The cells were washed twice with bHBSS solution (HBSS solution buffered with HEPES solution to achieve a pH 7.3), and then 0.5 mL bHBSS solution containing 0.1 Ci Na125I (Shanghai Hinko Co.) with 1 mol/L NaI were added into each well as reported by Weiss et al[7]. significantly enhanced by sodium butyrate, as up to 72% of SW1116 cells were infected with the CX-6258 hydrochloride hydrate virus at MOI of 400 and sodium butyrate at 0.5 mmol/L. No obvious cytotoxicity was observed under these conditions. Infection of SW1116 with Bac-NIS allowed uptake of131I in these tumor cells, which could be inhibited by sodium perchlorate. The viability of SW1116 cells infected with Bac-NIS was significantly lower than with Bac-GFP, suggesting that NIS gene-mediated131I uptake could specifically kill tumor cells. CONCLUSION: Baculovirus vector-mediated NIS gene therapy is a potential approach for treatment of colon cancer. Keywords:Colon cancer, Baculovirus, Sodium iodide symporter, Radionuclide therapy, Iodine radioisotopes == INTRODUCTION == Colon cancer is one of the most common malignant gastrointestinal tumors with a high morbidity and mortality. Currently, surgery and chemotherapy are the primary methods for treating colon cancer. However, surgical removal of the tumor is not an option for 30%-40% of these patients upon diagnosis because of metastasis; meanwhile, chemotherapy shows a relatively high incidence of severe side effects. Therefore, it is important to find alternative ways to improve the prognosis of individuals with colon cancer. The cloning of the sodium/iodide symporter (NIS) gene and subsequent studies of its properties have resulted in a new approach to targeted radiotherapy of malignant cancers. NIS is definitely a transmembrane glycoprotein located in the basal membrane of thyroid follicular cells. It can efficiently co-transport two Na+and one I-into cells[1], and this transportation of iodine is an active process against the chemical gradient. The uptake of radioactive131I can be achieved by the manifestation of the NIS protein in tumor cellsviavector-mediated gene transfer, and the radiotherapeutic damage of the tumor happens by emission of rays from131I. Recently, several researchers possess treated tumors by delivering the NIS geneviavectors based on retroviruses[2], adenoviruses[3] and lentiviruses[4], and accomplished a certain level of restorative effect. For the purpose of gene transfer, an ideal vector would have high transduction effectiveness and robust protein expression so that the uptake of radioactive131I in tumor cells will reach an effective radiotherapeutic dose. The adenovirus vector system does in fact possess high transduction effectiveness, but CX-6258 hydrochloride hydrate its high immunogenicity and cytotoxicity prevent its further applicationin vivo. The major concern concerning the retroviral or lentiviral system is the transgenes are easily integrated into the genomic DNA of sponsor cells, which may cause changes in the prospective genome manifestation and result in oncogenicityin vivo. Moreover, long-term gene manifestation is not necessary in restorative gene-mediated radiotherapy. We previously performed a pilot study using a baculovirus vector Smoc2 to deliver the NIS gene like a reporter[5]. Our results showed the large NIS gene could be inserted into the recombinant baculovirus. A follicular thyroid malignancy cell collection, FTC-133, infected with recombinant green fluorescent protein (GFP) baculovirus previously showed a high manifestation of GFP, which lasted approximately seven days[5]. Considering the above characteristics, we reasoned that a recombinant NIS gene baculovirus may be beneficial for mediating131I uptake and the killing of tumor cells. Furthermore, since baculoviruses can only infect arthropods, this vector is definitely highly safe for mammals. In fact, recombinant baculoviruses have been widely used in mammalian cells like a gene transfer vector because of these advantages[6]. However, no reports are available on baculovirus vector-mediated radiotherapy for malignancy. In this study, the recombinant sodium/iodide symporter gene baculovirus (Bac-NIS) was generated and used to infect colon cancer cells to investigate the manifestation and function of NIS in these cells and to provide an experimental basis for the NIS CX-6258 hydrochloride hydrate gene-mediated targeted radiotherapy of colorectal malignancy. == MATERIALS AND METHODS == == Building of baculovirus vectors == The baculovirus vector pFBGFPR (kindly provided by the Molecular Institute of the University or college of Hong Kong) was digested with restriction endonucleases Acc I and Age I. The digested ends of the vector were filled in with Klenow fragment, yielding a 6 kb pFB fragment which was then purified. The prospective gene was prepared by digesting the recombinant plasmid pCDNA-NIS (kindly provided by Professor Sissy Jhiang of Ohio University or college, USA) with restriction endonucleasesBamHI andXhoI and packed in with Klenow kinase to obtain the 2.3 kb NIS gene fragment. The purified NIS target gene fragment was ligated to the phosphatase-treated pFB vector using T4 bacteriophage DNA ligase. The producing plasmid was transformed into DH5 proficient bacteria (Tiangen Biotech Co., Ltd., Shanghai) and screened on ampicillin Luria Burtani (LB) tradition plates. White colored colonies were selected and screened for the correct orientation of the put target gene fragment by sequencing. The confirmed recombinant baculovirus pFBNIS plasmid DNA.