BACKGROUNDRev is necessary for exporting unspliced and incompletely spliced intron containing HIV mRNAs and for HIV replication. The aim of this study is to develop a kind of selective suicide construct that can specifically and directly induce HIV infected cells into apoptosis based on the high affinity of Rev and Rev response element (RRE).

METHODSMolecular-cloning technique was used to synthesis Rev dependent TNF-R1 expression construct pDM128-TNF-R1 (pT128) that contains RRE and TNFR1 gene. Restriction digestion, Polymerase Chain Reaction (PCR) and DNA sequencing were processed and the exactness and correctness of the inserted TNF-R1 gene in pT128 were confirmed repeatedly. The expression of pT128 co-transfected with different combination of other plasmids by calcium phosphate-DNA co-precipitation in Helas and by gene gun transfection in keratinocytes was further tested by flow-cytometry and cell counted under microscope.

RESULTSThe new plasmid specifically expressed TNF-R1 in Helas when co-transfected with pRev but did not when without pRev. Indirect expression of TNF-R1 from pT128 was slower than the direct expression of that from Hu p60 TNFR1 in pDC302 (pT60), but all those pT60 or pT128 transfected cells showed apoptosis at last while TNF-R1 was sufficiently expressed. Other kinds of Rev expression construct such as pAD8 and a chimeric HIV vaccine also can switched on the selective expression of pT128. Not only Rev-dependent expression in Helas, pT128 also normally expressed its TNF-R1 in keratinocytes. Co-transfected with pRev or pAD8 that expressed Rev, pT128 expressed TNF-R1 and induced apoptosis of green fluorescent keratinocytes in skin explant. The number of green fluorescent keratinocytes co-transfected by pT128 plus pRev or pAD8 was gradually outnumbered by that co-transfected by pT128 only. The difference was more significant after culturing for 72 hours.

CONCLUSIONSRev dependent pT128 is able to selectively induce apoptosis of HIV-infected or Rev-expressed target cells by expression of TNF-R1. The new strategy based on manipulation of the regulatory protein of HIV may be valuable in design of new HIV vaccine.

AIDS Vaccines ; immunology ; Apoptosis ; Biolistics ; Cell Line, Tumor ; Gene Products, rev ; physiology ; Genes, env ; physiology ; Genetic Vectors ; Humans ; Keratinocytes ; metabolism ; Plasmids ; Receptors, Tumor Necrosis Factor, Type I ; genetics

The Tagsk1 (Triticum asetium L. glycogen synthase kinase 1) gene derived from the genome of wheat salt-tolerance mutant RH8706-49 was cloned by PCR. The special primers designed according to full length cDNA sequence of Tagsk1 (AF525086). A binary expression vector pBI121-gsk1 containing Gus and Tagsk1 was constructed. And pBI121-gsk1 was introduced into the callus induced from mature embryos of salt-sensitive wheat H8706-34 and cv. China Spring by particle bombardment. The transformed callus were screened by Kanamycin and 0.5% NaCl. The salt-tolerance callus were obtained, which showed higher ability of salt-tolerance and could diffirentiate roots and buds on the medium containing 0.5% NaCl.
Adaptation, Physiological ; Biolistics ; DNA, Plant ; genetics ; Glycogen Synthase Kinases ; genetics ; Mutation ; Plant Proteins ; genetics ; Plants, Genetically Modified ; Salt-Tolerant Plants ; genetics ; Seeds ; genetics ; Sodium Chloride ; metabolism ; Transformation, Genetic ; Triticum ; enzymology ; genetics ; physiology

OBJECTIVETo investigate the inhibitory effect of DNA vaccine immunization on neu-overexpressed melanoma growth in prophylactic treatment and anti-lung-metastasis experiments in C57BL/6 mice.

METHODSpcDNA-neu transfected into B16F10 with transfection reagent Fugene 6, neu-overexpressed cell clone B16F10-neu was selected with limited dilution method. The growth curve was drawn to analyse its proliferating character in vitro. With Helios gene gun system, DNA vaccine pWRG-neu was immunized to 8-week-old C57BL/6 mice in the shaved abdominal skin for 3 times at two-weekly interval. After immunization, the life span was analyzed. Using MTT assay, the cytolysis activity of the DNA immunized mice spleen cells was compared.

RESULTSOne clone of neu-overexpressed B16F10-neu was selected and its proliferating character was the same as B16F10 and B16F10-pcDNA. In prophylactic, treatment and anti-lung-metastasis experiments, gene gun-mediated pWRG-neu immunization could exhibit antitumor effects. The growth and metastasis of neu-overexpressed melanoma was reduced dramatically. The spleen cells of the immuned mice showed cytotoxic T lymphocyte (CTL) activity.

CONCLUSIONGene gun-mediated gene transfer is effective and practicable. DNA vaccine pWRG-neu is potent in preventing subsequent tumor cells challenge, inhibiting the tumor growth and metastasis.

Animals ; Biolistics ; Cell Line, Tumor ; Cytotoxicity, Immunologic ; Genes, erbB-2 ; Immunization ; Lung Neoplasms ; prevention & control ; secondary ; Melanoma, Experimental ; metabolism ; pathology ; therapy ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Plasmids ; Receptor, ErbB-2 ; metabolism ; T-Lymphocytes, Cytotoxic ; immunology ; Vaccines, DNA