Mammalian cells are described that includes a recombination target site integrated within high integrating locus. Recombinant protein producer cell lines incorporating the mammalian cells and methods for forming the mammalian cells are also described. The high integrating loci have been developed through understanding and mapping of the three dimensional hierarchical structure of chromatin in mammalian cells. The high integrating loci are present in transcriptionally active environments that can provide both chromatin accessibility and epigenetic stability. As such, the recombinant mammalian cells can provide predictable and stable transgene production.

The present invention relates to a transfer vector for inserting a gene into a genetic locus of a baculovirus sequence. The transfer vector comprises an expression cassette comprising a eukaryotic promoter operably linked to the gene and a bipartite selection cassette. The present invention also relates to methods of using the transfer vector and derived bacmids and baculoviruses.

Disclosed is a mouse artificial chromosome vector, comprising: a natural centromere derived from a mouse chromosome; a mouse-chromosome-derived long-arm fragment formed by deleting a long-arm distal region at a mouse chromosome long-arm site proximal to the centromere; and a telomere sequence, wherein the vector is stably retained in a cell and/or tissue of a mammal. In addition, disclosed are cells or non-human animals comprising the vector, and use of the cells or non-human animals.

Circular nucleic acid vectors that provide for persistently high levels of protein expression are provided. The circular vectors of the subject invention are characterized by being devoid of expression-silencing bacterial sequences, where in many embodiments the subject vectors include a unidirectional site-specific recombination product hybrid sequence in addition to an expression cassette. Also provided are methods of using the subject vectors for introduction of a nucleic acid, e.g., an expression cassette, into a target cell, as well as preparations for use in practicing such methods. The subject methods and compositions find use in a variety of different applications, including both research and therapeutic applications. Also provided is a highly efficient and readily scalable method for producing the vectors employed in the subject methods, as well as reagents and kits/systems for practicing the same.

The present invention relates to a method based on the use of restriction enzyme digestion and ligation via cleavage sites, thereby to prepare two or more standardized expression cassettes.

Described herein are donor vectors and systems for use in dual recombinase-mediated cassette exchange. Also described herein are animal models and human cells for consistent, rigorous, and facile investigation of transgene expression. Further described herein are methods of screening for therapeutic drugs using these animal models, and methods of treatment.

The present disclosure provides methods and compositions for genetically modifying lymphocytes, for example T cells and/or NK cells, in shorter times than previously and/or in whole blood or a component thereof. In some embodiments a lymphodepletion filter assembly is used before or after forming a reaction mixture where lymphocytes are contacted with recombinant retroviral particles in a closed system, to genetically modify the lymphocytes.

The present invention is transgenic chickens obtained from long-term cultures of avian PGCs and techniques to produce and transgenic birds derived from prolonged PGC cultures. In some embodiments, these PGCs can be transfected with genetic constructs to modify the DNA of the PGC, specifically to introduce a transgene encoding an exogenous protein. When combined with a host avian embryo by known procedures, those modified PGCs are transmitted through the germline to yield transgenic offspring. This invention includes compositions comprising long-term cultures of PGCs and offspring derived from them that are genetically modified. The genetic modifications introduced into PGCs to achieve the gene inactivation may also include, but are not restricted to, random integrations of transgenes into the genome, transgenes inserted into the promoter region of genes, transgenes inserted into repetitive elements in the genome, site specific changes to the genome that are introduced using integrase, site specific changes to the genome introduced by homologous recombination, and conditional mutations introduced into the genome by excising DNA that is flanked by lox sites or other sequences that are substrates for site specific recombination.

An artificial recombinant chromosome produced by recombination of two or more chromosomes and the production of a transgenic animal using a cell including the same are disclosed. A method for producing a cell including one or more artificial recombinant chromosomes includes preparing a first targeted cell and a second targeted cell, producing one or more microcells using the second targeted cell, producing a fusion cell using the first targeted cell and the one or more microcells, and producing a cell including an artificial recombinant chromosome by treating the fusion cell with site specific recombinase (SSR).

Methods of constructing a cell comprising in its chromosome one or more copies of an open reading frame (ORF) or operon encoding at least one polypeptide of interest, each copy being under the transcriptional control of a heterologous promoter using a site specific recombinase and in vivo integration by recombination; resulting cells, and methods for producing a polypeptide of interest using the resulting cells.