ABSTRACT
We have constructed plasmid DNA vectors that contain Epstein-Barr virus (EBV) sequences and the human gene (SERPINA1) encoding alpha1-Antitrypsin (AAT). We demonstrate that a plasmid carrying the full SERPINA1 on a 19-kb genomic fragment and the EBV gene EBNA1 and its family of repeats binding sites undergoes efficient extrachromosomal replication in dividing mammalian tissue culture cells. Therefore, use of a whole genomic therapeutic gene to provide both replication and gene expression may be an effective gene therapy vector design, if the target cells are dividing. The efficacy of this same vector for expression of AAT in vivo in the nondividing cells of mouse liver was determined by hydrodynamic injection of naked plasmid DNA by means of the tail vein. A single injection of an EBV/genomic SERPINA1 vector provided >300 microg/ml of AAT, which approached normal plasma levels and persisted for the >9-month duration of the experiment. These data exceed most previously reported values, probably due to sequences in the genomic DNA that resist silencing of gene expression, possibly in combination with favorable effects on expression provided by the EBV sequences. These results demonstrate that plasmid DNA with the correct cis-acting sequences can provide in vivo long-term expression of protein at high levels that are therapeutically relevant for gene therapy.
Subject(s)
Gene Transfer Techniques , Genetic Vectors , Herpesvirus 4, Human/genetics , alpha 1-Antitrypsin/genetics , Animals , Blotting, Southern , Cell Line , DNA Replication/physiology , Herpesvirus 4, Human/metabolism , Humans , Liver/physiology , Mice , Mice, Inbred C57BL , Plasmids , Trypsin Inhibitors/biosynthesis , Trypsin Inhibitors/genetics , alpha 1-Antitrypsin/biosynthesisABSTRACT
We previously established that a unidirectional site-specific recombinase, the phage phiC31 integrase, can mediate integration into mammalian chromosomes. The enzyme directs integration of plasmids bearing the phage attB recognition site into pseudo attP sites, a set of native sequences related to the phage attP recognition site. Here we use two cycles of DNA shuffling and screening in Escherichia coli to obtain evolved integrases that possess significant improvements in integration frequency and sequence specificity at a pseudo attP sequence located on human chromosome 8, when measured in the native genomic environment of living human cells. Such integrases represent custom integration tools that will be useful for modifying the genomes of higher eukaryotic cells.
Subject(s)
Directed Molecular Evolution/methods , Integrases/genetics , Recombination, Genetic , Base Sequence , Binding Sites , Cell Line , DNA/genetics , DNA/metabolism , Genome, Human , Humans , Integrases/metabolism , Sequence Homology, Nucleic AcidABSTRACT
We have modified the tetracycline-regulatable system so that all components are present on a stable extrachromosomal vector that can replicate in a wide variety of mammalian cells. An EBV/human ori vector is used to carry the system, overcoming the species specificity of conventional Epstein-Barr virus vectors. By placing the transcriptional transactivator gene under autoregulation, better induction characteristics are obtained. This system offers greater speed and sensitivity than previously reported methods. It can be applied within 3-4 weeks and produces an induction range of several hundred-fold with a low background.
Subject(s)
Anti-Bacterial Agents/pharmacology , Doxycycline/pharmacology , Gene Expression Regulation , Genetic Vectors , Herpesvirus 4, Human/genetics , Animals , Cell Line , Humans , Luciferases/biosynthesis , Luciferases/genetics , Mice , Plasmids , Promoter Regions, Genetic , Regulatory Sequences, Nucleic Acid , Replication Origin , Trans-Activators/genetics , TransfectionABSTRACT
Vectors based on components of Epstein-Barr virus (EBV) have found increasingly wide applications in biotechnology. Three areas of recent advancement comprise the use of EBV vectors to improve the convenience of gene expression systems, the development of EBV vectors for gene transfer in gene therapy, and the use of EBV components to generate large vectors carrying sizable regions of genomic DNA.
Subject(s)
Genetic Vectors , Herpesvirus 4, Human/genetics , Animals , Biotechnology , Gene Expression , Gene Transfer Techniques , Genetic Therapy , HumansABSTRACT
OBJECTIVES: To use the protein-free medium blastocyst antihatching model to characterize physiological events that mediate hatching. DESIGN: In a series of four prospective experiments, our aims were to [1] test the efficacy of the antihatching model and assisted hatching; [2] exam the influence of initial in vivo developmental stage and late serum supplementation on hatching inhibition; [3] discount the role of zona hardness and physical expansion directly affecting hatching; and [4] provide evidence that the trophectoderm is directly responsible for secreting a zona lysin. SETTING: University-based research laboratory. RESULTS: Culturing two- to eight-cell mouse embryos in serum-free human tubal fluid (HTF) medium significantly reduced hatching levels to < or = 2%, however, hatching increased to 10.7% when initially culturing morula-stage embryos. Hatching was effectively rescued to control levels when embryos were placed in HTF with serum at the early blastocyst stage. There was no difference in blastocyst total cell numbers or zona pellucida digestion intervals between culture treatments. Finally, we showed that trophectodermal vesicles, devoid of inner cell mass, are capable of hatching under control conditions. CONCLUSIONS: The primary mechanism of blastocyst hatching is not physical expansion and abnormal zona hardness is not responsible for hatching inhibition. Certain extracellular precursors found in serum (e.g., amino acids) are required in culture medium upon cellular determination of trophectoderm (i.e., morula to blastocyst stage) to facilitate the intrinsic secretion of an undefined hatching factor.
