# US 5,707,830 A · Autonomous replicaton system for mammalian cells > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 5,707,830 A - Document ID: US5707830A - Status: Granted - Kind: A - Application number: 08/826,534 - Filed: 1997-04-02 - Granted: 1998-01-13 - Earliest claimed: 1994-05-20 - Assignees: The Board of Trustees of the Leland Stanford Junior University - Inventors: Michele Pamela Calos - Web page: https://patentgrep.com/patent/US5707830A ## CPC - C12N 15/85: for animal cells - C12N 2710/16222: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes - C12N 2800/108: episomal vectors - C12N 2830/006: tet repressible - C12N 2830/205: bidirectional - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract The present invention describes an expression vector useful for transfection of a selected mammalian host cell. The vector includes the following components: an Epstein Barr Virus (EBV) family of repeats; a copy of the EBV Nuclear Antigen-1 (EBNA-1) gene that can be functionally expressed in the host cell; a eucaryotic DNA fragment, which provides the ability of the vector to replicate in host cells; and an expression cassette which comprises a promoter functional in said host cell, a coding sequence having 5' and 3' ends, where said coding sequence is functionally linked to said promoter, where said 5' end is adjacent the promoter and said 3' end is adjacent transcription termination sequences. The vector of the present invention is useful in the transfection of mammalian cells, including rodent and human cells. The vector is stably retained and replicates in concert with genomic sequences of the host cell, that is, the vector is typically replicated once per cell cycle. The vector is useful in the follow methods involving mammalian cells: producing proteins, stable transformation, and gene therapy. ## Claims ### Claim 1 An expression useful for transfection of a host cell, comprising an Epstein Barr Virus (EBV) family of repeats, a copy of the EBV Nuclear Antigen-1 (EBNA-1) gene that can be functionally expressed in the host cell, a eucaryotic DNA fragment, which provides the ability of the vector to replicate in host cells, and an expression cassette which comprises a promoter functional in said host cell, a coding sequence having 5' and 3' ends, where said coding sequence is functionally linked to said promoter, where said 5' end is adjacent the promoter and said 3' end is adjacent transcription termination sequences. ### Claim 2 (depends on 1) The vector of claim 1, where said eucaryotic DNA fragment is randomly generated from human genomic DNA. ### Claim 3 (depends on 1) The vector of claim 1, which further includes DNA sequences that allow the propagation of the vector in a secondary host. ### Claim 4 (depends on 1) The vector of claim 1, where said vector further includes an expression cassette where said coding sequence encodes a gene useful for genetic selection in the host cell. ### Claim 5 (depends on 1) The vector of claim 1, where the coding sequence in said expression cassette is a reporter gene. ### Claim 6 (depends on 1) The vector of claim 1, where, in said expression cassette, said promoter is a cytomegalovirus promoter and said transcription termination sequences are upstream mouse sequences (UMS) or SV40 transcription termination sequences. ### Claim 7 (depends on 1) The vector of claim 1, wherein said coding sequence in said expression cassette encodes a polypeptide. ### Claim 8 (depends on 1) The vector of claim 1, wherein said coding sequence in said expression cassette encodes an RNA product. ### Claim 9 (depends on 1) A host cell transfected with the vector of claim 1. ### Claim 10 (depends on 2) The vector of claim 2, where said fragment is between about 8 and 20 kilobases. ### Claim 11 (depends on 3) The vector of claim 3, where said DNA sequences include an origin of replication and a selectable marker. ### Claim 12 (depends on 11) The vector of claim 11, where said secondary host is bacterial. ### Claim 13 (depends on 4) The vector of claim 4, where expression of the gene confers hygromycin resistance. ### Claim 14 (depends on 5) The vector of claim 5, where said reporter gene encodes chloramphenicol acetyl transferase, .beta.-galactosidase, or luciferase. ### Claim 15 (depends on 8) The vector of claim 8, wherein said coding sequence in said expression cassette encodes an antisense RNA. ### Claim 16 (depends on 8) The vector of claim 8, wherein said coding sequence in said expression cassette encodes a ribozyme. ### Claim 17 (depends on 9) The host cell of claim 9, where said host is a mammalian cell. ### Claim 18 (depends on 7) A method for expression of a protein in a host cell, comprising transfecting said host cell with the vector of claim 7, and culturing the host cell under suitable conditions so that the polypeptide is produced by said cell. ### Claim 19 (depends on 18) A method of claim 18, where said host cell is a mammalian cell. ### Claim 20 (depends on 18) A method of claim 18, which further includes isolating the polypeptide from the cell. ### Claim 21 A method of producing a stably transformed mammalian cell, comprising (i) selecting a mammalian host cell, (ii) preparing an expression vector which comprises an Epstein Barr Virus (EBV) family of repeats, a copy of the EBV Nuclear Antigen-1 (EBNA-1) gene that can be functionally expressed in the host cell, a eucaryotic DNA fragment, which provides the ability of the vector to replicate in host cells, and an expression cassette which comprises a promoter functional in said host cell, a coding sequence having 5' and 3' ends, where said coding sequence is functionally linked to said promoter, where said 5' end is adjacent to the promoter and said 3' end is adjacent to transcription termination sequences, (iii) introducing said expression vector into the host cell, and (iv) maintaining the host cell into which said expression vector has been introduced. ### Claim 22 (depends on 21) The method of claim 21, where said mammalian cell is a rodent cell. ### Claim 23 (depends on 21) The method of claim 21, where said cell is a human cell. ### Claim 24 (depends on 21) The method of claim 21, where said maintaining includes culturing the cell in vitro.