Rolling circle replicon expression vector

A method of making a rolling circle DNA replicon which replicates in a host eukaryotic cell, comprising combining:

• (a) a Rep gene open reading frame from a virus belonging to the viral taxonomic families Geminiviridae, Circoviridae or genus Nanovirus, said Rep gene open reading frame is placed under transcriptional control of a promoter, which promoter is placed 5′ of the gene;
• (b) any sequences that are required to be present in cis on the rolling circle DNA replicon in order that the Rep protein might promote replication of the rolling circle DNA replicon;
• (c) an expression cassette for expression of an ancillary protein that is capable of creating a cellular environment permissive for replication of the rolling circle DNA replicon in the host cell of interest;
• and
• (d) at least one expression cassette with an RNA polymerase II promoter, a multiple cloning site, and transcription termination and polyadenylation signals suitable for transcription of RNA molecules not normally intrinsic to a geminiviral, circoviral or nanoviral genome.

A method of making a rolling circle DNA replicon which replicates in a host eukaryotic cell which replicon has a truncated replication cycle, comprising combining:

• (a) a Rep gene open reading frame from a virus belonging to the viral taxonomic families Geminiviridae, Circoviridae or genus Nanovirus, said Rep gene open reading frame is placed under transcriptional control of a promoter, which promoter is placed 5′ of the gene;
• (b) sequences that are required to be present in cis on the rolling circle DNA replicon in order that the Rep protein might promote replication of the rolling circle DNA replicon;
• (c) an expression cassette for expression of an ancillary protein that is capable of creating a cellular environment permissive for replication of the rolling circle DNA replicon in the host cell of interest;
• and
• (d) at least one expression cassette with an RNA polymerase II promoter, a multiple cloning site, and transcription termination and polyadenylation signals suitable for transcription of RNA molecules not normally intrinsic to a geminiviral, circoviral or nanoviral genome.

A method of discovering the function of a gene or a gene segment in a host eukaryotic cell, the method comprising:

• (a) inserting a gene or a gene segment of interest into a rolling circle DNA replicon;
• (b) inserting a rolling circle DNA replicon into in a host eukaryotic cell;
• and
• (c) discovering a biochemical or phenotypic change in the in a host eukaryotic cell.

An RCR replicon comprising:

• (a) a Rep gene ORF from a virus belonging to the viral taxonomic families Geminiviridae, Circoviridae or genus Nanovirus, wherein said Rep gene ORF is placed under transcriptional control of a promoter, which promoter is placed 5′-end of the gene;
• (b) a sequence required to be present in cis on a RCR replicon in order that a Rep protein promotes replication of the RCR replicon;
• (c) an expression cassette for expression of an ancillary protein that is capable of creating a cellular environment permissive for replication of the RCR replicon in the host cell of interest;
• and
• (d) at least one expression cassette with a RNA polymerase II promoter, a multiple cloning site, and transcription termination and polyadenylation signals suitable for transcription of RNA molecules not normally intrinsic to a geminiviral, circoviral or nanoviral genome.