Two new MAR segments (M14 and M17) were cloned from tobacco genome. Both of the sequences contained several typical consensus sequences of MARs, which were different from the original MAR sequence, such as 90%AT-box, A-box, T-box, the base unpairing regions (BUR), autonomously replicating sequences (ARS), the consensus sequence for topoisomerase II, MAR recognition sequence (MRS), origin of replication (ORI), curved DNA motifs and ATATTT et al. To investigate the effects of these two sequences on gene expression in transgenic plants, 3 plant expression vectors were constructed with uidA gene coding beta-glucuronidase (GUS) which were flanked on one side and on both sides by the MARs we obtained. These plant expression vectors with one or two MARs were transformed into tobaccos via Agrobacterium-mediated transformation method, with the plant expression vector pCAMBIA2301 without MAR and wild type tobacco as controls. GUS histochemical staining results showed that the uidA gene expressed stably in transgenic tobaccos. Quantitative detection of GUS activity showed that the MARs could increase GUS expression levels in vivo in contrast to the controls, wherever they were flanked on one side or both sides of uidA gene. The vector ligated with MARs in the same direction on both sides of uidA could increase the GUS expression level much better than both vectors which just ligated with single MARs on one side. The former one increased the average GUS activity for 3.14 folds, but 1.56 and 2.43 folds for the latter two vectors with single MARs respectively contrasting to the pCAMBIA2301 control. But the expression differences among individual transformants were still obvious. Therefore, it was concluded that the DNA sequences we obtained in this experiment were two novel MARs and could enhance gene expression in vivo. In the meanwhile, although the numbers of the MARs typical motifs in M14 were more than in M17, especially the 90% AT box which had been considered to be the highest correlative motif with binding strength in vitro, the enhancement of gene expression was lower yet, which implied no correlation between improvement of gene expression and binding strength between MARs and nuclear matrix in vitro.
Cloning, Molecular ; DNA, Plant ; genetics ; Escherichia coli ; genetics ; metabolism ; Gene Expression Regulation, Plant ; Genetic Vectors ; Matrix Attachment Regions ; Nuclear Matrix ; metabolism ; Plants, Genetically Modified ; genetics ; Sequence Analysis, DNA ; Tobacco ; genetics

OBJECTIVETo construct an oncolytic adenovirus with the DD3 promoter regulation, expressing small hairpin RNA and targeting the SATB1 gene (SATBI-shRNA), and to evaluate its potential for inhibiting the growth of human prostatic carcinoma cells (LNCaP) in vitro.

METHODSSATB1-shRNA expression cassettes containing the H1 promoter were produced by PCR from pSilencer3. 1-SATB1 and inserted into the pZD55 vector, and the recombinant plasmid pZD55-SATB1-shRNA was constructed, pZD55SATB1-shRNA and pZXC2-DD3-E1A were digested with EcoRV and Xba I , and the obtained expression cassettes linked each other to construct the recombinant plasmid pDD3-ZD55-SATB1, which was cotransfected with the pBHGE3 packaging plasmids mixture into 293 cells by Effectence. The recombined adenoviruses DD3-ZD55-SATB1 were identified by PCR. Viruses were propagated on HEK293 cells and purified by standard techniques, and the functional PFU titers determined by plaque assay on 293 cells. The antitumor potential of DD3-ZD55-SATB1 to LNCaP was evaluated by the crystal violet dye method. The expression level of the E1A gene was detected by Western blot, and that of the SATB1 gene in the adenovirus-infected LNCaP cells by both Western blot and RT-PCR.

RESULTSAn oncolytic adenovirus expressing SATB1-shRNA with the DD3 promoter regulation, DD3-ZD55-SATB1, was constructed successfully, whose functional PFU titer was 1.2 x 10(10) PFU/ml. DD3-ZD55-SATB1 showed an obvious cytopathic effect and a selective expression of E1A in the adenovirus-infected LNCaP cells; it exhibited a high LNCaP-targetability and specific SATB1-silencing effect.

CONCLUSIONThe successful construction of the oncolytic adenovirus DD3-ZD55-SATB1 offers a new tool for researches on the gene therapy for human prostate cancer.

Adenoviridae ; genetics ; Carcinoma ; therapy ; Cell Line, Tumor ; Genetic Vectors ; Humans ; Male ; Matrix Attachment Region Binding Proteins ; genetics ; Oncolytic Virotherapy ; methods ; Oncolytic Viruses ; genetics ; Promoter Regions, Genetic ; Prostatic Neoplasms ; therapy ; RNA Interference ; RNA, Small Interfering ; genetics