Attb cell line, transgenic cell lines derived therefrom, and methods of making the same

A method of producing a genetically modified mammalian cell comprising a recombination sequence inserted in a target locus on a chromosome of the Vero cell genome, wherein the recombination sequence comprises Bxb1 attB sequence from Mycobacterium smegmatis, the method comprising:

• providing a complex comprising
• a guide RNA comprising an oligonucleotide sequence that hybridizes with a target site on the target locus, and
• a Cas9 endonuclease;
• providing a single stranded DNA sequence comprising the attB sequence;
• and
• introducing the complex and the single stranded DNA sequence into a Vero cell to obtain a genetically modified Vero cell comprising the Bxb1 attB sequence inserted in the target locus.

A method of propagating a single cycle infectious virus comprising a genome having a deletion of an essential gene, the method comprising:

• providing a transgenic mammalian cell comprising at least one copy of the essential gene inserted in a target locus on a chromosome of the mammalian cell genome, wherein the transgenic mammalian cell constitutively expresses a protein encoded by the essential gene;
• contacting the transgenic mammalian cell with the single cycle infectious virus;
• and
• complementing the single cycle infectious virus with the protein expressed by the transgenic mammalian cell to propagate the single cycle virus.

A method for detecting and/or quantifying infectious virus in a sample, the method comprising:

• providing transgenic mammalian cells comprising a heterologous nucleic acid stably integrated in a target locus on a chromosome of the genome of the mammalian cells, wherein the heterologous nucleic acid comprises a virus promoter operably linked to a reporter gene;
• contacting the transgenic mammalian cells with the sample, wherein infectious virus present in the sample transactivates the virus promoter and induces expression of the reporter gene in the transgenic mammalian cells;
• and
• quantifying the number of mammalian cells expressing protein encoded by the reporter gene to quantify the infectious virus.