-buffered saline; HRP, horseradish peroxidase; ECL, enhanced chemiluminescence; ER, endoplasmic reticulum; mRNA, messenger RNA. Analytical Biochemistry 400 (2010) 219–228 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio