The disclosure relates to a method of reprogramming one or more somatic cells, e.g., partially differentiated or fully/terminally differentiated somatic cells, to a less differentiated state, e.g., a pluripotent or multipotent state. In further embodiments the invention also relates to reprogrammed somatic cells produced by methods of the invention, to chimeric animals comprising reprogrammed somatic cells of the invention, to uses of said cells, and to methods for identifying agents useful for reprogramming somatic cells.

Disclosed is a new process for the production of recombinant proteins, by transient transfection of suspension-grown human embryonic kidney cells (293 cell line and its genetic variants) with an expression vector, using polyethylenimine (PEI) as a transfection reagent. In a preferred embodiment, the process uses 293E cells expressing the Epstein-Barr virus (EBV) EBNA 1 protein, in combination with an oriP-based episomal expression vector having an improved cytomegalovirus expression cassette comprising the CMV5 promoter. The process combines in a single step the cell growth, transfection and protein expression, is carried out without changing the culture medium, and allows to achieve high expression levels in a short period of time. The process may be carried out in a serum-free, low-protein culture medium, is easily scalable, compatible with continuous production processes, and fully adapted to high-throughput production of milligram quantities of recombinant proteins.

Non-conventional yeasts are disclosed herein comprising at least one RNA-guided endonuclease (RGEN) comprising at least one RNA component that does not have a 5-cap. This uncapped RNA component comprises a sequence complementary to a target site sequence in a chromosome or episome in the yeast. The RGEN can bind to, and optionally cleave, one or both DNA strands at the target site sequence. An example of an RGEN herein is a complex of a Cas9 protein with a guide RNA. A ribozyme is used in certain embodiments to provide an RNA component lacking a 5-cap. Further disclosed are methods of genetic targeting in non-conventional yeast.

Disclosed and described herein are nucleic acids encoding HIV broadly neutralizing antibodies. These antibodies are useful for treating or maintaining low viral loads in HIV infected individuals. The nucleic acids described herein are suitable for administration to individuals and provide long-lasting expression and secretion of the broadly neutralizing antibodies described into an individual's blood-stream.

Methods are disclosed for reprogramming a somatic cell, including an adherent cell and a cell in suspension, into an induced pluripotent stem comprising expressing exogenous Sox-2, exogenous Klf-4, exogenous Oct3/4 from DNA that has not integrated into the genome of the somatic cell, suppressing p53 activity within the somatic cell, and exposing the somatic cell to reprogramming-assistance factors comprising an exogenous Alk-5 inhibitor, an exogenous histone deacetylase inhibitor, and an exogenous activator of glycolysis. Compositions and kits for use in such methods are also disclosed as are cells made by such a method.

Methods of producing hepatocyte lineage cells are provided. The method can include transfecting a cell with one or more expression vectors. For example, a cell can be transfected with a first expression vector containing a first gene that encodes CCAAT/enhancer binding protein alpha (CEBPA), a second expression vector containing a second gene that encodes CCAAT/enhancer binding protein beta (CEBPB), a third expression vector containing a third gene that encodes forkhead box A1 (FOXA1), and a fourth expression vector containing a fourth gene that encodes forkhead box A3 (FOXA3). The method can include culturing the transfected cell obtained in a growth environment. The transfected cell can be cultured in Williams' E medium, ReproFF (feeder-free media maintaining pluripotency) medium, or both. Transfected and/or hepatocyte lineage cells obtained by a method of the present invention are also provided.

Disclosed is a new process for the production of recombinant proteins, by transient transfection of suspension-grown human embryonic kidney cells (293 cell line and its genetic variants) with an expression vector, using polyethylenimine (PEI) as a transfection reagent. In a preferred embodiment, the process uses 293E cells expressing the Epstein-Barr virus (EBV) EBNA 1 protein, in combination with an oriP-based episomal expression vector having an improved cytomegalovirus expression cassette comprising the CMV5 promoter. The process combines in a single step the cell growth, transfection and protein expression, is carried out without changing the culture medium, and allows to achieve high expression levels in a short period of time. The process may be carried out in a serum-free, low-protein culture medium, is easily scalable, compatible with continuous production processes, and fully adapted to high-throughput production of milligram quantities of recombinant proteins.

A Saccharomyces cerevisiae expression system and a construction method and application thereof, including an expression vector which includes, from 5 to 3, a YEplac195 plasmid backbone, an exogenous gene expression cassette, and a selective marker gene expression cassette. The exogenous gene expression cassette includes from upstream to downstream an rDNA promoter, an internal ribosome entry site (IRES) sequence, an exogenous gene expression cassette, a poly(T) sequence, and an rDNA terminator. The selective marker gene expression cassette includes a promoter, a selective marker gene, and a transcription terminator.

The invention provides compositions and methods for manufacturing pluripotent cells. In particular, the invention provides improved culture platforms for manufacturing pluripotent cells with ground state pluripotency.

A eukaryotic expression vector capable of displaying a multi-chain polypeptide on the surface of a host cell is provided, such that the biological activity of the multi-chain polypeptide is exhibited at the surface of the host cell. Such a vector allows for the display of complex biologically active polypeptides, e.g., biologically active multi-chain polypeptides such as immunoglobulin Fab fragments. The present invention describes and enables the successful display of a multi-chain polypeptide on the surface of a eukaryotic host cell. Preferred vectors are described for expressing the chains of a multi-chain polypeptide in a host cell separately and independently (e.g., under separate vector control elements, and/or on separate expression vectors, thus forming a matched vector set). The use of such matched vector sets provides flexibility and versatility in the generation of eukaryotic display libraries, for example the ability to generate and to display multi-chain polypeptides by combining and recombining vectors that express variegations of the individual chains of a multi-chain polypeptide. Entire repertoires of novel chain combinations can be devised using such vector sets.