Phage integrases for the construction and manipulation of transgenic mammals.

Phage integrases catalyze site-specific, unidirectional recombination between two short att recognition sites. Recombination results in integration when the att sites are present on two different DNA molecules and deletion or inversion when the att sites are on the same molecule. Here we demonstrate the ability of the phiC31 integrase to integrate DNA into endogenous sequences in the mouse genome following microinjection of donor plasmid and integrase mRNA into mouse single-cell embryos.

Epstein-Barr virus vectors provide prolonged robust factor IX expression in mice.

We demonstrate that vectors incorporating components from Epstein-Barr virus (EBV) for retention and from human genomic DNA for replication greatly enhance the level and duration of marker gene expression in dividing cultured cells. The same types of vectors were tested in vivo by high-pressure tail vein injection of naked DNA in mice, resulting in liver delivery and expression. The therapeutic gene was a human factor IX (hFIX) minigene comprising genomically derived 5', 3', and intronic sequences that provided relatively good gene expression in vivo.

Directed evolution of a recombinase for improved genomic integration at a native human sequence.

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.

Epstein-Barr virus/human vector provides high-level, long-term expression of alpha1-antitrypsin in mice.

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.

An extrachromosomal tetracycline-regulatable system for mammalian cells.

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.

Assaying extrachromosomal gene therapy vectors that carry replication/persistence elements.

Persistence in the cell is a desirable property for most gene therapy vectors. For extrachromosomal vectors, persistence is limited in most cell types. To address this problem, we have developed vectors with the ability to replicate and be retained in the nucleus.

Epstein-Barr virus vectors for gene expression and transfer.

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.

Physiological characterization of blastocyst hatching mechanisms by use of a mouse antihatching model.

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.