ABSTRACT: To construct lentiviruses carrying dominant negative mutant of Rac1-GTPase (Rac1N17) or the constitutive active mutant of Rac1-GTPase (Rac1L61) and expressing enhanced green fluorescent protein (EGFP) bicistronically.
The lentiviral expression plasmid Plenti6/v5-Rac1N17 and Plenti6/v5-Rac1L61 were constructed and identified by restriction enzyme digestion and DNA sequence analysis. The two plasmids were packaged using the ViraPowerTM lentiviral expression system to produce replication-incompetent lentiviruses Rac1L61 and Rac1N17, which were used to infect the prefrontal cortex neurons (PFCs) from neonatal SD rats. The transfection efficiency and biological activity of different Rac1 mutants were evaluated and the morphology of the transfected PFCs was observed.
The results of DNA sequencing and restriction enzyme analysis demonstrated correct plasmid construction. The packaged lentiviral titer was 1x10(6) TU/ml. Analysis of Rac1 biological activity showed that Rac1N17 lentivirus particles infection significantly inhibited epidermal growth factor-stimulated Rac1 activity in the PFCs, while Rac1L61 lentivirus particles enhanced the Rac1 activity. The transfection efficiency of these Rac1 mutant lentivirus particles exceeded 80% in the PFCs. Morphologically, the PFCs exhibited distinct dendritic branches after infection by these lentiviruses. CONCLSION: The lentiviruses carrying Rac1 dominant negative mutant and constitutive active mutant have been successfully constructed. The lentiviral particles can efficiently infect neonatal rat PFCs and lend important support for the study of Rac1-GTPase.
Nan fang yi ke da xue xue bao = Journal of Southern Medical University 02/2010; 30(2):197-201.
Sixth International Conference on Natural Computation, ICNC 2010, Yantai, Shandong, China, 10-12 August 2010; 01/2010
ABSTRACT: The purpose of this study was to establish a SYBR Green I real-time quantitative RT-PCR method for investigating the correlation between CML28 mRNA expression levels and relapse of leukemia after allo-hematopoietic stem cell transplantation (HSCT). pcDNA3.1HisA-CML28 plasmid had been constructed as the standard template. Serial monitoring of CML28 mRNA levels by SYBR Green I real-time quantitative RT-PCR technique was performed in 14 patients, including 10 patients with CML and 3 patients with AML, 1 patient with Ph(+) ALL. The results showed that the sensitivity of the established method was at 10(-4) (0.05 ng) level, with interassay variation and intraassay variation of standard samples both below 10%. The CML28 was highly expressed in AML and CML-BP or AP. CML28 level in newly diagnosed group was (6.58 +/- 2.34) x 10(-2), in pre-conditioning regimen group was (2.19 +/- 0.32) x 10(-2), in group that 1 month after HSCT was (1.35 +/- 1.28) x 10(-2), in group that 3 months after HSCT was (4.57 +/- 6.39) x 10(-3). CML28 can be detected 3 months after HSCT in 1 patient with CML-CP and 3 patients with CML-AP or BC. 2 of them with low level (<2 x 10(-2)) survived without relapse, the other 2 case with high level (>2 x 10(-2)) relapsed within one year, 1 case died and 1 case received the second time HSCT, CML28 level decreased rapidly after HSCT, but still higher than 2 x 10(-2) and relapse has taken place. The conclusions was made that CML28 mRNA level is obviously correlated with the development of leukemia. Serial quantification of CML28 mRNA levels are necessary for HSCT recipients, and more informative than a single detection. Using of this assay to evaluate MRD in the patients received HSCT is helpful for prediction of relapse.
Zhongguo shi yan xue ye xue za zhi / Zhongguo bing li sheng li xue hui = Journal of experimental hematology / Chinese Association of Pathophysiology 11/2005; 13(5):843-7.