OBJECTIVETo investigate the genetic stability during the serial passages of non-replicating recombinant adenoviruses based on the novel AdEasy system.

METHODSFour non-replicating adenoviruses expressing rotavirus antigens, which were generated by the AdEasy system, were used as models and continuously propagated on 293 cells for 20 passages. Samples of the infected cells were collected at every 5 passages for the PCR analysis of the inserted rotavirus genes, replication-competent adenoviruses (RCAs) as well as the Western blot examination of the expression of the certain rotavirus genes.

RESULTSThe adenoviruses can be stably propagated on 293 cells. The existence and the stable expression of inserted rotavirus genes were able to be detected during the serial passage. The RCAs were not found within the first 10 passages, but the rvAdG2VP7(o) at passage 15 and the rvAdG2VP7(o) and rvAdG1VP7(o) at passage 20.

CONCLUSIONThe non-replicating adenoviruses showed promising genetic stability during the continuous passage on 293 cells. The RCAs normally will not be detectable within 10 continuous passages. The results indicated the potential of such recombinant adenoviruses in the research and development of the gene therapies and adenoviral-vectored vaccines.

Adenoviridae ; genetics ; isolation & purification ; physiology ; Cell Line ; DNA, Recombinant ; genetics ; Humans ; Serial Passage ; Transgenes ; Virus Replication

Transgene silencing is one of two major obstacles in both basic biomedical research for transgene and clinical practice of gene therapy. Based on the model of HT1080 cell clones, which transduced single copy of retroviral vector MGPN2, the mechanism of transgene silencing was explored in this investigation by a serial molecular techniques. In the HT1080 cell clone that absence of GFP protein synthesized, no significant aberration of epigenetic modification was detected, but the transcript size and the sequence changed that resulted in the reading frame shift. In addition to chromosomal position effect leading to transgene silencing, the transcript reading frame shift associated with retroviral vector rearrangements could induce complete silencing of transgene.
Cell Line, Tumor ; Gene Rearrangement ; Gene Silencing ; physiology ; Genetic Engineering ; Genetic Vectors ; genetics ; Humans ; Retroviridae ; genetics ; metabolism ; Transgenes ; genetics

OBJECTIVE: Melittin, a cell-penetrating peptide, improves the efficiency of many non-viral gene delivery vectors, yet its application in viral vectors has not been well studied. The non-pathogenic recombinant adeno-associated virus (rAAV) vector is an ideal in vivo gene delivery vector. However, its full potential will only be achieved after improvement of its transduction efficiency. To improve the transduction efficiency of rAAV2 vectors, we attempted to develop a melittin-based rAAV2 vector delivery strategy. METHODS: The melittin peptide was inserted into the rAAV2 capsid either in the loop VIII of all viral proteins (VPs) or at the N terminus of VP2. Various rAAV2-gfp or -fluc vectors were subjected to quantitative real-time polymerase chain reaction and Western blot assays to determine their titers and integrity of capsid proteins, respectively. Alternatively, the vectors based on wild-type capsid were pre-incubated with melittin, followed by transduction of cultured cells or tail vein administration of the mixture to C57BL/6 and BALB/c nude mice. In vivo bioluminescence imaging was performed to evaluate the transgene expression. RESULTS: rAAV2 vectors with melittin peptide inserted in the loop VIII of VPs had low transduction efficiency, probably due to dramatically reduced ability to bind to the target cells. Fusing the melittin peptide at the N-terminus of VP2 produced vectors without the VP2 subunit. Interestingly, among the commonly used rAAV vectors, pre-incubation of rAAV2 and rAAV6 vectors with melittin significantly enhanced their transduction efficiency in HEK293 and Huh7 cells in vitro. Melittin also had the ability to increase the rAAV2-mediated transgene expression in mouse liver in vivo. Mechanistically, melittin did not change the vector-receptor interaction. Moreover, cell counting kit-8 assays of cultured cells and serum transaminase levels indicated melittin had little cytotoxicity. CONCLUSION Pre-incubation with melittin, but not insertion of melittin into the rAAV2 capsid, significantly enhanced rAAV2-mediated transgene expression. Although further in vivo evaluations are required, this research not only expands the pharmacological potential of melittin, but also provides a new strategy to improve gene therapy mediated by rAAV vectors.
Mice ; Animals ; Humans ; Melitten/genetics* ; Dependovirus/genetics* ; Serogroup ; HEK293 Cells ; Mice, Nude ; Mice, Inbred C57BL ; Transgenes ; Genetic Vectors/genetics*

BACKGROUNDThe sodium-iodide symporter (NIS) protein can mediate the active radioiodine uptake. The human telomerase reverse transcriptase (hTERT) promoter is known to be selectively reactivated in majority of tumors and hence could be used for tumor targeting. We constructed a recombinant adenovirus containing the human sodium iodide symporter (hNIS) gene directed by the hTERT promoter, characterized the ability of infected cells in uptaking iodide, and explored the therapeutic efficacy of (131)I in a lung cancer cell line in vitro.

METHODSThe hTERT promoter was amplified by PCR from DNA isolated from log-phase HepG2 cells, subcloned into lineralized FL*-hNIS/pcDNA3, and then the hTERT-hNIS sequence was subcloned into the shuttle plasmid pAdTrack. The recombinant adenovirus Ad-hTERT-hNIS was constructed by AdEasy system. A positive control adenovirus Ad-CMV-hNIS and a negative control adenovirus Ad-CMV were created similarly. A549 cells were transduced with recombinant adenoviruses. (125)I uptake studies and sodium perchlorate suppression studies were used to confirm hNIS expression and function. Toxic effects of (131)I on tumor cells were studied by in vitro clonogenic assay.

RESULTSWe first successfully constructed an adenovirus mediated transgene expression system of the hNIS under the control of hTERT promoter. When infected with recombinant adenovirus constructs expressing hNIS directed by hTERT- and CMV-promoters (Ad-hTERT-hNIS and Ad-CMV-hNIS, respectively), the lung cancer cell line A549 had increased ability to uptake radioiodide up to 23- and 30-fold compared to the control parental cells, respectively. The radioiodide uptake ability of both the Ad-CMV-hNIS and Ad-hTERT-hNIS transduced cell lines were repressed 11-fold by sodium perchlorate (NaClO4). The subsequent in vitro clonogenic assay of the infected A549 cell line was further repressed to 23% (Ad-CMV-hNIS) and 30% (Ad-hTERT-hNIS) of the control group after receiving radioiodide for 7 hours (P < 0.001).

CONCLUSIONOur preliminary study indicates that an adenovirus mediated transgene expression system of the hNIS under the control of hTERT promoter has the potential to become an effective wide-spectrum yet highly specific anti-cancer strategy.

Adenoviridae ; genetics ; Cell Line, Tumor ; Genetic Vectors ; genetics ; Humans ; Lung Neoplasms ; genetics ; therapy ; Promoter Regions, Genetic ; genetics ; Symporters ; genetics ; Telomerase ; genetics ; Transgenes ; genetics

OBJECTIVETo clone the full-length cDNA of a uridine diphosphate glycosyltransferase (UGT) gene which may be involved in saikosaponin biosynthesis of Bupleurum chinense, and construct the transgenic vectors for over expression and RNAi of the cloned UGT. These works will provide foundation for further its function analysis by transgene study.

METHODRAGE and LD-PCR were used to clone the full-length cDNA of the UGT, on the basis of its partial cDNA sequence obtained from our previous 454-sequenced dataset. The ORF and partial sequences of the UGT were PCR cloned using primers with corresponding restriction enzymes cutting sites. The PCR products were digested with corresponding restriction enzymes and then were inserted into pCAMBIA-SUPER 1 300 and pHANNIBAL. The recombinant pHANNIBAL were digested with Not I and then were inserted into a binary vector, pART27. Finally, the transgenic vectors for over expression and RNAi of the cloned UGT were constructed.

RESULTThe full-length cDNA of a UGT were cloned from B. chinense. The recombinant vectors for over expression and RNAi of the UGT were obtained.

CONCLUSIONOur works on full-length cDNA cloning and transgenic vectors construction provide a substantial foundation for follow-up biofunction analysis of the UGT through transgenic research.

Amino Acid Sequence ; Bupleurum ; genetics ; Cloning, Molecular ; Genetic Vectors ; Glucuronosyltransferase ; chemistry ; genetics ; Molecular Sequence Data ; RNA Interference ; Transgenes

To investigate the transgenic expressing efficacy of helper-dependent adenoviral vector (HDAd) in vitro, we constructed a HDAd encoding enhanced green fluorescent protein (EGFP), denominated as HDAd/EGFP, performed large scale preparation and purification, and then identified the purified HDAd/EGFP under fluorescent microscope and electron microscope. After the concentration of HDAd/EGFP was determined by spectrophotometer, the transgenic expression efficiency of HDAd/EGFP was compared with first generation adenoviral vector encoding EGFP (FGAd/EGFP) in vitro. Therefore, we infected A549 cells with 2000 virus particles (vp) per cell by HDAd/EGFP and FGAd/EGFP respectively and analyzed EGFP expressing level by flow cytometry. Consequently, the fluorescent expression rate and fluorescent intensity of EGFP were higher in early infected A549 cells by HDAd/EGFP than by FGAd/EGFP. HDAd, capable of expressing transgene instantly and efficiently in vitro, is a potential vaccine vector.
Adenoviridae ; genetics ; metabolism ; Cell Line, Tumor ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; Helper Viruses ; genetics ; metabolism ; Humans ; Transgenes ; Viral Fusion Proteins ; genetics ; metabolism

Animals ; CD40 Ligand ; genetics ; metabolism ; Carcinoma, Hepatocellular ; genetics ; metabolism ; therapy ; Genetic Therapy ; Liver Neoplasms ; genetics ; metabolism ; therapy ; Mice ; Neoplasms, Multiple Primary ; genetics ; metabolism ; therapy ; Transgenes ; genetics

OBJECTIVETo establish a chiro chromatography for studying the stereoselective metabolism of propranolol (PL) in S(9) incubates prepared from transgenic cell lines expressing human cytochrome P450.

METHODSThe concentration of each enantiomer in S(9) incubates was determined through precolumn derivatization with GITC, followed by RP-HPLC assay using S-(+)-propafenone as internal standard.

RESULTSBaseline separations among the diastereomers of S(-)-P, internal standard and R(+)-PL were achieved on Shimpack CLC C(18)ODS column, with UV detection and methanol:water:glacial acetic acid (67/33/0.05,v/v/v) as mobile phase. The assay was simple, accurate, precise and specific. The linear range was from 5 to 500 micromol/L for each enantiomer. The limit of quantitation (LOQ) for the method was 5 micromol/L for the S(-)-and R(+)-PL, respectively (n=5, RSD<10%). The analytical method afforded average recoveries of 98.7 and 98.1% for S(-)- and R(+)-PL, respectively. The reproducibility of the assay was good (RSD<10%). The time-dependent studies showed that PL had the stereoselectivity of S-(-)-isomer in metabolism via CYP2C18 and the stereoselectivity of R-(+)-isomer in metabolism via CYP2C9.

CONCLUSIONThe method allows to study of stereoselective metabolism of PL in vitro.

Chromatography, High Pressure Liquid ; Cytochrome P-450 Enzyme System ; genetics ; physiology ; Humans ; Propranolol ; analysis ; metabolism ; Reproducibility of Results ; Stereoisomerism ; Transgenes

Gene transfer technology is being used to enhance agronomic performance or improve quality traits in a wide variety of crop species. However, it is sometimes severely handicapped by difficulty in obtaining material in which transgene expression is predictable and stable over many generations. Because integration seemed to occur randomly in the plant genome, it was thought that some transgenes would be integrated in a relatively uncondensed, transcriptionally active chromatin environment, while others in a condensed, transcriptionally inert chromatin structure. Nuclear matrix attachment regions (MARs) are defined as DNA sequences that bind preferentially to the proteins of the nuclear matrix. They typically are localized at the borders of gene domains, implicating them in the formation of individual loops of higher order chromatin structure and transcription regulation. When MARs are positioned on either side of a transgene their presence usually results in higher and more stable espression in transgenic plants, most likely by minimizing gene silencing. In this review, we focus mainly on novel findings and our observations concerning the function of MARs in transcription regulation. Our objective is not only to summarize the current data and present several possible models to explain MAR effects on the transcription regulation, but also to point out some open questions involving the utilization of MARs in constructing high efficient expression vectors.
Chromatin ; physiology ; DNA ; metabolism ; Gene Expression Regulation, Plant ; Models, Genetic ; Nuclear Matrix ; metabolism ; Nuclear Proteins ; metabolism ; Transcription, Genetic ; Transgenes ; genetics

With the development of various biotechnology,the research on molecular genetics of medicinal plants has gradually deepened. In this paper,the research system of molecular genetics of medicinal plants was proposed for the first time,which was elaborated from the aspects of genetic resources,genome,gene function and research methods. The application fields of medicinal plant mainly contain species identification,molecular breeding and biosynthesis. The research directions of molecular genetics of medicinal plants in genetic resources,model platform,synthetic biology and molecular breeding were put forward,which include 1 000 genome projects of medicinal plants,model species and mutant libraries,gene original libraries of heterologous synthetic systems,construction gene original library and specific chassis cells in heterologous synthesis system of active ingredient,breeding of new varieties of medicinal plants with high active ingredient and high resistance based on molecular markers andtransgenes.
Biotechnology ; Gene Library ; Genetic Markers ; Genome, Plant ; Molecular Biology ; trends ; Plant Breeding ; Plants, Medicinal ; genetics ; Research ; Transgenes