Abstract: This disclosure provides methods and landing pad constructs for generation of parental cell lines suitable for targeted integration. A method is provided by the parental cell line development; this is, the introduction of binding sites of BPV1 E2 protein to landing pad vectors so that expressed BPV1 E2 protein could locate the vector to transcriptionally active region in the genome. Cells with high expression level of reporter genes are selected for the next stage and will be used in the development of cell lines expressing another recombinant protein by recombination mediated cassette exchange (RMCE). Landing pad constructs include recombination target sites for site-specific recombinases, and therefore, it could be replaced with gene-of-interest expression construct containing the same set of recombination target sites. This yields the generation of producer cell lines with less effort compared to traditional cell line development by random integration.

Abstract: This disclosure provides methods and landing pad constructs for generation of parental cell lines suitable for targeted integration. A method is provided by the parental cell line development; this is, the introduction of binding sites of BPV1 E2 protein to landing pad vectors so that expressed BPV1 E2 protein could locate the vector to transcriptionally active region in the genome. Cells with high expression level of reporter genes are selected for the next stage and will be used in the development of cell lines expressing another recombinant protein by recombination mediated cassette exchange (RMCE). Landing pad constructs include recombination target sites for site-specific recombinases, and therefore, it could be replaced with gene-of-interest expression construct containing the same set of recombination target sites. This yields the generation of producer cell lines with less effort compared to traditional cell line development by random integration.

Abstract: A U2OS-based model system using luciferase reporters to monitor the genome replication of alpha and beta HPVs is provided. A modified U2OS cell line is disclosed that expresses Firefly luciferase to measure toxicity of the screened compounds. In addition, provided are HPV18, HPV 16 and HPV5 marker genomes that express Renilla luciferase under the control of viral promoters used to measure changes in the viral copy number. This ready-to-use model system is capable of being used in high-throughput screens to identify compounds inhibiting initial amplification and stable maintenance as well as vegetative phase of various HPV subtypes.

Abstract: This disclosure provides methods and landing pad constructs for generation of parental cell lines suitable for targeted integration. A method is provided by the parental cell line development; this is, the introduction of binding sites of BPV1 E2 protein to landing pad vectors so that expressed BPV1 E2 protein could locate the vector to transcriptionally active region in the genome. Cells with high expression level of reporter genes are selected for the next stage and will be used in the development of cell lines expressing another recombinant protein by recombination mediated cassette exchange (RMCE). Landing pad constructs include recombination target sites for site-specific recombinases, and therefore, it could be replaced with gene-of-interest expression construct containing the same set of recombination target sites. This yields the generation of producer cell lines with less effort compared to traditional cell line development by random integration.

Abstract: A U2OS-based model system using luciferase reporters to monitor the genome replication of alpha and beta HPVs is provided. A modified U2OS cell line is disclosed that expresses Firefly luciferase to measure toxicity of the screened compounds. In addition, provided are HPV18, HPV 16 and HPV5 marker genomes that express Renilla luciferase under the control of viral promoters used to measure changes in the viral copy number. This ready-to-use model system is capable of being used in high-throughput screens to identify compounds inhibiting initial amplification and stable maintenance as well as vegetative phase of various HPV subtypes.