ABSTRACT
Lentivirus-derived vectors are among the most promising viral vectors for gene therapy which is currently available, but their use in clinical practice is limited due to associated risk of insertional mutagenesis. Gene targeting is an ideal method for gene therapy, but it has low efficiency in comparison to viral vector methods. In this study, we are going to design and construct an integrase-minus lentiviral vector. This vector is suitable for transient expression of gene and gene targeting with viral vector. In this experimental study, three missense mutations were induced in the catalytic domain of Integrase gene in the pLP1 plasmid and resulted D64V, D116A and E152G changes in the amino acid sequence through site directed mutagenesis. The pLenti6.2-GW/EmGFP transfer vector, associated with native and mutated packaging mix, was transfected into 293T cell line. In order to titer the lentivirus stock, the viruses were harvested. Finally, the viruses transduced into COS-7 cell line to assess green fluorescent protein [GFP] gene expression by a fluorescence microscopy. Recombinant and wild lentiviruses titer was about 58×10[6] transducing units/ml in COS-7 cell line. The number of GFP-positive cells transduced with native viruses was decreased slightly during two weeks after viral transduction. In contrast, in the case of integrase-minus viruses, a dramatic decrease in the number of GFP positive cells was observed. This study was conducted to overcome the integration of lentiviral genome into a host genome. Nonintegrating lentiviral vectors can be used for transient gene expression and gene targeting if a Target gene cassette is placed in the lentivirus gene structure. This combination method decreases disadvantages of both processes, such as random integration of lentiviruses and low efficiency of gene targeting
Subject(s)
Disease Vectors , Genetic Therapy , Gene Expression , IntegrasesABSTRACT
Each year, about 50,000 new cases of cancer occur in Iran and the most common being the gastrointestinal [GI] tract [38%]. Colorectal cancers account for the 3[rd] and 4[th] most prevalent cancers in Iranian men and women, respectively. Since genetic and environmental factors lead to differences in colorectal cancer occurrence in different geographic regions and races, we designed a study to assess characteristics of this cancer, in the Guilak race of Mazandaranian people. The records of all colorectal cancer patients who were referred to in a private GI clinic or admitted in hospitals and were studied during 1999 to 2007. Data was analyzed using SPSS software, Chi- Square and T-Tests. 296 cases of colorectal cancers [CRCs] were enrolled in the study. There were 2 cases of colon lymphoma and 1 case of cervical cancer with metastasis to colon. The remaining 293 patients had colorectal Aden carcinoma, of which 152 were males and 141 were females. The mean age of patients was 52.6 +/- 15.2 years. 98 patients [33.5%] were under 45 years of age. A total of 70 cases [24%] had familial history of CRC. Moreover, 47% of patients under 45 years had positive familial history for CRC, of whom 43 patients [14.6%] were classified as having Hereditary Non- Polyposis Colorectal Cancer [HNPCC]. 125 patients had rectal, while 168 cases had colon cancers. Right- sided colon cancers were 2 times more prevalent than the left- sided involvements. In addition, right- sided colon cancers were relatively more prevalent in those with positive familial history of CRC. Importantly, about half of all patients showed advanced stages of the disease [Lymph node involvement or distal metastases] at the time of diagnosis. Regarding the younger age of involvement, the significant positive familial history, the more prevalent proximal colon cancers and advanced stages of the disease at the time of diagnosis, we recommend to design screening programs for earlier detection of CRCS in this geographic region